free full text journal articles: molecular and cellular biology




Recent Articles in Molecular Biology of the Cell

Badowski C, Pawlak G, Grichine A, Chabadel A, Oddou C, Jurdic P, Pfaff M, Albigčs-Rizo C, Block MR
Paxillin Phosphorylation Controls Invadopodia/Podosomes Spatiotemporal Organization.
Mol Biol Cell. 2007 Nov 28; .
Monitoring Editor: Mark Ginsberg In RSV-transformed BHK cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins while the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118 which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self organization. Similar mechanism is unraveled in osteoclasts using paxillin knockdown. Lack of paxillin phosphorylation, calpain or Erk inhibition, result in similar phenotype suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and transmigration through a cell monolayer. [Abstract/Link to Full Text]

Casey L, Patterson EE, Müller U, Fox CA
Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S-Phase Cyclin.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Orna Cohen-Fix Silencing of the mating-type locus HMR in S. cerevisiae requires DNA elements called silencers. To establish HMR silencing the Origin Recognition Complex binds the HMR-E silencer and recruits the Sir1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1Delta cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1(hc)), a conserved forkhead transcription factor, or by deletion of the S-phase cyclin CLB5 (clb5Delta). FKH1(hc) caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1(hc) prolonged the cell cycle in a manner distinct from deletion of its close paralog FKH2, and created a cell cycle phenotype more reminiscent to that caused by a clb5Delta. Unexpectedly, and in contrast to SIR1, both FKH1(hc) and clb5Delta established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRDeltaE::ARS). HMRDeltaE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRDeltaE::ARS1 was reduced by clb5Delta or FKH1(hc), while ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRDeltaE::ARS1 did not result from formation of Sir2-4 chromatin since sir2Delta did not rescue origin firing in clb5Delta cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway. [Abstract/Link to Full Text]

Granell S, Baldini G, Mohammad S, Nicolin V, Narducci P, Storrie B, Baldini G
Sequestration of Mutated {alpha}1-Antitrypsin Into Inclusion Bodies Is a Cell Protective Mechanism to Maintain Endoplasmic Reticulum Function.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Thomas Sommer A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers and is associated with liver disease. In the hepatocyte of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell-type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulphide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway. [Abstract/Link to Full Text]

Ansbach AB, Noguchi C, Klansek IW, Heidlebaugh M, Nakamura TM, Noguchi E
RFCCtf18 and the Swi1-Swi3 Complex Function in Separate and Redundant Pathways Required for the Stabilization of Replication Forks to Facilitate Sister Chromatid Cohesion in Schizosaccharomyces pombe.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Mark Solomon Sister chromatid cohesion is established during S-phase near the replication fork. However, how DNA replication is coordinated with chromosomal cohesion pathway is largely unknown. Here we report studies of fission yeast Ctf18, a subunit of the RFC(Ctf18) replication factor C complex, and Chl1, a putative DNA helicase. We show that RFC(Ctf18) is essential in the absence of the Swi1-Swi3 replication fork protection complex required for the S-phase stress response. Loss of Ctf18 leads to an increased sensitivity to S-phase stressing agents, a decreased level of Cds1 kinase activity, and accumulation of DNA damage during S-phase. Ctf18 associates with chromatin during S-phase and is required for the proper resumption of replication after fork arrest. We also show that chl1Delta is synthetically lethal with ctf18Delta and that a dosage increase of chl1(+) rescues sensitivities of swi1Delta to S-phase stressing agents, indicating that Chl1 is involved in the S-phase stress response. Finally, we demonstrate that inactivation of Ctf18, Chl1 or Swi1-Swi3 leads to defective centromere cohesion, suggesting the role of these proteins in chromosome segregation. We propose that RFC(Ctf18) and the Swi1-Swi3 complex function in separate and redundant pathways essential for replication fork stabilization to facilitate sister chromatid cohesion in fission yeast. [Abstract/Link to Full Text]

Paroni G, Cernotta N, Russo CD, Gallinari P, Pallaoro M, Foti C, Talamo F, Orsatti L, Steinkühler C, Brancolini C
PP2A Regulates HDAC4 Nuclear Import.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Karsten Weis Different signal-regulated serine/threonine kinases phosphorylate class II HDACs to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme Calpha, Aalpha, B/PR55alpha. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid (OA) or RNAi have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import. [Abstract/Link to Full Text]

Roth L, Nasarre C, Dirrig-Grosch S, Aunis D, Crémel G, Hubert P, Bagnard D
Transmembrane Domain Interactions Control Biological Functions Neuropilin-1.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Carl-Henrik Heldin Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling. [Abstract/Link to Full Text]

Peng Y, Liu X, Schoenberg DR
Hsp90 Stabilizes the PMR1 mRNA Endonuclease to Degradation by the 26S Proteasome.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: A. Gregory Matera The PMR1 mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that Hsp90 is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes. [Abstract/Link to Full Text]

Kodani A, Sütterlin C
The Golgi Protein GM130 Regulates Centrosome Morphology and Function.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Vivek Malhotra The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNAi-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. While GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase. [Abstract/Link to Full Text]

Cammarato A, Dambacher CM, Knowles AF, Kronert WA, Bodmer R, Ocorr K, Bernstein SI
Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Thomas Pollard Striated muscle myosin is a multi-domain ATP-dependent molecular motor. Alterations to various domains affect the motor's chemomechanical properties and are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here we helped define the transducer's role by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function as well as skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility while Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but induces degradation of the myofibrillar apparatus, likely as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing myosin's chemomechanical properties and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. [Abstract/Link to Full Text]

Klein RM, Spofford LS, Abel EV, Ortiz A, Aplin AE
B-RAF Regulation of Rnd3 Participates in Actin Cytoskeletal and Focal Adhesion Organization.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Jean Schwarzbauer The actin cytoskeleton controls multiple cellular functions including cell morphology, movement and growth. Accumulating evidence indicates that oncogenic activation of the MEK/ERK1/2 pathway is accompanied by actin cytoskeletal reorganization. The signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation, however, are largely unknown. Mutant B-RAF is found in a wide variety of cancers including melanoma, and enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with siRNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/ROCK/LIM kinase-2 signaling pathway cumulating in the inactivation of the actin depolymerizing/severing protein, cofilin. The expression of Rnd3, a Rho antagonist, was attenuated following B-RAF knockdown or MEK inhibition, but was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Taken together our results identify Rnd3 as a regulator of cross-talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions. [Abstract/Link to Full Text]

Cheeseman IM, Hori T, Fukagawa T, Desai A
KNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates.
Mol Biol Cell. 2007 Nov 28;
Monitoring Editor: Kerry Bloom Chromosome segregation during mitosis requires the assembly of a large proteinaceous structure termed the kinetochore. In Caenorhabditis elegans, KNL-1 is required to target multiple outer kinetochore proteins. Here, we demonstrate that the vertebrate KNL1 counterpart is essential for chromosome segregation and is required to localize a subset of outer kinetochore proteins. However, unlike in C. elegans, depletion of vertebrate KNL1 does not abolish kinetochore localization of the microtubule-binding Ndc80 complex. Instead, we show that KNL1 and CENP-K, a subunit of a constitutively centromere-associated complex that is missing from C. elegans, coordinately direct Ndc80 complex localization. Simultaneously reducing both hKNL1 and CENP-K function abolishes all aspects of kinetochore assembly downstream of centromeric chromatin and causes catastrophic chromosome segregation defects. These findings explain discrepancies in kinetochore assembly pathways between different organisms and reveal a surprising plasticity in the assembly mechanism of an essential cell division organelle. [Abstract/Link to Full Text]

Blish KR, Wang W, Willingham MC, Du W, Birse CE, Krishnan SR, Brown JC, Hawkins GA, Garvin AJ, D'Agostino RB, Torti FM, Torti SV
A Human Bone Morphogenetic Protein Antagonist is Down-Regulated in Renal Cancer.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Carl-Henrik Heldin We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a BMP antagonist, SOSTDC1 (SclerOSTin Domain-Containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p <0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7-induced phosphorylation of R-Smads-1, -5 and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma. [Abstract/Link to Full Text]

Galvan C, Camoletto PG, Cristofani F, Van Veldhoven PP, Ledesma MD
Anomalous Surface Distribution of GPI-anchored Proteins in Neurons Lacking Acid Sphingomyelinase.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sean Munro Acid sphingomyelinase (ASM) converts sphingomyelin (SM) into ceramide. Mutations in the ASM gene cause the mental retardation syndrome Niemann Pick type A (NPA) characterized as a lysosomal disorder because of the SM accumulation in these organelles. We here report that neurons from mice lacking ASM (ASMKO) present increased plasma membrane SM levels evident in detergent resistant membranes. Paralleling this lipidic alteration, GPI-anchored proteins show an aberrant distribution in both axons and dendrites instead of the axonal enrichment observed in neurons from wild type mice. Trafficking analysis suggests that this is due to defective internalization from dendrites. Increasing the SM content in wild type neurons mimics these defects while SM reduction in ASMKO neurons prevents their occurrence. Moreover, expression of active RhoA, which membrane attachment is affected by SM accumulation, rescues internalization rates in ASMKO neurons. These data unveil an unexpected role for ASM in neuronal plasma membrane organization and trafficking providing insight on the molecular mechanisms involved. They also suggest that deficiencies in such processes could be key pathological events in NPA disease. Keywords: sphingomyelin; lipid microdomains; Niemann Pick type A; GPI-anchored proteins; endocytosis. [Abstract/Link to Full Text]

Oganesian A, Armstrong LC, Migliorini MM, Strickland DK, Bornstein P
Thrombospondins Use the VLDL Receptor and a Non-Apoptotic Pathway to Inhibit Cell Division in Microvascular Endothelial Cells.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Josephine Adams TSPs 1 and 2 function as endogenous inhibitors of angiogenesis. Although TSPs have been shown to induce apoptosis in HMVEC, we reasoned that a homeostatic mechanism would also be needed to inhibit EC growth without causing cell death, e.g., in the maintenance of a normal vascular endothelium. HMVEC, cultured in low serum, responded to VEGF with an increase in [(3)H]thymidine incorporation that was inhibited by TSPs and was accompanied by decreases in the phosphorylation of Akt and MAPK, without an increase in apoptosis. RAP, an inhibitor of the LDL family of endocytic receptors, and blocking antibodies to VLDLR, were as effective as TSPs in the inhibition of thymidine uptake in response to VEGF, and the effects of these agents were not additive. Supportive evidence for the role of the VLDLR in mediating this inhibition was provided by the demonstration of a high-affinity interaction between TSPs and the VLDLR. We propose that TSP1 and TSP2, together with the VLDLR, initiate a nonapoptotic pathway for maintenance of the normal adult vascular endothelium in a quiescent state, similar to that invoked for the regulation of mitogenesis by PDGF, but involving signaling via the VLDLR rather than LRP1. [Abstract/Link to Full Text]

Rue SM, Mattei S, Saksena S, Emr SD
Novel Ist1-Did2 Complex Functions at a Late Step in MVB Sorting.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sean Munro In S. cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport (ESCRTs). Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Delta is synthetic with vta1Delta and vps60Delta, indicating that Ist1 is also a positive component of the MVB sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Delta mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB sorting pathway. [Abstract/Link to Full Text]

Froget B, Blaisonneau J, Lambert S, Baldacci G
Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Wendy Bickmore During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed. [Abstract/Link to Full Text]

Dimaano C, Jones CB, Hanono A, Curtiss M, Babst M
Ist1 Regulates Vps4 Localization and Assembly.
Mol Biol Cell. 2007 Nov 21;
Monitoring Editor: Sandra Lemmon The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities. [Abstract/Link to Full Text]

Lam AD, Tryoen-Toth P, Tsai B, Vitale N, Stuenkel EL
SNARE-catalyzed Fusion Events Are Regulated by Syntaxin1A Lipid Interactions.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Patrick Brennwald Membrane fusion is a process that intimately involves both proteins and lipids. While the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events. [Abstract/Link to Full Text]

Kim J, Shao Y, Kim SY, Kim S, Song HK, Jeon JH, Woo Suh H, Chung JW, Yoon SR, Kim YS, Choi I
Hypoxia-induced IL-18 Increases Hypoxia-inducible Factor-1{alpha} Expression through a Rac1-dependent NF-{kappa}B Pathway.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: John Cleveland Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Mechanistically, IL-18 induces HIF-1alpha through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor beta (IL-18Rbeta) subunit, via a PI3K-AKT-NF-kappaB-dependent pathway. Importantly, the knockdown of the IL-18Rbeta subunit inhibited IL-18-driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1alpha-mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1alpha and tumor cell metastasis." [Abstract/Link to Full Text]

Manolea F, Claude A, Chun J, Rosas J, Melançon P
Distinct Functions for Arf Nucleotide Exchange Factors at the Golgi Complex: GBF1 and BIGs Are Required for Assembly and Maintenance of the Golgi Stack and TGN, Respectively.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Benjamin Glick We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, while BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the ER, but caused its accumulation into peripheral vesiculo-tubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, BIGs knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, while GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization and cargo progression to the cell surface. [Abstract/Link to Full Text]

Avezov E, Frenkel Z, Ehrlich M, Herscovics A, Lederkremer GZ
ER Mannosidase I Is Compartmentalized and Required for N-Glycan Trimming to Man5 6GlcNAc2 in Glycoprotein ER-associated Degradation.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Reid Gilmorez We had previously shown that ER-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of 3-4 mannose residues from the N-linked oligosaccharide Man9GlcNAc2. A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to 4 alpha1,2-linked mannose residues. Using siRNA knock-down of ERManI, we show that this enzyme is required for trimming to Man5-6GlcNAc2 and for ERAD in cells in vivo, leading to the accumulation of Man9GlcNAc2 and Glc1Man9GlcNAc2 on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI appears strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knock-down prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly-made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation. [Abstract/Link to Full Text]

Takeshita N, Higashitsuji Y, Konzack S, Fischer R
Apical Sterol-rich Membranes Are Essential for Localizing Cell End Markers that Determine Growth Directionality in the Filamentous Fungus Aspergillus nidulans.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: David Drubin In filamentous fungi hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell-end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell-end factor destinations and thereby polarized growth. [Abstract/Link to Full Text]

Dickinson BL, Claypool SM, D'Angelo JA, Aiken ML, Venu N, Yen EH, Wagner JS, Borawski JA, Pierce AT, Hershberg R, Blumberg RS, Lencer WI
Ca2+-dependent Calmodulin-binding to FcRn Affects IgG Transport in the Transcytotic Pathway.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Keith Mostov The Fcgamma receptor FcRn transports IgG so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic alpha-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route. [Abstract/Link to Full Text]

Chen D, Wilkinson CR, Watt S, Penkett CJ, Toone WM, Jones N, Bähler J
Multiple Pathways Differentially Regulate Global Oxidative Stress Responses in Fission Yeast.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Jonathan Weissman Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage. [Abstract/Link to Full Text]

D'Silva PR, Schilke B, Hayashi M, Craig EA
Interaction of the J-Protein Heterodimer, Pam18/Pam16, of the Mitochondrial Import Motor with the Translocon of the Inner Membrane.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Jeffrey Brodsky Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial Hsp70 (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N-terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18IMS) with Tim17, and the direct interaction of Pam18's J-domain with Pam16's J-like domain. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in Pam16's N-terminus were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon. [Abstract/Link to Full Text]

Wiese C
Distinct Dgrip84 Isoforms Correlate with Distinct {gamma}-Tubulins in Drosophila.
Mol Biol Cell. 2007 Nov 15;
Monitoring Editor: Sandra Schmid gamma-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, gamma-tubulin exists in a multi-protein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). gamma-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the gamma-tubulin ring complex (gammaTuRC). Curiously, many organisms (including humans) have two distinct gamma-tubulin genes. In Drosophila, where the two gamma-tubulin isotypes have been studied most extensively, the gamma-tubulin genes are developmentally regulated: the 'maternal' gamma-tubulin isotype (named gammaTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second gamma-tubulin isotype (gammaTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both gamma-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes (Raynaud-Messina et al., 2001. Eur. J. Cell Biol. 80, 643-649): gammaTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of gammaTub23C at the centrosome increases at the beginning of mitosis, and gammaTub23C also associates with spindle pole microtubules. Here, we show that gammaTub23C forms discrete complexes that closely resemble the complexes formed by gammaTub37CD. Surprisingly, however, gammaTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the gamma-tubulin complexes formed with gammaTub37CD and gammaTub23C. [Abstract/Link to Full Text]

Abdi KM, Bennett V
Adducin Promotes Micron-Scale Organization of {beta}2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells.
Mol Biol Cell. 2007 Nov 14;
Monitoring Editor: Ben Margolis Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta2-spectrin. We use a Tet-on regulated inducible siRNA system to deplete alpha-adducin from confluent HBE cells. Depletion of alpha-adducin resulted in increased detergent solubility of spectrin following normal membrane biogenesis during mitosis. Conversely, depletion of beta2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant alpha-adducin prevented loss of lateral membrane, but only if alpha-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at PKC phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 microns. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells. [Abstract/Link to Full Text]

Barkus RV, Klyachko O, Horiuchi D, Dickson BJ, Saxton WM
Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides.
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Adam Linstedt A screen for genes required in Drosophila eye development identified a UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of GFP-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are likely transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism. [Abstract/Link to Full Text]

Liu P, Lu J, Cardoso WV, Vaziri C
The SPARC-related Factor SMOC-2 Promotes Growth Factor-induced Cyclin D1 Expression and DNA Synthesis via Integrin-linked Kinase (ILK).
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Jean Schwarzbauer Secreted Modular Calcium-binding Protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed following mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of PDGFbetaR, a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and bFGF). Conversely, SMOC-2 ablation using siRNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis following SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of MAPK and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, Integrin-Linked Kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control. [Abstract/Link to Full Text]

Jin R, Dobry CJ, McCown PJ, Kumar A
Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression.
Mol Biol Cell. 2007 Nov 7;
Monitoring Editor: Charles Boone Under certain conditions of nutrient stress, the budding yeast S. cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known MAPK and PKA signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Sigma1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth. [Abstract/Link to Full Text]

Recent Articles in Molecular and Cellular Biology

Chan MC, Nguyen PH, Davis BN, Ohoka N, Hayashi H, Du K, Lagna G, Hata A
A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor.
Mol Cell Biol. 2007 Aug;27(16):5776-89.
Bone morphogenetic protein (BMP) signaling regulates many different biological processes, including cell growth, differentiation, and embryogenesis. BMPs bind to heterogeneous complexes of transmembrane serine/threonine (Ser/Thr) kinase receptors known as the BMP type I and II receptors (BMPRI and BMPRII). BMPRII phosphorylates and activates the BMPRI kinase, which in turn activates the Smad proteins. The cytoplasmic region of BMPRII contains a "tail" domain (BMPRII-TD) with no enzymatic activity or known regulatory function. The discovery of mutations associated with idiopathic pulmonary artery hypertension mapping to BMPRII-TD underscores its importance. Here, we report that Tribbles-like protein 3 (Trb3) is a novel BMPRII-TD-interacting protein. Upon BMP stimulation, Trb3 dissociates from BMPRII-TD and triggers degradation of Smad ubiquitin regulatory factor 1 (Smurf1), which results in the stabilization of BMP receptor-regulated Smads and potentiation of the Smad pathway. Downregulation of Trb3 inhibits BMP-mediated cellular responses, including osteoblast differentiation of C2C12 cells and maintenance of the smooth muscle phenotype of pulmonary artery smooth muscle cells. Thus, Trb3 is a critical component of a novel mechanism for regulation of the BMP pathway by BMPRII. [Abstract/Link to Full Text]

Pfaff KL, Straub CT, Chiang K, Bear DM, Zhou Y, Zon LI
The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization.
Mol Cell Biol. 2007 Aug;27(16):5887-97.
A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly. [Abstract/Link to Full Text]

Jones NP, Katan M
Role of phospholipase Cgamma1 in cell spreading requires association with a beta-Pix/GIT1-containing complex, leading to activation of Cdc42 and Rac1.
Mol Cell Biol. 2007 Aug;27(16):5790-805.
The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase Cgamma1 (PLCgamma1) is critical for integrin-mediated cell spreading and motility (N. Jones et al., J. Cell Sci. 118:2695-2706, 2005). In the current study we show that, on a basement membrane-type matrix, PLCgamma1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor beta-Pix; GIT1 and beta-Pix form tight complexes independently of PLCgamma1. The association of PLCgamma1 with the complex requires both GIT1 and beta-Pix and the specific array region (gammaSA) of PLCgamma1. Mutations of PLCgamma1 within the gammaSA region reveal that association with this complex is essential for the phosphorylation of PLCgamma1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either beta-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLCgamma1. Furthermore, siRNA depletion of PLCgamma1, beta-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RhoA, were able to rescue the elongation of these cells. Signaling of the PLCgamma1/GIT1/beta-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLCgamma1 with complexes containing GIT1 and beta-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLCgamma1 is also involved in the activation of Cdc42 and Rac1. [Abstract/Link to Full Text]

Chen LY, Liu D, Songyang Z
Telomere maintenance through spatial control of telomeric proteins.
Mol Cell Biol. 2007 Aug;27(16):5898-909.
The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance. [Abstract/Link to Full Text]

Scaglione KM, Bansal PK, Deffenbaugh AE, Kiss A, Moore JM, Korolev S, Cocklin R, Goebl M, Kitagawa K, Skowyra D
SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo.
Mol Cell Biol. 2007 Aug;27(16):5860-70.
One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF. [Abstract/Link to Full Text]

Yoshida H, Ichikawa H, Tagata Y, Katsumoto T, Ohnishi K, Akao Y, Naoe T, Pandolfi PP, Kitabayashi I
PML-retinoic acid receptor alpha inhibits PML IV enhancement of PU.1-induced C/EBPepsilon expression in myeloid differentiation.
Mol Cell Biol. 2007 Aug;27(16):5819-34.
PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPepsilon expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPepsilon gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor alpha fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia. [Abstract/Link to Full Text]

Bilanges B, Argonza-Barrett R, Kolesnichenko M, Skinner C, Nair M, Chen M, Stokoe D
Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner.
Mol Cell Biol. 2007 Aug;27(16):5746-64.
The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1(-/-) or Tsc2(-/-) MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected approximately 40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5' and 3' untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology. [Abstract/Link to Full Text]

Bell EL, Klimova TA, Eisenbart J, Schumacker PT, Chandel NS
Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia.
Mol Cell Biol. 2007 Aug;27(16):5737-45.
Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span. [Abstract/Link to Full Text]

Chauvin C, Salhi S, Jean-Jean O
Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway.
Mol Cell Biol. 2007 Aug;27(16):5619-29.
Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity. [Abstract/Link to Full Text]

Ai D, Wang J, Amen M, Lu MF, Amendt BA, Martin JF
Nuclear factor 1 and T-cell factor/LEF recognition elements regulate Pitx2 transcription in pituitary development.
Mol Cell Biol. 2007 Aug;27(16):5765-75.
Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives. [Abstract/Link to Full Text]

Huang FT, Yu K, Balter BB, Selsing E, Oruc Z, Khamlichi AA, Hsieh CL, Lieber MR
Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region.
Mol Cell Biol. 2007 Aug;27(16):5921-32.
The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Smu locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Smu R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Smu repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Smu repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Smu repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence. [Abstract/Link to Full Text]

Yu EY, Steinberg-Neifach O, Dandjinou AT, Kang F, Morrison AJ, Shen X, Lue NF
Regulation of telomere structure and functions by subunits of the INO80 chromatin remodeling complex.
Mol Cell Biol. 2007 Aug;27(16):5639-49.
ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex. [Abstract/Link to Full Text]

Tateno H, Li H, Schur MJ, Bovin N, Crocker PR, Wakarchuk WW, Paulson JC
Distinct endocytic mechanisms of CD22 (Siglec-2) and Siglec-F reflect roles in cell signaling and innate immunity.
Mol Cell Biol. 2007 Aug;27(16):5699-710.
Sialic acid-binding immunoglobulin-like lectins (siglecs) are predominately expressed on immune cells. They are best known as regulators of cell signaling mediated by cytoplasmic tyrosine motifs and are increasingly recognized as receptors for pathogens that bear sialic acid-containing glycans. Most siglec proteins undergo endocytosis, an activity tied to their roles in cell signaling and innate immunity. Here, we investigate the endocytic pathways of two siglec proteins, CD22 (Siglec-2), a regulator of B-cell signaling, and mouse eosinophil Siglec-F, a member of the rapidly evolving CD33-related siglec subfamily that are expressed on cells of the innate immune system. CD22 exhibits hallmarks of clathrin-mediated endocytosis and traffics to recycling compartments, consistent with previous reports demonstrating its localization to clathrin domains. Like CD22, Siglec-F mediates endocytosis of anti-Siglec-F and sialoside ligands, a function requiring intact tyrosine-based motifs. In contrast, however, we find that Siglec-F endocytosis is clathrin and dynamin independent, requires ADP ribosylation factor 6, and traffics to lysosomes. The results suggest that these two siglec proteins have evolved distinct endocytic mechanisms consistent with roles in cell signaling and innate immunity. [Abstract/Link to Full Text]

Tsai KW, Tarn WY, Lin WC
Wobble splicing reveals the role of the branch point sequence-to-NAGNAG region in 3' tandem splice site selection.
Mol Cell Biol. 2007 Aug;27(16):5835-48.
Alternative splicing involving the 3' tandem splice site NAGNAG sequence may play a role in the structure-function diversity of proteins. However, how 3' tandem splice site utilization is determined is not well understood. We previously demonstrated that 3' NAGNAG-based wobble splicing occurs mostly in a tissue- and developmental stage-independent manner. Bioinformatic analysis reveals that the nucleotide preceding the AG dinucleotide may influence 3' splice site utilization; this is also supported by an in vivo splicing assay. Moreover, we found that the intron sequence plays an important role in 3' splice site selection for NAGNAG wobble splicing. Mutations of the region between the branch site and the NAGNAG 3' splice site, indeed, affected the ratio of the distal/proximal AG selection. Finally, we found that single nucleotide polymorphisms around the NAGNAG motif could affect the splice site choice, which may lead to a change in mRNA patterns and influence protein function. We conclude that the NAGNAG motif and its upstream region to the branch point sequence are required for 3' tandem splice site selection. [Abstract/Link to Full Text]

Johns L, Grimson A, Kuchma SL, Newman CL, Anderson P
Caenorhabditis elegans SMG-2 selectively marks mRNAs containing premature translation termination codons.
Mol Cell Biol. 2007 Aug;27(16):5630-8.
Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed "nonsense-mediated mRNA decay" (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with ("marks") those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD. [Abstract/Link to Full Text]

Ho AT, Li QH, Okada H, Mak TW, Zacksenhaus E
XIAP activity dictates Apaf-1 dependency for caspase 9 activation.
Mol Cell Biol. 2007 Aug;27(16):5673-85.
The current model for the intrinsic apoptotic pathway holds that mitochondrial activation of caspases in response to cytotoxic drugs requires both Apaf-1-induced dimerization of procaspase 9 and Smac/Diablo-mediated sequestration of inhibitors of apoptosis proteins (IAPs). Here, we showed that either pathway can independently promote caspase 9 activation in response to apoptotic stimuli. In drug-treated Apaf-1(-/-) primary myoblasts, but not fibroblasts, Smac/Diablo accumulates in the cytosol and sequesters X-linked IAP (XIAP), which is expressed at lower levels in myoblasts than in fibroblasts. Consequently, caspase 9 activation proceeds in Apaf-1(-/-) myoblasts; concomitant ablation of Apaf-1 and Smac is required to prevent caspase 9 activation and the onset of apoptosis. Conversely, in stimulated Apaf-1(-/-) fibroblasts, the ratio of XIAP to Smac/Diablo is high compared to that for myoblasts and procaspase 9 is not activated. Suppressing XIAP with exogenous Smac/Diablo or a pharmacological inhibitor can still induce caspase 9 in drug-treated Apaf-1-null fibroblasts. Thus, caspase 9 activation in response to intrinsic apoptotic stimuli can be uncoupled from Apaf-1 in vivo by XIAP antagonists. [Abstract/Link to Full Text]

Sautel CF, Cannella D, Bastien O, Kieffer S, Aldebert D, Garin J, Tardieux I, Belrhali H, Hakimi MA
SET8-mediated methylations of histone H4 lysine 20 mark silent heterochromatic domains in apicomplexan genomes.
Mol Cell Biol. 2007 Aug;27(16):5711-24.
Posttranslational histone modifications modulate chromatin-templated processes in various biological systems. H4K20 methylation is considered to have an evolutionarily ancient role in DNA repair and genome integrity, while its function in heterochromatin function and gene expression is thought to have arisen later during evolution. Here, we identify and characterize H4K20 methylases of the Set8 family in Plasmodium and Toxoplasma, two medically important members of the protozoan phylum Apicomplexa. Remarkably, parasite Set8-related proteins display H4K20 mono-, di-, and trimethylase activities, in striking contrast to the monomethylase-restricted human Set8. Structurally, few residues forming the substrate-specific channel dictate enzyme methylation multiplicity. These enzymes are cell cycle regulated and focally enriched at pericentric and telomeric heterochromatin in both parasites. Collectively, our findings provide new insights into the evolution of Set8-mediated biochemical pathways, suggesting that the heterochromatic function of the marker is not restricted to metazoans. Thus, these lower eukaryotes have developed a diverse panel of biological stages through their high capacity to differentiate, and epigenetics only begins to emerge as a strong determinant of their biology. [Abstract/Link to Full Text]

Smith FM, Holt LJ, Garfield AS, Charalambous M, Koumanov F, Perry M, Bazzani R, Sheardown SA, Hegarty BD, Lyons RJ, Cooney GJ, Daly RJ, Ward A
Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life.
Mol Cell Biol. 2007 Aug;27(16):5871-86.
The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health. [Abstract/Link to Full Text]

Heidt AB, Rojas A, Harris IS, Black BL
Determinants of myogenic specificity within MyoD are required for noncanonical E box binding.
Mol Cell Biol. 2007 Aug;27(16):5910-20.
The MyoD family of basic helix-loop-helix (bHLH) transcription factors has the remarkable ability to induce myogenesis in vitro and in vivo. This myogenic specificity has been mapped to two amino acids in the basic domain, an alanine and threonine, referred to as the myogenic code. These essential determinants of myogenic specificity are conserved in all MyoD family members from worms to humans, yet their function in myogenesis is unclear. Induction of the muscle transcriptional program requires that MyoD be able to locate and stably bind to sequences present in the promoter regions of critical muscle genes. Recent studies have shown that MyoD binds to noncanonical E boxes in the myogenin gene, a critical locus required for myogenesis, through interactions with resident heterodimers of the HOX-TALE transcription factors Pbx1A and Meis1. In the present study, we show that the myogenic code is required for MyoD to bind to noncanonical E boxes in the myogenin promoter and for the formation of a tetrameric complex with Pbx/Meis. We also show that these essential determinants of myogenesis are sufficient to confer noncanonical E box binding to the E12 basic domain. Thus, these data show that noncanonical E box binding correlates with myogenic potential, and we speculate that the myogenic code residues in MyoD function as myogenic determinants via their role in noncanonical E box binding and recognition. [Abstract/Link to Full Text]

Ura S, Nishina H, Gotoh Y, Katada T
Activation of the c-Jun N-terminal kinase pathway by MST1 is essential and sufficient for the induction of chromatin condensation during apoptosis.
Mol Cell Biol. 2007 Aug;27(15):5514-22.
Chromatin condensation is the most recognizable nuclear hallmark of apoptosis. Cleavage and activation of MST1 by caspases induce chromatin condensation. It was previously reported that, during apoptosis, activated MST1 induced c-Jun N-terminal kinase (JNK) activation and also phosphorylated histone H2B. However, which of these mechanisms underlies MST1's induction of chromatin condensation has yet to be clarified. Here, we report that MST1-mediated activation of JNK is both essential and sufficient for chromatin condensation. MST1 activation did not result in chromatin condensation in mitogen-activate protein kinase kinase 4 (MKK4)/MKK7 double knockout (MKK4/7 DKO) embryonic stem (ES) cells, which genetically lack the ability to activate JNK. On the other hand, constitutively active JNK was able to induce chromatin condensation in MKK4/7 DKO ES cells. In contrast, histone H2B phosphorylation did not correlate with chromatin condensation in wild-type ES cells. Finally, inhibition of JNK as well as inhibitor of caspase-activated DNase blocked chromatin condensation during Fas-mediated apoptosis of Jurkat cells. Taken together, our results indicate that caspase-mediated cleavage of MST1, followed by MST1-mediated activation of the JNK pathway, is the mechanism responsible for inducing chromatin condensation during apoptosis. [Abstract/Link to Full Text]

Fisher AE, Hochegger H, Takeda S, Caldecott KW
Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase.
Mol Cell Biol. 2007 Aug;27(15):5597-605.
Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1. [Abstract/Link to Full Text]

Rahmani M, Davis EM, Crabtree TR, Habibi JR, Nguyen TK, Dent P, Grant S
The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress.
Mol Cell Biol. 2007 Aug;27(15):5499-513.
Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib. [Abstract/Link to Full Text]

Tank EM, Harris DA, Desai AA, True HL
Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.
Mol Cell Biol. 2007 Aug;27(15):5445-55.
Mammalian prion diseases are fatal neurodegenerative disorders dependent on the prion protein PrP. Expansion of the oligopeptide repeats (ORE) found in PrP is associated with inherited prion diseases. Patients with ORE frequently harbor PrP aggregates, but other factors may contribute to pathology, as they often present with unexplained phenotypic variability. We created chimeric yeast-mammalian prion proteins to examine the influence of the PrP ORE on prion properties in yeast. Remarkably, all chimeric proteins maintained prion characteristics. The largest repeat expansion chimera displayed a higher propensity to maintain a self-propagating aggregated state. Strikingly, the repeat expansion conferred increased conformational flexibility, as observed by enhanced phenotypic variation. Furthermore, the repeat expansion chimera displayed an increased rate of prion conversion, but only in the presence of another aggregate, the [RNQ+] prion. We suggest that the PrP ORE increases the conformational flexibility of the prion protein, thereby enhancing the formation of multiple distinct aggregate structures and allowing more frequent prion conversion. Both of these characteristics may contribute to the phenotypic variability associated with PrP repeat expansion diseases. [Abstract/Link to Full Text]

Dormoy-Raclet V, Ménard I, Clair E, Kurban G, Mazroui R, Di Marco S, von Roretz C, Pause A, Gallouzi IE
The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-rich-element-dependent manner.
Mol Cell Biol. 2007 Aug;27(15):5365-80.
A high expression level of the beta-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the beta-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the beta-actin mRNA by associating with a uridine-rich element within its 3' untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the beta-actin mRNA. HuR depletion in HeLa cells alters key beta-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated beta-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation. [Abstract/Link to Full Text]

Shetty S, Velusamy T, Idell S, Shetty P, Mazar AP, Bhandary YP, Shetty RS
Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA.
Mol Cell Biol. 2007 Aug;27(16):5607-18.
We found that p53-deficient (p53(-/-)) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53(-/-) cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3' untranslated region (3'UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3'UTR sequence, and insertion of the p53 binding sequence into beta-globin mRNA destabilized beta-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric beta-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells. [Abstract/Link to Full Text]

Shimotsuma M, Matsuzaki H, Tanabe O, Campbell AD, Engel JD, Fukamizu A, Tanimoto K
Linear distance from the locus control region determines epsilon-globin transcriptional activity.
Mol Cell Biol. 2007 Aug;27(16):5664-72.
Enhancer elements modulate promoter activity over vast chromosomal distances, and mechanisms that ensure restrictive interactions between promoters and enhancers are critical for proper control of gene expression. The human beta-globin locus control region (LCR) activates expression of five genes in erythroid cells, including the proximal embryonic epsilon- and the distal adult beta-globin genes. To test for possible distance sensitivity of the genes to the LCR, we extended the distance between the LCR and genes by 2.3 kbp within the context of a yeast artificial chromosome, followed by the generation of transgenic mice (TgM). In these TgM lines, epsilon-globin gene expression decreased by 90%, while the more distantly located gamma- or beta-globin genes were not affected. Remarkably, introduction of a consensus EKLF binding site into the epsilon-globin promoter rendered its expression distance insensitive; when tested in an EKLF-null genetic background, expression of the mutant epsilon-globin gene was severely compromised. Thus, the epsilon-globin gene differs in its distance sensitivity to the LCR from the other beta-like globin genes, which is, at least in part, determined by the transcription factor EKLF. [Abstract/Link to Full Text]

Taniguchi N, Yoshida K, Ito T, Tsuda M, Mishima Y, Furumatsu T, Ronfani L, Abeyama K, Kawahara K, Komiya S, Maruyama I, Lotz M, Bianchi ME, Asahara H
Stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification.
Mol Cell Biol. 2007 Aug;27(16):5650-63.
High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1(-/-) mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1(-/-) mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis. [Abstract/Link to Full Text]

Tillmanns S, Otto C, Jaffray E, Du Roure C, Bakri Y, Vanhille L, Sarrazin S, Hay RT, Sieweke MH
SUMO modification regulates MafB-driven macrophage differentiation by enabling Myb-dependent transcriptional repression.
Mol Cell Biol. 2007 Aug;27(15):5554-64.
During the execution of differentiation programs, lineage-specific transcription factors are in competition with antagonistic factors that drive progenitor proliferation. Thus, the myeloid transcription factor MafB promotes macrophage differentiation of myeloid progenitors, but a constitutively active Myb transcription factor (v-Myb) can maintain proliferation and block differentiation. Little is known, however, about the regulatory mechanisms that control such competing activities. Here we report that the small ubiquitin-like protein SUMO-1 can modify MafB in vitro and in vivo on lysines 32 and 297. The absence of MafB SUMO modification increased MafB-driven transactivation and macrophage differentiation potential but inhibited cell cycle progression and myeloid progenitor growth. Furthermore, we observed that direct repression of MafB transactivation by v-Myb was strictly dependent on MafB SUMO modification. Consequently, a SUMOylation-deficient MafB K32R K297R (K32,297R) mutant could specify macrophage fate even after activation of inducible Myb alleles and resist their differentiation-inhibiting activity. Our findings suggest that SUMO modification of MafB affects the balance between myeloid progenitor expansion and terminal macrophage differentiation by controlling MafB transactivation capacity and susceptibility to Myb repression. SUMO modification of lineage-specific transcription factors may thus modulate transcription factor antagonism to control tissue homeostasis in the hematopoietic system. [Abstract/Link to Full Text]

Limpert AS, Karlo JC, Landreth GE
Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein.
Mol Cell Biol. 2007 Aug;27(16):5686-98.
Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation. [Abstract/Link to Full Text]

Pérez-Valle J, Jenkins H, Merchan S, Montiel V, Ramos J, Sharma S, Serrano R, Yenush L
Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters.
Mol Cell Biol. 2007 Aug;27(16):5725-36.
K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves. [Abstract/Link to Full Text]

Recent Articles in Journal of Cell Science

Francavilla C, Loeffler S, Piccini D, Kren A, Christofori G, Cavallaro U
Neural cell adhesion molecule regulates the cellular response to fibroblast growth factor.
J Cell Sci. 2007 Dec 15;120(Pt 24):4388-94.
Neural cell adhesion molecule (NCAM) mediates cell-cell adhesion and signaling in the nervous system, yet NCAM is also expressed in non-neural tissues, in which its function has in most parts remained elusive. We have previously reported that NCAM stimulates cell-matrix adhesion and neurite outgrowth by activating fibroblast growth factor receptor (FGFR) signaling. Here, we investigated whether the interplay between NCAM and FGFR has any impact on the response of FGFR to its classical ligands, FGFs. To this end, we employed two fibroblast cell lines, NCAM-negative L cells and NCAM-positive NIH-3T3 cells, in which the expression of NCAM was manipulated by means of transfection or RNAi technologies, respectively. The results demonstrate that NCAM expression reduces FGF-stimulated ERK1/2 activation, cell proliferation and cell-matrix adhesion, in both L and NIH-3T3 cells. Furthermore, our data show that NCAM inhibits the binding of FGF to its high-affinity receptor in a competitive manner, providing the mechanisms for the NCAM-mediated suppression of FGF function. In this context, a small peptide that mimics the binding of NCAM to FGFR was sufficient to block FGF-dependent cell proliferation. These findings point to NCAM as being a major regulator of FGF-FGFR interaction, thus introducing a novel type of control mechanism for FGFR activity and opening new therapeutic perspectives for those diseases characterized by aberrant FGFR function. [Abstract/Link to Full Text]

Shapira I, Charuvi D, Elkabetz Y, Hirschberg K, Bar-Nun S
Distinguishing between retention signals and degrons acting in ERAD.
J Cell Sci. 2007 Dec 15;120(Pt 24):4377-87.
Endoplasmic reticulum-associated degradation (ERAD) eliminates aberrant proteins from the secretory pathway. Such proteins are retained in the endoplasmic reticulum and targeted for degradation by the ubiquitin-proteasome system. Cis-acting motifs can function in ERAD as retention signals, preventing vesicular export from the endoplasmic reticulum, or as degrons, targeting proteins for degradation. Here, we show that mustp, the C-terminal 20-residue tailpiece of the secretory IgM mus heavy chain, functions both as a portable retention signal and as an ERAD degron. Retention of mustp fusions of secreted versions of thyroid peroxidase and yellow fluorescent protein in the endoplasmic reticulum requires the presence of the penultimate cysteine of mustp. In its role as a portable degron, the mustp targets the retained proteins for ERAD but does not serve as an obligatory ubiquitin-conjugation site. Abolishing mustp glycosylation accelerates the degradation of both mustpCys-fused substrates, yet absence of the N-glycan eliminates the requirement for the penultimate cysteine in the retention and degradation of the unglycosylated yellow fluorescent protein. Hence, the dual role played by the mustpCys motif as a retention signal and as a degron can be attributed to distinct elements within this sequence. [Abstract/Link to Full Text]

Rohlfs M, Arasada R, Batsios P, Janzen J, Schleicher M
The Ste20-like kinase SvkA of Dictyostelium discoideum is essential for late stages of cytokinesis.
J Cell Sci. 2007 Dec 15;120(Pt 24):4345-54.
The genome of the social amoeba Dictyostelium discoideum encodes approximately 285 kinases, which represents approximately 2.6% of the total genome and suggests a signaling complexity similar to that of yeasts and humans. The behavior of D. discoideum as an amoeba and during development relies heavily on fast rearrangements of the actin cytoskeleton. Here, we describe the knockout phenotype of the svkA gene encoding severin kinase, a homolog of the human MST3, MST4 and YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to multinucleated cells sometimes with >30 nuclei. The defect arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage furrow and to sever the last cytosolic connection. We demonstrate that GFP-SvkA is enriched at the centrosome and localizes to the midzone during the final stage of cell division. This distribution is mediated by the C-terminal half of the kinase, whereas a rescue of the phenotypic changes requires the active N-terminal kinase domain as well. The data suggest that SvkA is part of a regulatory pathway from the centrosome to the midzone, thus regulating the completion of cell division. [Abstract/Link to Full Text]

Mseka T, Bamburg JR, Cramer LP
ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization.
J Cell Sci. 2007 Dec 15;120(Pt 24):4332-44.
How formation of the front and rear of a cell are coordinated during cell polarization in migrating cells is not well understood. Time-lapse microscopy of live primary chick embryo heart fibroblasts expressing GFP-actin show that, prior to cell polarization, polymerized actin in the cell body reorganizes to form oriented actin-filament bundles spanning the entire cell body. Within an average of 5 minutes of oriented actin bundles forming, localized cell-edge retraction initiates at either the side or at one end of the newly formed bundles and then elaborates around the nearest end of the bundles to form the cell rear, the first visual break in cell symmetry. Localized net protrusion occurs at the opposing end of the bundles to form the cell front and lags formation of the rear of the cell. Consequently, cells acquire full polarity and start to migrate in the direction of the long axis of the bundles, as previously documented for already migrating cells. When ADF/cofilin family protein activity or actin-filament disassembly is specifically blocked during cell polarization, reorganization of polymerized actin to form oriented actin-filament bundles in the cell body fails, and formation of the cell rear and front is inhibited. We conclude that formation of oriented actin-filament bundles in the cell body requires ADF/cofilin family proteins, and is an early event needed to coordinate the spatial location of the cell rear and front during fibroblast polarization. [Abstract/Link to Full Text]

Kobayashi T, Hearing VJ
Direct interaction of tyrosinase with Tyrp1 to form heterodimeric complexes in vivo.
J Cell Sci. 2007 Dec 15;120(Pt 24):4261-8.
Mutations of the critical and rate-limiting melanogenic enzyme tyrosinase (Tyr) result in hypopigmentation of the hair, skin and eyes. Two other related enzymes, Tyrp1 and Dct, catalyze distinct post-Tyr reactions in melanin biosynthesis. Tyr, Tyrp1 and Dct have been proposed to interact with and stabilize each other in multi-enzyme complexes, and in vitro, Tyr activity is more stable in the presence of Tyrp1 and/or Dct. We recently reported that Tyr is degraded more quickly in mutant Tyrp1 mouse melanocytes than in wild-type Tyrp1 melanocytes, and that decreased stability of Tyr can be partly rescued by infection with wild-type Tyrp1. Although interactions between Tyr and Tyrp1 have been demonstrated in vitro, there is no direct evidence for Tyr interaction with Tyrp1 in vivo. In this study, we use in vivo chemical crosslinking to stabilize the association of Tyr with other cellular proteins. Western blot analysis revealed that Tyrp1, but not Dct, associates with Tyr in murine melanocytes in vivo, and more specifically, in melanosomes. Two-dimensional SDS-PAGE analysis detected heterodimeric species of Tyr and Tyrp1. Taken together, these data demonstrate that Tyrp1 interacts directly with Tyr in vivo, which may regulate the stability and trafficking of melanogenic enzymes and thus pigment synthesis. [Abstract/Link to Full Text]

Miyamoto Y, Yamauchi J, Chan JR, Okada A, Tomooka Y, Hisanaga S, Tanoue A
Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin.
J Cell Sci. 2007 Dec 15;120(Pt 24):4355-66.
Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs) in order to form myelin, which is required for the rapid propagation of action potentials in the vertebrate nervous system. In spite of the considerable clinical importance of myelination, little is known about the basic molecular mechanisms underlying OL differentiation and myelination. Here, we show that cyclin-dependent kinase (Cdk) 5 is activated following the induction of differentiation, and that the Cdk5 inhibitor roscovitine inhibits OL differentiation. The complexity of the OL processes is also diminished after knocking down endogenous Cdk5 using RNAi. We also show that the focal adhesion protein paxillin is directly phosphorylated at Ser244 by Cdk5. Transfection of a paxillin construct harboring a Ser244 to Ala mutation dramatically inhibits its morphological effects. Importantly, phosphorylation of paxillin at Ser244 reduces its interaction with focal adhesion kinase (FAK). Taken together, these results suggest that phosphorylation of paxillin by Cdk5 is a key mechanism in OL differentiation and may ultimately regulate myelination. [Abstract/Link to Full Text]

Graser S, Stierhof YD, Nigg EA
Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion.
J Cell Sci. 2007 Dec 15;120(Pt 24):4321-31.
The centrosome duplicates during the cell cycle but functions as a single microtubule-organising centre until shortly before mitosis. This raises the question of how centrosome cohesion is maintained throughout interphase. One dynamic model proposes that parental centrioles are held together through centriole-associated, entangling filaments. Central to this model are C-Nap1, a putative centriolar docking protein and rootletin, a fibrous component. Here we identify two novel proteins, Cep68 and Cep215, as required for centrosome cohesion. Similar to rootletin, Cep68 decorates fibres emanating from the proximal ends of centrioles and dissociates from centrosomes during mitosis. Furthermore, Cep68 and rootletin depend both on each other and on C-Nap1 for centriole association. Unlike rootletin, overexpression of Cep68 does not induce extensive fibre formation, but Cep68 is readily recruited to ectopic rootletin fibres. These data suggest that Cep68 cooperates with rootletin and C-Nap1 in centrosome cohesion. By contrast, Cep215 associates with centrosomes throughout the cell cycle and does not appear to interact with Cep68, rootletin or C-Nap1. Instead, our data suggest that Cep215 functionally interacts with pericentrin, suggesting that both proteins influence centrosome cohesion through an indirect mechanism related to cytoskeletal dynamics. [Abstract/Link to Full Text]

Kirik V, Herrmann U, Parupalli C, Sedbrook JC, Ehrhardt DW, Hülskamp M
CLASP localizes in two discrete patterns on cortical microtubules and is required for cell morphogenesis and cell division in Arabidopsis.
J Cell Sci. 2007 Dec 15;120(Pt 24):4416-25.
In animals and yeast, CLASP proteins are microtubule plus-end tracking proteins (+TIPS) involved in the regulation of microtubule plus-end dynamics and stabilization. Here we show that mutations in the Arabidopsis CLASP homolog result in various plant growth reductions, cell form defects and reduced mitotic activity. Analysis of Arabidopsis plants that carry a YFP:AtCLASP fusion construct regulated by the AtCLASP native promoter showed similarities to the described localization of the animal CLASP proteins, but also prominent differences including punctate and preferential localization along cortical microtubules. Colocalization studies of YFP:AtCLASP and CFP:EB1b also showed that AtCLASP is enriched at the plus ends of microtubules where it localizes behind the AtEB1b protein. Moreover, AtCLASP overexpression causes abnormal cortical microtubule bundling and array organization. Cortical microtubule arrays have evolved to be prominent in plants, and our findings suggest that plant CLASP proteins may have adopted specific functions in regulating cortical microtubule properties and cell growth. [Abstract/Link to Full Text]

Carvalho RL, Itoh F, Goumans MJ, Lebrin F, Kato M, Takahashi S, Ema M, Itoh S, van Rooijen M, Bertolino P, Ten Dijke P, Mummery CL
Compensatory signalling induced in the yolk sac vasculature by deletion of TGF receptors in mice.
J Cell Sci. 2007 Dec 15;120(Pt 24):4269-77.
Vascular development depends on transforming growth factor beta (TGFbeta), but whether signalling of this protein is required for the development of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) or both is unclear. To address this, we selectively deleted the type I (ALK5, TGFBR1) and type II (TbetaRII, TGFBR2) receptors in mice. Absence of either receptor in ECs resulted in vascular defects in the yolk sac, as seen in mice lacking receptors in all cells, causing embryonic lethality at embryonic day (E)10.5. Deletion of TbetaRII specifically in VSMCs also resulted in vascular defects in the yolk sac; however, these were observed at later stages of development, allowing the embryo to survive to E12.5. Because TGFbeta can also signal in ECs via ALK1 (ACVRL1), we replaced ALK5 by a mutant defective in SMAD2 and SMAD3 (SMAD2/3) activation that retained the ability to transactivate ALK1. This again caused defects in the yolk sac vasculature with embryonic lethality at E10.5, demonstrating that TGFbeta/ALK1 signalling in ECs cannot compensate for the lack of TGFbeta/ALK5-induced SMAD2/3 signalling in vivo. Unexpectedly, SMAD2 phosphorylation and alpha-smooth muscle actin (SMAalpha, ACTA2) expression occurred in the yolk sacs of ALK5(-/-) embryos and ALK5(-/-) embryonic stem cells undergoing vasculogenesis, and these processes could be blocked by an ALK4 (ACVR1B)/ALK5 inhibitor. Together, the data show that ALK5 is required in ECs and VSMCs for yolk sac vasculogenesis; in the absence of ALK5, ALK4 mediates SMAD2 phosphorylation and consequently SMAalpha expression. [Abstract/Link to Full Text]

Chibalina MV, Seaman MN, Miller CC, Kendrick-Jones J, Buss F
Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment.
J Cell Sci. 2007 Dec 15;120(Pt 24):4278-88.
Myosin VI is an actin-based retrograde motor protein that plays a crucial role in both endocytic and secretory membrane trafficking pathways. Myosin VI's targeting to and function in these intracellular pathways is mediated by a number of specific binding partners. In this paper we have identified a new myosin-VI-binding partner, lemur tyrosine kinase 2 (LMTK2), which is the first transmembrane protein and kinase that directly binds to myosin VI. LMTK2 binds to the WWY site in the C-terminal myosin VI tail, the same site as the endocytic adaptor protein Dab2. When either myosin VI or LMTK2 is depleted by siRNAs, the transferrin receptor (TfR) is trapped in swollen endosomes and tubule formation in the endocytic recycling pathway is dramatically reduced, showing that both proteins are required for the transport of cargo, such as the TfR, from early endosomes to the endocytic recycling compartment. [Abstract/Link to Full Text]

Itoh G, Yumura S
A novel mitosis-specific dynamic actin structure in Dictyostelium cells.
J Cell Sci. 2007 Dec 15;120(Pt 24):4302-9.
Cell division of various animal cells depends on their attachment to a substratum. Dictyostelium cells deficient in type II myosin, analogous to myosin in muscle, can divide on a substratum without the contractile ring. To investigate the mechanism of this substratum-dependent cytokinesis, the dynamics of actin in the ventral cortex were observed by confocal and total internal reflection fluorescence microscopy. Specifically during mitosis, we found novel actin-containing structures (mitosis-specific dynamic actin structures, MiDASes) underneath the nuclei and centrosomes. When the nucleus divided, the MiDAS also split in two and followed the movement of the daughter nuclei. At that time, the distal ends of astral microtubules reached mainly the MiDAS regions of the ventral cortex. An inhibitor of microtubules induced disappearance of MiDASes, leading to aborted cytokinesis, suggesting that astral microtubules are required for the formation and maintenance of MiDASes. Fluorescence recovery after photobleaching experiments revealed that the MiDAS was highly dynamic and comprised small actin-containing dot-like structures. Interference reflection microscopy and assays blowing away the cell bodies by jet streaming showed that MiDASes were major attachment sites of dividing cells. Thus, the MiDASes are strong candidates for scaffolds for substratum-dependent cytokinesis, serving to transmit mechanical force to the substratum. [Abstract/Link to Full Text]

Spiller MP, Reijns MA, Beggs JD
Requirements for nuclear localization of the Lsm2-8p complex and competition between nuclear and cytoplasmic Lsm complexes.
J Cell Sci. 2007 Dec 15;120(Pt 24):4310-20.
Sm-like (Lsm) proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many RNAs. In eukaryotes, a hetero-heptameric complex of seven Lsm proteins (Lsm2-8) affects the processing of small stable RNAs and pre-mRNAs in the nucleus, whereas a different hetero-heptameric complex of Lsm proteins (Lsm1-7) promotes mRNA decapping and decay in the cytoplasm. These two complexes have six constituent proteins in common, yet localize to separate cellular compartments and perform apparently disparate functions. Little is known about the biogenesis of the Lsm complexes, or how they are recruited to different cellular compartments. We show that, in yeast, the nuclear accumulation of Lsm proteins depends on complex formation and that the Lsm8p subunit plays a crucial role. The nuclear localization of Lsm8p is itself most strongly influenced by Lsm2p and Lsm4p, its presumed neighbours in the Lsm2-8p complex. Furthermore, overexpression and depletion experiments imply that Lsm1p and Lsm8p act competitively with respect to the localization of the two complexes, suggesting a potential mechanism for co-regulation of nuclear and cytoplasmic RNA processing. A shift of Lsm proteins from the nucleus to the cytoplasm under stress conditions indicates that this competition is biologically significant. [Abstract/Link to Full Text]

Moscatelli A, Ciampolini F, Rodighiero S, Onelli E, Cresti M, Santo N, Idilli A
Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold.
J Cell Sci. 2007 Nov 1;120(Pt 21):3804-19.
In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes - positively or negatively charged nanogold - were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes. [Abstract/Link to Full Text]

Friedland JC, Lakins JN, Kazanietz MG, Chernoff J, Boettiger D, Weaver VM
alpha6beta4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-kappaB-dependent resistance to apoptosis in 3D mammary acini.
J Cell Sci. 2007 Oct 15;120(Pt 20):3700-12.
Malignant transformation and multidrug resistance are linked to resistance to apoptosis, yet the molecular mechanisms that mediate tumor survival remain poorly understood. Because the stroma can influence tumor behavior by regulating the tissue phenotype, we explored the role of extracellular matrix signaling and tissue organization in epithelial survival. We report that elevated (alpha6)beta4 integrin-dependent Rac-Pak1 signaling supports resistance to apoptosis in mammary acini by permitting stress-dependent activation of the p65 subunit of NF-kappaB through Pak1. We found that inhibiting Pak1 through expression of N17Rac or PID compromises NF-kappaB activation and renders mammary acini sensitive to death, but that resistance to apoptosis could be restored to these structures by overexpressing wild-type NF-kappaB p65. We also observed that acini expressing elevated levels of Pak1 can activate p65 and survive death treatments, even in the absence of activated Rac, yet will die if activation of NF-kappaB is simultaneously inhibited through expression of IkappaBalphaM. Thus, mammary tissues can resist apoptotic stimuli by activating NF-kappaB through alpha6beta4 integrin-dependent Rac-Pak1 signaling. Our data emphasize the importance of the extracellular matrix stroma in tissue survival and suggest that alpha6beta4 integrin-dependent Rac stimulation of Pak1 could be an important mechanism mediating apoptosis-resistance in some breast tumors. [Abstract/Link to Full Text]

Chen CL, Liu IH, Fliesler SJ, Han X, Huang SS, Huang JS
Cholesterol suppresses cellular TGF-beta responsiveness: implications in atherogenesis.
J Cell Sci. 2007 Oct 15;120(Pt 20):3509-21.
Hypercholesterolemia is a major causative factor for atherosclerotic cardiovascular disease. The molecular mechanisms by which cholesterol initiates and facilitates the process of atherosclerosis are not well understood. Here, we demonstrate that cholesterol treatment suppresses or attenuates TGF-beta responsiveness in all cell types studied as determined by measuring TGF-beta-induced Smad2 phosphorylation and nuclear translocation, TGF-beta-induced PAI-1 expression, TGF-beta-induced luciferase reporter gene expression and TGF-beta-induced growth inhibition. Cholesterol, alone or complexed in lipoproteins (LDL, VLDL), suppresses TGF-beta responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-beta receptors and facilitating rapid degradation of TGF-beta and thus suppressing TGF-beta-induced signaling. Conversely, cholesterol-lowering agents (fluvastatin and lovastatin) and cholesterol-depleting agents (beta-cyclodextrin and nystatin) enhance TGF-beta responsiveness by increasing non-lipid raft microdomain accumulation of TGF-beta receptors and facilitating TGF-beta-induced signaling. Furthermore, the effects of cholesterol on the cultured cells are also found in the aortic endothelium of ApoE-null mice fed a high-cholesterol diet. These results suggest that high cholesterol contributes to atherogenesis, at least in part, by suppressing TGF-beta responsiveness in vascular cells. [Abstract/Link to Full Text]

Feigin ME, Malbon CC
RGS19 regulates Wnt-beta-catenin signaling through inactivation of Galpha(o).
J Cell Sci. 2007 Oct 1;120(Pt 19):3404-14.
The Wnt-beta-catenin pathway controls numerous cellular processes, including differentiation, cell-fate decisions and dorsal-ventral polarity in the developing embryo. Heterotrimeric G-proteins are essential for Wnt signaling, and regulator of G-protein signaling (RGS) proteins are known to act at the level of G-proteins. The functional role of RGS proteins in the Wnt-beta-catenin pathway was investigated in mouse F9 embryonic teratocarcinoma cells. RGS protein expression was investigated at the mRNA level, and each RGS protein identified was overexpressed and tested for the ability to regulate the canonical Wnt pathway. Expression of RGS19 specifically was found to attenuate Wnt-responsive gene transcription in a time- and dose-dependent manner, to block cytosolic beta-catenin accumulation and Dishevelled3 (Dvl3) phosphorylation in response to Wnt3a and to inhibit Wnt-induced formation of primitive endoderm (PE). Overexpression of a constitutively active mutant of Galpha(o) rescued the inhibition of Lef-Tcf-sensitive gene transcription caused by RGS19. By contrast, expression of RGS19 did not inhibit activation of Lef-Tcf gene transcription when induced in response to Dvl3 expression. However, knockdown of RGS19 by siRNA suppressed canonical Wnt signaling, suggesting a complex role for RGS19 in regulating the ability of Wnt3a to signal to the level of beta-catenin and gene transcription. [Abstract/Link to Full Text]

Christova R, Jones T, Wu PJ, Bolzer A, Costa-Pereira AP, Watling D, Kerr IM, Sheer D
P-STAT1 mediates higher-order chromatin remodelling of the human MHC in response to IFNgamma.
J Cell Sci. 2007 Sep 15;120(Pt 18):3262-70.
Transcriptional activation of the major histocompatibility complex (MHC) by IFNgamma is a key step in cell-mediated immunity. At an early stage of IFNgamma induction, chromatin carrying the entire MHC locus loops out from the chromosome 6 territory. We show here that JAK/STAT signalling triggers this higher-order chromatin remodelling and the entire MHC locus becomes decondensed prior to transcriptional activation of the classical HLA class II genes. A single point mutation of STAT1 that prevents phosphorylation is sufficient to abolish chromatin remodelling, thus establishing a direct link between the JAK/STAT signalling pathway and human chromatin architecture. The onset of chromatin remodelling corresponds with the binding of activated STAT1 and the chromatin remodelling enzyme BRG1 at specific sites within the MHC, and is followed by RNA-polymerase recruitment and histone hyperacetylation. We propose that the higher-order chromatin remodelling of the MHC locus is an essential step to generate a transcriptionally permissive chromatin environment for subsequent activation of classical HLA genes. [Abstract/Link to Full Text]

Moss DK, Bellett G, Carter JM, Liovic M, Keynton J, Prescott AR, Lane EB, Mogensen MM
Ninein is released from the centrosome and moves bi-directionally along microtubules.
J Cell Sci. 2007 Sep 1;120(Pt 17):3064-74.
Cell-to-cell contact and polarisation of epithelial cells involve a major reorganisation of the microtubules and centrosomal components. The radial microtubule organisation is lost and an apico-basal array develops that is no longer anchored at the centrosome. This involves not only the relocation of microtubules but also of centrosomal anchoring proteins to apical non-centrosomal sites. The relocation of microtubule minus-end-anchoring proteins such as ninein to the apical sites is likely to be essential for the assembly and stabilisation of the apico-basal arrays in polarised epithelial cells. In this study, we establish that ninein is highly dynamic and that, in epithelial cells, it is present not only at the centrosome but also in the cytoplasm as distinct speckles. Live-cell imaging reveals that GFP-ninein speckles are released from the centrosome and move in a microtubule-dependent manner within the cytoplasm and thus establishes that epithelial cells possess the mechanical means for relocation of ninein to non-centrosomal anchoring sites. We also provide evidence for the deployment of ninein speckles to apical anchoring sites during epithelial differentiation in both an in situ tissue and an in vitro culture system. In addition, the findings suggest that the non-centrosomal microtubule anchoring sites associate with adherens junctions in polarised epithelial cells. [Abstract/Link to Full Text]

Caballero R, Setien F, Lopez-Serra L, Boix-Chornet M, Fraga MF, Ropero S, Megias D, Alaminos M, Sanchez-Tapia EM, Montoya MC, Esteller M, Gonzalez-Sarmiento R, Ballestar E
Combinatorial effects of splice variants modulate function of Aiolos.
J Cell Sci. 2007 Aug 1;120(Pt 15):2619-30.
The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function. [Abstract/Link to Full Text]

Sone M, Hayashi T, Tarui H, Agata K, Takeichi M, Nakagawa S
The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons.
J Cell Sci. 2007 Aug 1;120(Pt 15):2498-506.
Recent transcriptome analyses have revealed that a large body of noncoding regions of mammalian genomes are actually transcribed into RNAs. Our understanding of the molecular features of these noncoding RNAs is far from complete. We have identified a novel mRNA-like noncoding gene, named Gomafu, which is expressed in a distinct set of neurons in the mouse nervous system. Interestingly, spliced mature Gomafu RNA is localized to the nucleus despite its mRNA-like characteristics, which usually act as potent export signals to the cytoplasm. Within the nucleus, Gomafu RNA is detected as numerous spots that do not colocalize with known nuclear domain markers. Gomafu RNA is extremely insoluble and remains intact after nuclear matrix preparation. Furthermore, heterokaryon assays revealed that Gomafu RNA does not shuttle between the nucleus and cytoplasm, but is retained in the nucleus after its transcription. We propose that Gomafu RNA represents a novel family of mRNA-like noncoding RNA that constitutes a cell-type-specific component of the nuclear matrix. [Abstract/Link to Full Text]

Chen PS, Wang MY, Wu SN, Su JL, Hong CC, Chuang SE, Chen MW, Hua KT, Wu YL, Cha ST, Babu MS, Chen CN, Lee PH, Chang KJ, Kuo ML
CTGF enhances the motility of breast cancer cells via an integrin-alphavbeta3-ERK1/2-dependent S100A4-upregulated pathway.
J Cell Sci. 2007 Jun 15;120(Pt 12):2053-65.
Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-alphavbeta3-ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-alphavbeta3-ERK1/2-S100A4 pathway. [Abstract/Link to Full Text]

Popoff V, Mardones GA, Tenza D, Rojas R, Lamaze C, Bonifacino JS, Raposo G, Johannes L
The retromer complex and clathrin define an early endosomal retrograde exit site.
J Cell Sci. 2007 Jun 15;120(Pt 12):2022-31.
Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes. [Abstract/Link to Full Text]

Bujny MV, Popoff V, Johannes L, Cullen PJ
The retromer component sorting nexin-1 is required for efficient retrograde transport of Shiga toxin from early endosome to the trans Golgi network.
J Cell Sci. 2007 Jun 15;120(Pt 12):2010-21.
The mammalian retromer complex is a multi-protein complex that regulates retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR) from early endosomes to the trans Golgi network (TGN). It consists of two subcomplexes: a membrane-bound coat comprising sorting nexin-1 (SNX1) and possibly sorting nexin-2 (SNX2), and a cargo-selective subcomplex, composed of VPS26, VPS29 and VPS35. In addition to the retromer, a variety of other protein complexes has been suggested to regulate endosome-to-TGN transport of not only the CI-MPR but a wide range of other cargo proteins. Here, we have examined the role of SNX1 and SNX2 in endosomal sorting of Shiga and cholera toxins, two toxins that undergo endosome-to-TGN transport en route to their cellular targets located within the cytosol. By using small interfering RNA (siRNA)-mediated silencing combined with single-cell fluorescent-toxin-uptake assays and well-established biochemical assays to analyze toxin delivery to the TGN, we have established that suppression of SNX1 leads to a significant reduction in the efficiency of endosome-to-TGN transport of the Shiga toxin B-subunit. Furthermore, we show that for the B subunit of cholera toxin, retrograde endosome-to-TGN transport is less reliant upon SNX1. Overall, our data establish a role for SNX1 in the endosome-to-TGN transport of Shiga toxin and are indicative for a fundamental difference between endosomal sorting of Shiga and cholera toxins into endosome-to-TGN retrograde transport pathways. [Abstract/Link to Full Text]

Kadler KE, Baldock C, Bella J, Boot-Handford RP
Collagens at a glance.
J Cell Sci. 2007 Jun 15;120(Pt 12):1955-8. [Abstract/Link to Full Text]

Clark KA, Bland JM, Beckerle MC
The Drosophila muscle LIM protein, Mlp84B, cooperates with D-titin to maintain muscle structural integrity.
J Cell Sci. 2007 Jun 15;120(Pt 12):2066-77.
Muscle LIM protein (MLP) is a cytoskeletal LIM-only protein expressed in striated muscle. Mutations in human MLP are associated with cardiomyopathy; however, the molecular mechanism by which MLP functions is not established. A Drosophila MLP homolog, mlp84B, displays many of the same features as the vertebrate protein, illustrating the utility of the fly for the study of MLP function. Animals lacking Mlp84B develop into larvae with a morphologically intact musculature, but the mutants arrest during pupation with impaired muscle function. Mlp84B displays muscle-specific expression and is a component of the Z-disc and nucleus. Preventing nuclear retention of Mlp84B does not affect its function, indicating that Mlp84B site of action is likely to be at the Z-disc. Within the Z-disc, Mlp84B is colocalized with the N-terminus of D-titin, a protein crucial for sarcomere organization and stretch mechanics. The mlp84B mutants phenotypically resemble weak D-titin mutants. Furthermore, reducing D-titin activity in the mlp84B background leads to pronounced enhancement of the mlp84B muscle defects and loss of muscle structural integrity. The genetic interactions between mlp84B and D-titin reveal a role for Mlp84B in maintaining muscle structural integrity that was not obvious from analysis of the mlp84B mutants themselves, and suggest Mlp84B and D-titin cooperate to stabilize muscle sarcomeres. [Abstract/Link to Full Text]

Coultas L, Terzano S, Thomas T, Voss A, Reid K, Stanley EG, Scott CL, Bouillet P, Bartlett P, Ham J, Adams JM, Strasser A
Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis.
J Cell Sci. 2007 Jun 15;120(Pt 12):2044-52.
The pro-apoptotic BH3-only members of the Bcl2 family, crucial initiators of cell death, are activated by a diverse array of developmental cues or experimentally applied stress stimuli. We have investigated, through gene targeting in mice, the biological roles for the BH3-only family member HRK (also known as DP5) in apoptosis regulation. Hrk gene expression was found to be restricted to cells and tissues of the central and peripheral nervous systems. Sensory neurons from mice lacking Hrk were less sensitive to apoptosis induced by nerve growth factor (NGF) withdrawal, consistent with the induction of Hrk following NGF deprivation. By contrast, cerebellar granule neurons that upregulate Hrk upon transfer to low-K+ medium underwent apoptosis normally under these conditions in the absence of Hrk. Furthermore, loss of Hrk was not sufficient to rescue the neuronal degeneration in lurcher mutant mice. Despite previous reports, no evidence was found for Hrk expression or induction in growth-factor-dependent haematopoietic cell lines following withdrawal of their requisite cytokine, and haematopoietic progenitors lacking HRK died normally in response to cytokine deprivation. These results demonstrate that HRK contributes to apoptosis signalling elicited by trophic factor withdrawal in certain neuronal populations but is dispensable for apoptosis of haematopoietic cells. [Abstract/Link to Full Text]

Jeong Y, Lee J, Kim K, Yoo JC, Rhee K
Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule stabilization.
J Cell Sci. 2007 Jun 15;120(Pt 12):2106-16.
Nek2 is a mitotic kinase whose activity varies during the cell cycle. It is well known that Nek2 is involved in centrosome splitting, and a number of studies have indicated that Nek2 is crucial for maintaining the integrity of centrosomal structure and microtubule nucleation activity. In the present study, we report that NIP2, previously identified as centrobin, is a novel substrate of Nek2. NIP2 was daughter-centriole-specific, but was also found in association with a stable microtubule network of cytoplasm. Ectopic NIP2 formed aggregates but was dissolved by Nek2 into small pieces and eventually associated with microtubules. Knockdown of NIP2 showed significant reduction of microtubule organizing activity, cell shrinkage, defects in spindle assembly and abnormal nuclear morphology. Based on our results, we propose that NIP2 has a role in stabilizing the microtubule structure. Phosphorylation may be crucial for mobilization of the protein to a new microtubule and stabilizing it. [Abstract/Link to Full Text]

Takenaga M, Fukumoto M, Hori Y
Regulated Nodal signaling promotes differentiation of the definitive endoderm and mesoderm from ES cells.
J Cell Sci. 2007 Jun 15;120(Pt 12):2078-90.
Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-alpha indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine. [Abstract/Link to Full Text]

Goossens S, Janssens B, Bonné S, De Rycke R, Braet F, van Hengel J, van Roy F
A unique and specific interaction between alphaT-catenin and plakophilin-2 in the area composita, the mixed-type junctional structure of cardiac intercalated discs.
J Cell Sci. 2007 Jun 15;120(Pt 12):2126-36.
Alpha-catenins play key functional roles in cadherin-catenin cell-cell adhesion complexes. We previously reported on alphaT-catenin, a novel member of the alpha-catenin protein family. alphaT-catenin is expressed predominantly in cardiomyocytes, where it colocalizes with alphaE-catenin at the intercalated discs. Whether alphaT- and alphaE-catenin have specific or synergistic functions remains unknown. In this study we used the yeast two-hybrid approach to identify specific functions of alphaT-catenin. An interaction between alphaT-catenin and plakophilins was observed and subsequently confirmed by co-immunoprecipitation and colocalization. Interaction with the amino-terminal part of plakophilins appeared to be specific for the central ;adhesion-modulation' domain of alphaT-catenin. In addition, we showed, by immuno-electron microscopy, that desmosomal proteins in the heart localize not only to the desmosomes in the intercalated discs but also at adhering junctions with hybrid composition. We found that in the latter junctions, endogenous plakophilin-2 colocalizes with alphaT-catenin. By providing an extra link between the cadherin-catenin complex and intermediate filaments, the binding of alphaT-catenin to plakophilin-2 is proposed to be a means of modulating and strengthening cell-cell adhesion between cardiac muscle cells. This could explain the devastating effect of plakophilin-2 mutations on cell junction stability in intercalated discs, which lead to cardiac muscle malfunction. [Abstract/Link to Full Text]

Hirai Y, Nelson CM, Yamazaki K, Takebe K, Przybylo J, Madden B, Radisky DC
Non-classical export of epimorphin and its adhesion to alphav-integrin in regulation of epithelial morphogenesis.
J Cell Sci. 2007 Jun 15;120(Pt 12):2032-43.
Epimorphin (also known as syntaxin 2) acts as an epithelial morphogen when secreted by stromal cells of the mammary gland, lung, liver, colon, pancreas and other tissues, but the same molecule functions within the cell to mediate membrane fusion. How this molecule, which lacks a signal sequence and contains a transmembrane domain at the C-terminus, translocates across the plasma membrane and is secreted to become a morphogen, and how it initiates morphogenic events is not clear. Here, we show that epimorphin is secreted through a non-classical mechanism, similar to that previously described for secretion of the leaderless protein FGF1, and we identify the key molecular elements responsible for translocation and secretion from the cell. We also show that secreted epimorphin binds to alphav-integrin-containing receptors on target epithelial cells, leading to activation of specific downstream signaling pathways and induction of epithelial morphogenesis. These findings provide key insight into how epimorphin functions as an epithelial morphogen. [Abstract/Link to Full Text]

Recent Articles in Journal of Cellular and Molecular Medicine

Song ZF, Ji XP, Li XX, Wang SJ, Wang SH
Inhibition of the activity of Poly(ADP-Ribose) Polymerase reduces heart ischemia/reperfusion injury via suppressing JNK mediated AIF translocation.
J Cell Mol Med. 2007 Dec 6; .
Poly (ADP-ribose) polymerase (PARP) has been proposed to play an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. However, the mechanisms of PARP mediated heart I/R injury in vivo are still not throughly understood. Therefore, in this study, we investigate the effect of PARP inhibition on heart I/R injury and try to elucidate the underlying mechanisms. Studies were performed with I/R rats' hearts in vivo. Ischemia followed by reperfusion caused a significant increase in Poly (ADP-ribose) (PAR), c-Jun NH(2)-terminal kinase (JNK) and Apoptosis-inducing factor (AIF) activity. Administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)- isoquinolinone (DPQ), an inhibitor of PARP, decreased myocardial infarction size from 61.11+/-7.46% to 38.83+/-5.67% (P<0.05) and cells apoptosis from 35 +/- 5.3% to 20 +/- 4.1% (P<0.05) and simultaneously improved the cardiac function. Western blot analysis showed that administration of DPQ reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. The results of the present study demonstrated that the inhibition of PARP was able to reduce heart I/R injury in vivo. Our results also suggested that JNK may be downstream of PARP activation and be required for PARP mediated AIF translocation. Inhibition of the activity of PARP may reduce heart I/R injury via suppressing AIF translocation mediated by JNK. [Abstract/Link to Full Text]

Botos E, Klumperman J, Oorschot V, Igyártó B, Magyar A, Oláh M, Kiss AL
Caveolin-1 is transported to multivesicular bodies after albumin-induced endocytosis of caveolae in HepG2 cells.
J Cell Mol Med. 2007 Dec 5;
Caveolae-mediated endocytosis is a highly regulated endocytic pathway that exists in parallel to other forms of clathrin-dependent and -independent endocytosis. Internalized caveolae accumulate in intermediate organelles called caveosomes. Here we addressed the further fate of internalized caveolae by inducing caveolae-mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin-1 by immunogold labeling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Long term (1 and 3 hours) albumin treatment resulted in the appearance of albumin containing caveolae in special multicaveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome-like structures in the cytoplasm. In addition, numerous CD63 (LIMP-1) positive late endosomes/multivesicular bodies were found positive for caveolin-1, suggesting that upon albumin incubation caveolin-1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long term albumin uptake caveolin-1 travels to late endosomes and is replaced by newly synthesized caveolin-1 at the plasma membrane. [Abstract/Link to Full Text]

Kowluru A
Protein Prenylation in Glucose-Induced Insulin Secretion from the Pancreatic Islet beta Cell: A Perspective.
J Cell Mol Med. 2007 Dec 5;
Insulin secretion from the pancreatic beta-cell is regulated principally by the ambient concentration of glucose. However, the molecular and cellular mechanisms underlying the stimulus-secretion coupling of glucose-stimulated insulin secretion [GSIS] remain only partially understood. Emerging evidence from multiple laboratories suggests key regulatory roles for GTP-binding proteins [G-proteins] in the cascade of events leading to GSIS. This class of signaling proteins undergo a series of requisite post-translational modifications [e.g., prenylation] at their C-terminal cysteines, which appear to be necessary for their targeting to respective membranous sites for optimal interaction with their respective effector proteins. This communication represents a perspective on potential regulatory roles for protein prenylation steps [i.e., protein farnesylation and protein geranylgeranylation] in GSIS from the islet beta cell. Possible consequences of protein prenylation and potential mechanisms underlying glucose-induced regulation of prenylation, specifically in the context of GSIS are also discussed. [Abstract/Link to Full Text]

Lee HY, Yea K, Kim J, Lee BD, Chae YC, Kim HS, Lee DW, Kim SH, Cho JH, Jin CJ, Koh DS, Park KS, Suh PG, Ryu SH
Epidermal Growth Factor Increases Insulin Secretion and Lowers Blood Glucose in Diabetic Mice.
J Cell Mol Med. 2007 Dec 5;
Epidermal growth factor (EGF) is synthesized in the pancreas and diabetic animals have low levels of EGF. However, the role of EGF in regulating the major function of the pancreas, insulin secretion, has not been studied. Here, we show that EGF rapidly increased insulin secretion in mouse pancreatic islets, as well as in a pancreatic beta-cell line. These events were dependent on a Ca(2+) influx and phospholipase D (PLD) activity, particularly PLD2, as determined using pharmacological blockers and molecular manipulations such as overexpression and siRNA of PLD isozymes. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in normal and diabetic mice. Here, for the first time, we provide evidence that EGF is a novel secretagogue that regulates plasma glucose levels and a candidate for the development of therapeutics for diabetes. [Abstract/Link to Full Text]

Gao JX
Cancer stem cells: the lessons from precancerous stem cells.
J Cell Mol Med. 2007 Dec 5;
How a cancer is initiated and established remains elusive despite all the advances in decades of cancer research. Recently the cancer stem cell (CSC) hypothesis has been revived, challenging the long-standing model of "clonal evolution" for cancer development and implicating the dawning of a potential cure for cancer [1]. The recent identification of precancerous stem cells (pCSCs) in cancer, an early stage of CSC development, however, implicates that the "clonal evolution" is not contradictory to the CSC hypothesis, but is rather an aspect of the process of CSC development [2]. The discovery of pCSC has revealed and will continue to reveal the volatile properties of CSC with respects to their phenotype, differentiation and tumorigenic capacity during initiation and progression. Both pCSC and CSC might also serve as precursors of tumor stromal components such as tumor vasculogenic stem/progenitor cells (TVPCs). Thus, the CSC hypothesis covers the developing process of tumor-initiating cells (TIC) --> pCSC --> CSC --> cancer, a cellular process that should parallel the histological process of hyperplasia/metaplasia (TIC) --> precancerous lesions (pCSC) --> malignant lesions (CSC --> cancer). The embryonic stem (ES) cell and germline stem (GS) cell genes are subverted in pCSCs. Especially the GS cell protein piwil2 may play an important role during the development of TIC --> pCSC --> CSC, and this protein may be used as a common biomarker for early detection, prevention, and treatment of cancer. As cancer stem cell research is yet in its infancy, definitive conclusions regarding the role of pCSC can not be made at this time. However this review will discuss what we have learned from pCSC and how this has led to innovative ideas that may eventually have major impacts on the understanding and treatment of cancer. [Abstract/Link to Full Text]

Susick L, Veluthakal R, Suresh MV, Hadden T, Kowluru A
Regulatory roles for histone deacetylation in IL-1beta-induced nitric oxide release in pancreatic beta-cells.
J Cell Mol Med. 2007 Dec 5;
Histone [de]acetylases control gene transcription via modification of the chromatin structure. Herein, we investigated potential roles for histone deacetylation [or hypoacetylation] in interleukin-1beta [IL-1beta]-mediated inducible nitric oxide synthase [iNOS] and nitric oxide [NO] release in insulin-secreting INS 832/13 [INS] cells. Western blot analysis suggested localization of members of Class 1 and Class 2 families of histone deacetylases [HDACs] in these cells. Trichostatin A [TSA], a known inhibitor of HDACs, markedly reduced IL-1beta-mediated iNOS expression and NO release from these cells in a concentration-dependent manner. TSA also promoted hyperacetylation of histone H4 under conditions in which it inhibited IL-1beta-mediated effects on isolated beta cells. Rottlerin, a known inhibitor of protein kinase Cdelta, also increased histone H4 acetylation, and inhibited IL-1beta-induced iNOS expression and NO release in these cells. It appears that the putative mechanism underlying the stimulatory effects of rottlerin on acetylation status of histone H4 are distinct from the HDAC inhibitory property of TSA, since rottlerin failed to inhibit HDAC activity in nuclear extracts isolated from INS cells. These data are suggestive of potential regulatory effects of rottlerin at the level of increasing the histone acetyltransferase activity in these cells. Together, our studies present the first evidence to suggest a PKCdelta-mediated signaling step, which promotes hypoacetylation of candidate histones culminating in IL-1beta-induced metabolic dysfunction of the isolated beta-cell. [Abstract/Link to Full Text]

Kumar S, Reusch HP, Ladilov Y
Acidic preconditioning suppresses apoptosis and increases expression of Bcl-xL in coronary endothelial cells under simulated ischemia.
J Cell Mol Med. 2007 Dec 5;
Ischemic preconditioning has a powerful protective potential against ischemia-induced cell death, and acidosis is an important feature of ischemia and can lead to apoptosis. Here we tested whether preconditioning with acidosis, i.e. acidic preconditioning (APC), may protect coronary endothelial cells (EC) against apoptosis induced by simulated ischemia. For preconditioning, EC were exposed for 40 min to acidosis (pH 6.4) followed by a 14 hrs recovery period (pH 7.4) and finally treated for 2 hrs with simulated ischemia (glucose free anoxia at pH 6.4). Cells undergoing apoptosis were visualized by chromatin staining or by determination of caspase-3 activity. Simulated ischemia in untreated EC increased caspase-3 activity and the number of apoptotic cell (31.3+/-1.3 %, vs. 3.9+/-0.6 %, in control). APC significantly reduced the rate of apoptosis (14.2+/-1.3 %) and caspase-3 activity. Western blot analysis exploring the underlying mechanism leading to this protection revealed suppression of the endoplasmic reticulum- (reduced cleavage of caspase-12) and mitochondria-mediated (reduced cytochrome C release) pathways of apoptosis. These effects were associated with an overexpression of the anti-apoptotic protein Bcl-xL 14 hrs after APC, whereas no effect on the expression of Bcl-2, Bax, Bak, procaspase-12, reticulum-localized chaperons (GRP78, calreticulin), HSP70, HSP32 and HSP27 could be detected. Knock-down of Bcl-xL by siRNA-treatment prevented the protective effect of APC. In conclusion, short acidic pre-treatment can protect EC against ischemic apoptosis. The mechanism of this protection consists of suppression of the endoplasmic reticulum- and mitochondria-mediated pathways. Overexpression of the anti-apoptotic protein Bcl-xL is responsible for the increased resistance to apoptosis during ischemic insult. [Abstract/Link to Full Text]

Baugé C, Beauchef G, Leclercq S, Kim SJ, Pujol JP, Galéra P, Boumédiene K
NFkappaB mediates IL-1beta-induced down-regulation of TbetaRII through the modulation of Sp3 expression.
J Cell Mol Med. 2007 Dec 5;
We previously showed that interleukin-1beta (IL-1beta) down-regulation of type II TGFbeta receptor (TbetaRII) involves NFkappaB pathway and requires de novo synthesis of a yet unknown protein. Here, we demonstrate that this effect is mediated through Sp1 site located at position -25 of human TbetaRII promoter. Inhibition of transcription factors binding (decoy oligonucleotides or mithramycin) abolished IL-1beta effect. EMSA and ChIP revealed that this treatment induced Sp3 binding to cis-sequence whereby IL-1beta exerts its transcriptional effects, whereas it decreased that of Sp1. Moreover, although the cytokine did not modulate Sp1 expression, it increased that of Sp3 via NFkappaB pathway. Experiments of gain and loss of function clearly showed that Sp3 inhibited TbetaRII expression whereas its silencing abolished IL-1beta effect. In addition, both Sp1 and Sp3 were found to interact with NFkappaB which therefore may indirectly interacts with TbetaRII promoter. All together, these data suggest that IL-1beta decreases TbetaRII expression by inducing Sp3 via NFkappaB and its binding on core promoter at the expense of Sp1, what could explain the loss of cell responsiveness in certain conditions. These findings bring new insights in the knowledge of the interference between two antagonistic transduction pathways involved in multiple physiopathological processes. [Abstract/Link to Full Text]

Patarroyo ME, Cifuentes G, Bermúdez A, Patarroyo MA
Strategies for developing multi-epitope, subunit-based, chemically-synthesized antimalarial vaccines.
J Cell Mol Med. 2007 Dec 5;
An anti-malarial vaccine against the extremely lethal P. falciparum is desperately needed. Peptides from this parasite's proteins involved in invasion and having high red blood cell binding ability were identified; these conserved peptides were not immunogenic or protection-inducing when used for immunizing Aotus monkeys. Modifying some critical binding residues in these high activity binding peptides' (HABPs) attachment to red blood cells (RBC) allowed them to induce immunogenicity and protection against experimental challenge and acquire the ability to bind to specific HLA-DRbeta1* alleles. These modified HABPs adopted certain characteristic structural configurations as determined by circular dichroism (CD) and (1)H-nuclear magnetic resonance (NMR) associated with certain HLA-DR haplotype binding activities and characteristics, such as a 2A distance difference between amino acids fitting into HLA-DRbeta1* Pockets 1 to 9, residues participating in binding to HLA-DR pockets and residues making contact with the TCR, suggesting haplotype- and allele-conscious TCR. This data has been demonstrated in HLA-DR-like genotyped monkeys and provided the basis for designing highly-effective, subunit-based, multi-antigen, multistage, synthetic vaccines, for immediate human use, malaria being one of them. [Abstract/Link to Full Text]

Yan WH, Lin A, Chen BG, Luo WD, Dai MZ, Chen XJ, Xu HH, Li BL
Unfavorable clinical implications for HLA-G expression in acute myeloid leukemia.
J Cell Mol Med. 2007 Dec 5;
Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions which have been suggested to contribute to the immune evasion of tumor cells. Studies on HLA-G expression in malignant hematopoietic diseases are controversial and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analyzed in different types of patients: de novo acute myeloid leukemia (AML, n=54), B cell acute lymphoblastic leukemia (B-ALL, n=13), chronic myeloid leukemia (CML, n=9) and myelodysplastic syndrome (MDS, n=11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukemic blast cell percentage when compared with that of HLA-G negative patients (p<0.01). Total T cell percentage was dramatically decreased in HLA-G positive patients (p<0.05). Cytogenetic karyotyping results showed that all HLA-G positive AML patients (n=5) were cytogenetically abnormal which was markedly different from that of HLA-G negative patients (p<0.01). ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukemic cells could directly inhibit NK cell cytolysis (p<0.01). These findings indicated that HLA-G expression in AML is of unfavorable clinical implications, and that HLA-G could be a potential target for therapy. [Abstract/Link to Full Text]

Bussolati G, Marchiň C, Gaetano L, Lupo R, Sapino A
Pleomorphism of the Nuclear Envelope in breast cancer: a new approach to an old problem.
J Cell Mol Med. 2007 Dec 5;
In routine practice nuclear pleomorphism of tumours is assessed by Hematoxylin staining of the membrane-bound heterochromatin. However, decoration of the Nuclear Envelope (NE) through the immunofluorescence staining of NE proteins such as Lamin B and Emerin can provide a more objective appreciation of the nuclear shape. In breast cancer nuclear pleomorphism is one of the least reproducible parameters to score histological grade, thus we sought to use NE proteins to improve the reproducibility of nuclear grading. First, immunofluorescence staining of NE as well as confocal microscopy and 3D reconstruction of nuclei in cultured cells showed a smooth and uniform NE of normal breast epithelium in contrast to an irregular foldings of the membrane and the presence of deep invaginations leading to the formation of an intranuclear scaffold of NE-bound tubules in breast cancer cells. Following the above methods and criteria, we recorded the degree of NE pleomorphism (NEP) in a series of 273 invasive breast cancers tested by immunofluorescence. A uniform nuclear shape with few irregularities (Low NEP) was observed in 135 cases or, alternatively, marked folds of the NE and an intranuclear tubular scaffold (High NEP cases) were observed in 138 cases. The latter features were significantly correlated (p-value <0.002) with lymph node metastases in 54 histological Grade 1 and in 173 cancers with low mitotic count. Decoration of the NE might thus be regarded as a novel diagnostic parameter to define the grade of malignancy, which parallels and enhances that provided by routine histological procedures. [Abstract/Link to Full Text]

Moutsatsou P, Papavassiliou AG
The glucocorticoid receptor signaling in breast cancer.
J Cell Mol Med. 2007 Dec 5;
Glucocorticoids are provided as co-medication with chemotherapy in breast cancer, albeit several lines of evidence indicate that their use may have diverse effects and in fact may inhibit chemosensitivity. The molecular basis of glucocorticoid-induced resistance to chemotherapy in breast cancer remains poorly defined-. Recent researchers, in an attempt to clarify some aspects of the underlying pathways, provide convincing evidence that glucocorticoids induce effects that are dependent upon the glucocorticoid receptor -mediated transcriptional regulation of specific genes known to play key roles in cellular/tissue functions, including growth, apoptosis, differentiation, metastasis and cell survival. In this review, we focus on how glucocorticoid-induced chemoresistance in breast cancer is mediated by the glucocorticoid receptor, unraveling the molecular interplay of glucocorticoid receptor signaling with other signaling cascades prevalent in breast cancer. We also include a detailed description of glucocorticoid receptor structure and function, summarizing data gained during recent years into the mechanism(s) of the cross-talk between the glucocorticoid receptor and other signaling cascades and secondary messengers, via which glucocorticoids exert their pleiotropic effects. [Abstract/Link to Full Text]

Ueberham E, Lindner R, Kamprad M, Hiemann R, Hilger N, Woithe B, Mahn D, Cross M, Sack U, Gebhardt R, Arendt T, Ueberham U
Oval cell proliferation in p16(INK4a) expressing mouse liver is triggered by chronic growth stimuli.
J Cell Mol Med. 2007 Dec 5;
Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a). Like other organs adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells. For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli. We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function. [Abstract/Link to Full Text]

de Almeida SF, de Sousa M
The unfolded protein response in Hereditary Hemochromatosis.
J Cell Mol Med. 2007 Dec 5;
To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signaling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signaling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in Hereditary Hemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH. [Abstract/Link to Full Text]

Marcus AJ, Coyne TM, Black IB, Woodbury D
Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation.
J Cell Mol Med. 2007 Dec 5;
We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 GFP ADSCs migrated to a number of brain regions. Examination, at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumor formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit. [Abstract/Link to Full Text]

Yang CS, Ko SR, Cho BG, Shin DM, Yuk JM, Li S, Kim JM, Evans RM, Jung JS, Song DK, Jo EK
The Ginsenoside Metabolite Compound K, a Novel Agonist of Glucocorticoid Receptor, Induces Tolerance to Endotoxin-induced Lethal Shock.
J Cell Mol Med. 2007 Dec 5;
Compound K (C-K), a protopanaxadiol ginsenoside metabolite, was previously shown to have immunomodulatory effects. Here, we describe a novel therapeutic role for C-K in the treatment of lethal sepsis through the modulation of Toll-like receptor (TLR) 4-associated signaling via glucocorticoid receptor (GR) binding. In mononuclear phagocytes, C-K significantly repressed the activation of TLR4/lipopolysaccharide (LPS)-induced NF-kappaB and mitogen-activated protein kinases (MAPKs), as well as the secretion of proinflammatory cytokines. However, C-K did not affect the TLR3-mediated expression of interferon-beta or the nuclear translocation of IRF-3. C-K competed with the synthetic glucocorticoid dexamethasone for binding to GR and activated GRE-containing reporter plasmids in a dose-dependent manner. In addition, the blockade of GR with either the GR antagonist RU486 or a siRNA against GR substantially reversed the anti-inflammatory effects of C-K. Furthermore, TLR4-dependent repression of inflammatory response genes by C-K was mediated through the disruption of p65/interferon regulatory factor complexes. Importantly, pre- or posttreatment with C-K significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock by lowering their systemic inflammatory cytokine levels and by reversing the lethal sequelae of sepsis. Collectively, these results demonstrate that C-K, as a functional ligand of GR, regulates distinct TLR4-mediated inflammatory responses, and suggest a novel therapy for Gram-negative septic shock. [Abstract/Link to Full Text]

Kirk MJ, Hayward RM, Sproull M, Scott T, Smith S, Cooley-Zgela T, Crouse NS, Citrin DE, Camphausen K
Non-patient related variables affecting levels of vascular endothelial growth factor in urine biospecimens.
J Cell Mol Med. 2007 Dec 5;
Vascular endothelial growth factor (VEGF) is an angiogenic protein proposed to be an important biomarker for the prediction of tumor growth and disease progression. Recent studies suggest that VEGF measurements in biospecimens, including urine, may have predictive value across a range of cancers. However, the reproducibility and reliability of urinary VEGF measurements have not been determined. We collected urine samples from patients receiving radiation treatment for glioblastoma multiforme (GBM) and examined the effects of five variables on measured VEGF levels using an ELISA assay. To quantify the factors affecting the precision of the assay, two variables were examined: the variation between ELISA kits with different lot numbers and the variation between different technicians. Three variables were tested for their effects on measured VEGF concentration: the time the specimen spent at room temperature prior to assay, the addition of protease inhibitors prior to specimen storage, and the alteration of urinary pH. This study found that VEGF levels were consistent across three different ELISA kit lot numbers. However, significant variation was observed between results obtained by different technicians. VEGF concentrations were dependent on time at room temperature before measurement, with higher values observed 3-7 hrs after removal from the freezer. No significant difference was observed in VEGF levels with the addition of protease inhibitors, and alteration of urinary pH did not significantly affect VEGF measurements. In conclusion, this determination of the conditions necessary to reliably measure urinary VEGF levels will be useful for future studies related to protein biomarkers and disease progression. [Abstract/Link to Full Text]

Radulescu RT, Boenisch T
Blocking endogenous peroxidases: a cautionary note for immunohistochemistry.
J Cell Mol Med. 2007 Dec 5; [Abstract/Link to Full Text]

Wan L, Lin CW, Lin YJ, Sheu JJ, Chen BH, Liao CC, Tsai Y, Lin WY, Lai CH, Tsai FJ
Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2 : STAT6 heterodimer.
J Cell Mol Med. 2007 Nov 26;
The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signaling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2:STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signaling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra. [Abstract/Link to Full Text]

Gong Q, Huntsman C, Ma D
Clathrin-independent internalization and recycling.
J Cell Mol Med. 2007 Nov 26;
The functionality of receptor and channel proteins depends directly upon their expression level on the plasma membrane. Therefore, the ability to selectively adjust the surface level of a particular receptor or channel protein is pivotal to many cellular signaling events. The internalization and recycling pathway plays a major role in the regulation of protein surface level, and thus has been a focus of research for many years. Although several endocytic pathways have been identified, most of our knowledge has come from the clathrin-dependent pathway, while the other pathways remain much less well defined. Considering that clathrin-independent internalization may account for as much as 50% of the total endocytic activity in the cell, the lack of such knowledge constitutes a major gap in our efforts to understand how different internalization pathways are utilized and coordinated. Recent studies have provided valuable insights into this area, yet many more questions still remain. In this review, we will give a panoramic introduction to the current knowledge of various internalization and recycling pathways, with an emphasis on the latest findings that have broadened our view of the clathrin-independent pathways. We will also dedicate one section to the emerging studies of the clathrin-independent internalization pathways in neuronal cells. [Abstract/Link to Full Text]

Popescu BO
Still debating a cause and diagnostic criteria for Alzheimer's disease.
J Cell Mol Med. 2007 Nov 20; [Abstract/Link to Full Text]

Lockshin RA, Zakeri Z
Cell death in health and disease.
J Cell Mol Med. 2007 Nov 20;
Cell death is clearly an important factor in development, homeostasis, pathology, and in aging, but medical efforts based on controlling cell death have not become major aspects of medicine. There are several reasons why hopes have been slow to be fulfilled, and they present indications for new directions in research. Most effort has focused on the machinery of cell death, or the proximate effectors of apoptosis and their closely-associated and interacting proteins. But cells have many options other than apoptosis. These include autophagy, necrosis, atrophy, and stepwise or other alternate means of self-disassembly. The response of a cell to a noxious or otherwise intimidating signal will depend heavily on the history, lineage, and current status of the cell. Many metabolic and other processes adjust the sensitivity of cells to signals, and viruses aggressively attempt to regulate the death of their host cells. Another complicating factor is that many death-associated proteins may have functions totally unrelated to their role in cell death, generating the possibility of undesirable side effects if one interferes with them. In the future, the challenge will be more to understand the challenge to the cell from a more global standpoint, including many more aspects of metabolism, and work toward alleviating or provoking the challenge in a targeted fashion. [Abstract/Link to Full Text]

Price ND, Foltz G, Madan A, Hood L, Tian Q
Systems Biology and Cancer Stem Cells.
J Cell Mol Med. 2007 Nov 20;
The identification, purification, and characterization of cancer stem cells holds tremendous promise for improving the treatment of cancer. Mounting evidence is demonstrating that only certain tumor cells (i.e. the cancer stem cells) can give rise to tumors when injected and that these purified cell populations generate heterogeneous tumors. While the cell of origin is still not determined definitively, specific molecular markers for populations containing these cancer stem cells have been found for leukemia, brain cancer, and breast cancer, among others. Systems approaches, particularly molecular profiling, have proven to be of great utility for cancer diagnosis and characterization. These approaches also hold significant promise for identifying distinctive properties of the cancer stem cells, and progress is already being made. [Abstract/Link to Full Text]

Mangieri D, Nico B, Benagiano V, De Giorgis M, Vacca A, Ribatti D
Angiogenic activity of multiple myeloma endothelial cells in vivo in the chick embryo chorioallantoic membrane assay is associated to a down-regulation in the expression of endogenous endostatin.
J Cell Mol Med. 2007 Nov 20;
We have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells (MMEC) by using the chick embryo chorioallantoic membrane (CAM) assay and by reverse transcriptase-polymerase chain reaction (RT-PCR). Results showed that in the CAM assay MMEC induced an angiogenic response comparable to that of a well known angiogenic cytokine, namely fibroblast growth factor-2 (FGF-2), while RT-PCR demonstrated that the expression of endostatin mRNA detected in MM treated CAM was significantly lower respect to control CAM. These data suggest that angiogenic switch in MM may involve loss of an endogenous angiogenesis inhibitor, such as endostatin. [Abstract/Link to Full Text]

Knizetova P, Darling JL, Bartek J
Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy.
J Cell Mol Med. 2007 Nov 20;
Malignant astrogliomas are among the most aggressive, highly vascular and infiltrating tumours bearing a dismal prognosis, mainly due to their resistance to current radiation treatment and chemotherapy. Efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells, their biological properties, interplay with their local microenvironment or 'niche', and their role in tumour progression and recurrence. Both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor (VEGF) are emerging as key factors that determine astroglioma cell fate. Here, we review these recent rapid advances in astroglioma research, with emphasis on the significance of VEGF in astroglioma stem-like cell biology. Furthermore, we highlight the unique DNA damage checkpoint properties of the CD133-marker-positive astroglioma stem-like cells, discuss their potential involvement in astroglioma radioresistance, and consider the implications of this new knowledge for designing combinatorial, more efficient therapeutic strategies. [Abstract/Link to Full Text]

Zuba-Surma EK, Kucia M, Abdel-Latif A, Dawn B, Hall B, Singh R, Lillard JW, Ratajczak MZ
Morphological characterization of very small embryonic- like stem cells (VSELs) by image stream system analysis.
J Cell Mol Med. 2007 Nov 20;
Background: Recently, our group purified a rare population of primitive Sca1(+)/Lin(-)/CD45(-) cells from murine bone marrow by employing multi-parameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). Objectives: In order to better characterize VSELs, we focused on their morphological parameters (e.g., diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. Methods: To examine the morphological features of VSELs, we employed a multidimensional approach, including i) traditional flow cytometry, ii) a novel approach, which is ImageStream (IS) cytometry, and iii) confocal microscopy. Results: We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than hematopoetic stem cells (HSC) (3.63+/-0.09 vs. 6.54+/-0.17 mum in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Conclusion: Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbor small primitive cells that are larger than platelets and smaller than erythrocytes. [Abstract/Link to Full Text]

Wang H, Chen C, Song X, Chen J, Zhen Y, Sun K, Hui R
Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene.
J Cell Mol Med. 2007 Nov 16;
The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is a both unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter and CARK transcription level can be downregulated by MEF2C antisence. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays. [Abstract/Link to Full Text]

Cruz-Gonzalez I, Pabón P, Rodríguez-Barbero A, Martín-Moreiras J, Pericacho M, Sánchez PL, Ramirez V, Sánchez-Ledesma M, Martín-Herrero F, Jiménez-Candil J, Maree AO, Sánchez-Rodríguez A, Martín-Luengo C, López-Novoa JM
Identification of serum endoglin as a novel prognostic marker after acute myocardial infarction.
J Cell Mol Med. 2007 Nov 16;
Endoglin is a proliferation-associated and hypoxia-inducible protein expressed in endothelial cells. The levels of soluble circulating endoglin and their prognostic significance in patients with acute myocardial infarction (AMI) are not known. In this observational prospective study serum endoglin levels were measured by ELISA in 183 AMI patients upon admission to hospital and 48 hours later and in 72 healthy controls. Endoglin levels in AMI patients on admission were significantly lower than in healthy controls (4.25 +/- 0.99 ng/ml vs. 4.59 +/- 0.87 ng/mL; p=0.013), and decreased further in the first 48 hours (3.65 +/- 0.76 ng/ml, p<0.001). Upon follow-up (median 319 days), patients who died had a significantly greater decrease in serum endoglin level over the first 48 hours than those who survived (1.03+/-0.91 vs. 0.54+/-0.55 ng/ml; p=0.025). Endoglin decrease was an independent predictor of short term (30 days) (hazard ratio 2.33; 95% CI= 1.27-4.23; p=0.006) cardiovascular mortality, and also predicts overall cardiovascular mortality during the follow-up (median 319 days) in AMI patients (hazard ratio 2.13; 95% CI= 1.20-3.78; p=0.01). In conclusion, early changes in serum endoglin may predict mortality after acute myocardial infarction. [Abstract/Link to Full Text]

Hamaguchi Y, Matsubara T, Amano T, Uetani T, Asano H, Iwamoto T, Furukawa K, Murohara T, Nakayama S
Na(+)-independent Mg(2+) Transport Sensitive to 2-Aminoethoxydiphenyl Borate (2-APB) in Vascular Smooth Muscle Cells: Involvement of TRPM-like Channels.
J Cell Mol Med. 2007 Nov 16;
Magnesium is associated with several important cardiovascular diseases. There is an accumulating body of evidence verifying the important roles of Mg(2+)-permeable channels. In the present study, we estimated the intracellular free Mg(2+) concentration ([Mg(2+)](i)) using 31P-NMR in porcine carotid arteries. pH(i) and intracellular phosphorus compounds were simultaneously monitored. Removal of extracellular divalent cations (Ca(2+) and Mg(2+)) in the absence of Na(+) caused a gradual decrease in [Mg(2+)](i) to approximately 60% of the control value after 125 min. On the other hand, the simultaneous removal of extracellular Ca(2+) and Na(+) in the presence of Mg(2+) gradually increased [Mg(2+)](i) in an extracellular Mg(2+)-dependent manner. 2-aminoethoxydiphenyl borate (2-APB) attenuated both [Mg(2+)](i) load and depletion caused under Na(+)- and Ca(2+)-free conditions. Neither [ATP](i) nor pH(i) correlated with changes in [Mg(2+)](i). RT-PCR detected transcripts of both TRPM6 and TRPM7, although TRPM7 was predominant. In conclusion, the results suggest the presence of Mg(2+)-permeable channels of TRPM family that contribute to Mg(2+) homeostasis in vascular smooth muscle cells. The low, basal [Mg(2+)](i) level in vascular smooth muscle cells is attributable to the relatively low activity of this Mg(2+) entry pathway. [Abstract/Link to Full Text]

Pedrola L, Espert A, Valdés-Sánchez T, Sánchez-Piris M, Sirkowski EE, Scherer SS, Farińas I, Palau F
Cell expression of GDAP1 in the nervous system and pathogenesis of Charcot-Marie-Tooth type 4A disease.
J Cell Mol Med. 2007 Nov 16;
Mutations in the mitochondrial protein GDAP1 are the cause of Charcot-Marie-Tooth type 4A disease (CMT4A), a severe form of peripheral neuropathy associated with either demyelinating, axonal or intermediate phenotypes. GDAP1 is located in the outer mitochondrial membrane and it seems that may be related with the mitochondrial network dynamics. We are interested to define cell expression in the nervous system and the effect of mutations in mitochondrial morphology and pathogenesis of the disease. We investigated GDAP1 expression in the nervous system and dorsal root ganglia (DRG) neuron cultures. GDAP1 is expressed in motor and sensory neurons of the spinal cord and other large neurons such as cerebellar Purkinje neurons, hippocampal pyramidal neurons, mitral neurons of the olfactory bulb, and cortical pyramidal neurons. The lack of GDAP1 staining in the white matter and nerve roots suggested that glial cells do not express GDAP1. In DRG cultures satellite cells and Schwann cells were GDAP1-negative. Overexpression of GDAP1 induced fragmentation of mitochondria suggesting a role of GDAP1 in the fission pathway of the mitochondrial dynamics. Missense mutations showed two different patterns: most of them induced mitochondrial fragmentation but the T157P mutation showed an aggregation pattern. Whereas null mutations of GDAP1 should be associated with loss of function of the protein, missense mutations may act through different pathogenic mechanisms including a dominant-negative effect, suggesting that different molecular mechanisms may underlay the pathogenesis of CMT4A. [Abstract/Link to Full Text]

Recent Articles in Molecules and Cells

Lee K, Lee SM, Park SR, Jung J, Moon JK, Cheong JJ, Kim M
Overexpression of Arabidopsis homogentisate phytyltransferase or tocopherol cyclase elevates vitamin E content by increasing gamma-tocopherol level in lettuce (Lactuca sativa L.).
Mol Cells. 2007 Oct 31;24(2):301-6.
Tocopherols, essential components of the human diet, are synthesized exclusively by photosynthetic organisms. To increase tocopherol content by increasing total flux to the tocopherol biosynthetic pathway, genes encoding Arabidopsis homogentisate phytyltransferase (HPT/V-TE2) and tocopherol cyclase (TC/VTE1) were constitutively overexpressed in lettuce (Lactuca sativa L.). Total tocopherol content of the transgenic plants overexpressing either of the genes was increased by more than 2-fold mainly due to an increase in gamma-tocopherol. However, chlorophyll content in the HPT/VTE2 and TC/VTE1 transgenic lines decreased by up to 20% and increased by up to 35%, respectively (P < 0.01). These results demonstrate that manipulation of the tocopherol biosynthetic pathway can increase or decrease chlorophyll content depending on the gene introduced. [Abstract/Link to Full Text]

Kim YU, Hong Y
Functional analysis of the first mannosyltransferase (PIG-M) involved in glycosylphosphatidylinositol synthesis in Plasmodium falciparum.
Mol Cells. 2007 Oct 31;24(2):294-300.
The mammalian glycosylphosphatidylinositol (GPI) anchor consists of three mannoses attached to acylated GlcN-(acyl)PI to form Man(3)-GlcN-(acyl)PI. The first of the three mannose groups is attached to an intermediate to generate Man-GlcN-(acyl)PI by the first mannosyltransferase (GPI-MT-I). Mammalian and protozoan GPI-MT-I have different substrate specificities. PIG-M encodes the mammalial GPI-MT-I which has 423 amino acids and multiple transmembrane domains. In this work we cloned PIG-M homologues from humans, Plasmodium falciparum (PfPIG-M), and Saccharomyces cerevisiae (GPI14), to test whether they could complement GPI-MT-I-deficient mammalian cells, since this biosynthetic step is likely to be a good target for selective screening of inhibitors against many pathogenic organisms. PfPIG-M partially restored cell surface expression of the GPI-anchored protein CD59 in PIG-M deficient mammalian cells, and first mannose transfer activity in vitro; however, this was not the case for GPI14. [Abstract/Link to Full Text]

Lee K, Shim JH, Kang MJ, Kim JH, Ahn JS, Yoo SJ, Kim Kwon Y, Kwon H
Association of BAF53 with mitotic chromosomes.
Mol Cells. 2007 Oct 31;24(2):288-93.
The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis. [Abstract/Link to Full Text]

Choi SH, Seo GY, Nam EH, Jeon SH, Kim HA, Park JB, Kim PH
Opposing effects of Arkadia and Smurf on TGFbeta1-induced IgA isotype expression.
Mol Cells. 2007 Oct 31;24(2):283-7.
TGF-beta1 induces Ig germ-line alpha (GLalpha) transcription and subsequent class switching recombination (CSR) to IgA. In the present study, we investigated the roles of two E3-ubiquitin ligases, Smurfs (HECT type) and Arkadia (RING finger type) on TGFbeta1-induced IgA CSR. We found that over-expression of Smurf1 and Smurf2 decreased TGFbeta1-induced GLalpha promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Further, over-expression of Smurf1 and Smurf2 decreased both Smad3/4-mediated and Runx3-mediated GLalpha promoter activities, suggesting that the Smurfs can down-regulate the major TGF-beta1 signaling pathway and decrease GLalpha gene expression. In parallel, the over-expressed Smurf1 decreased the expression of endogenous IgA CSR-predictive transcripts (GLT(alpha), PST(alpha), and CT(alpha)) and also TGFbeta1-induced IgA secretion. Conversely over-expression of Arkadia abolished the inhibitory effect of Smad7 on TGFbeta1-induced GLT(alpha) expression and IgA secretion. Similar results were obtained in the presence of over-expressed Smad7 and Smurf1. These results indicate that Arkadia can amplify TGFbeta1-induced IgA CSR by degrading Smad7, which interacts with Smurf1. We conclude that Smurf and Arkadia have opposite roles in the regulation of TGFbeta1-induced IgA isotype expression. [Abstract/Link to Full Text]

Jeong JC, Shin D, Lee J, Kang CH, Baek D, Cho MJ, Kim MC, Yun DJ
Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis.
Mol Cells. 2007 Oct 31;24(2):276-82.
Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling. [Abstract/Link to Full Text]

Kwon SJ, Choi EY, Seo JB, Park OK
Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach.
Mol Cells. 2007 Oct 31;24(2):268-75.
Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach. [Abstract/Link to Full Text]

Na KS, Park BC, Jang M, Cho S, Lee do H, Kang S, Lee CK, Bae KH, Park SG
Protein disulfide isomerase is cleaved by caspase-3 and -7 during apoptosis.
Mol Cells. 2007 Oct 31;24(2):261-7.
Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action. [Abstract/Link to Full Text]

Tokalov SV, Gruener S, Schindler S, Iagunov AS, Baumann M, Abolmaali ND
A number of bone marrow mesenchymal stem cells but neither phenotype nor differentiation capacities changes with age of rats.
Mol Cells. 2007 Oct 31;24(2):255-60.
Bone marrow (BM) derived mesenchymal stem cells (MSC) are pluripotent cells which can differentiate into osteogenic, adipogenic and other lineages. In spite of the broad interest, the information about the changes in BM cell composition, in particularly about the variation of MSC number and their properties in relation to the age of the donor is still controversial. The aim of this study was to investigate the age associated changes in variations of BM cell composition, phenotype and differentiation capacities of MSC using a rat model. Cell populations were characterized by flow cytometry using light scattering parameters, DNA content and a set of monoclonal antibodies. Single cell analysis was performed by conventional fluorescent microscopy. In vitro culture of MSC was established and their phenotype and capability for in vitro differentiation into osteogenic and adipogenic cells was shown. Age related changes in tibiae and femurs, amount of BM tissue, BM cell composition, proportions of separated MSC and yield of MSC in 2 weeks of in vitro culture were found. At the same time, neither change in phenotype no in differentiation capacities of MSC was registered. Age-related changes of the number of MSC should be taken into account whenever MSC are intended to be used for investigations. [Abstract/Link to Full Text]

Kim JY, Choung S, Lee EJ, Kim YJ, Choi YC
Immune activation by siRNA/liposome complexes in mice is sequence- independent: lack of a role for Toll-like receptor 3 signaling.
Mol Cells. 2007 Oct 31;24(2):247-54.
Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon alpha, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics. [Abstract/Link to Full Text]

Gang J, Choi J, Lee JH, Nham SU
Identification of critical residues for plasminogen binding by the alphaX I-domain of the beta2 integrin, alphaXbeta2.
Mol Cells. 2007 Oct 31;24(2):240-6.
The beta2 integrins on leukocytes play important roles in cell adhesion, migration and phagocytosis. One of the beta2 integrins, alphaXbeta2 (CD11c/CD18), is known to bind ligands such as fibrinogen, Thy-1 and iC3b, but its function is not well characterized. To understand its biological roles, we attempted to identify novel ligands. The functional moiety of alphaXbeta2, the alphaX I-domain, was found to bind plasminogen, the zymogen of plasmin, with moderate affinity (1.92 X 10-(6) M) in the presence of Mg(2+) or Mn(2+). The betaD-alpha5 loop of the alphaX I-domain proved to be responsible for binding, and lysine residues (Lys(242), Lys(243)) in the loop were the most important for recognizing plasminogen. An excess amount of the lysine analog, 6-aminohexanoic acid, inhibited alphaX I-domain binding to plasminogen, indicating that binding is lysine-dependent. The results of this study indicate that leukocytes regulate plasminogen activation, and consequently plasmin activities, through an interaction with alphaXbeta2 integrin. [Abstract/Link to Full Text]

Sohn SI, Kim YH, Kim BR, Lee SY, Lim CK, Hur JH, Lee JY
Transgenic tobacco expressing the hrpN(EP) gene from Erwinia pyrifoliae triggers defense responses against botrytis cinerea.
Mol Cells. 2007 Oct 31;24(2):232-9.
HrpN(EP), from the gram-negative pathogen, Erwinia pyrifoliae, is a member of the harpin group of proteins, inducing pathogen resistance and hypersensitive cell death in plants. When the hrpN(EP) gene driven by the OsCc1 promoter was introduced into tobacco plants via Agrobacterium-mediated transformation, their resistance to the necrotrophic fungal pathogen, Botrytis cinerea, increased. Resistance to B. cinerea was correlated with enhanced induction of SA-dependent genes such as PR-1a, PR2, PR3 and Chia5, of JA-dependent genes such as PR-1b, and of genes related to ethylene production, such as NT-EFE26, NT-1A1C, DS321, NT-ACS1 and NT-ACS2. However the expression of NPR1, which is thought to be essential for multiple-resistance, did not increase. Since the pattern of expression of defense-related genes in hrpN(EP)-expressing tobacco differed from that in plants expressing hpaG(Xoo) from Xanthomonas oryzae pv. Oryzae, these results suggest that different harpins can affect the expression of different defense-related genes, as well as resistance to different plant pathogens. [Abstract/Link to Full Text]

Cieslak D, Lazou A
Regulation of BAD protein by PKA, PKCdelta and phosphatases in adult rat cardiac myocytes subjected to oxidative stress.
Mol Cells. 2007 Oct 31;24(2):224-31.
H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases. [Abstract/Link to Full Text]

Jang CS, Jung JH, Yim WC, Lee BM, Seo YW, Kim W
Divergence of genes encoding non-specific lipid transfer proteins in the poaceae family.
Mol Cells. 2007 Oct 31;24(2):215-23.
The genes encoding non-specific lipid transfer proteins (nsLTPs), members of a small multigene family, show a complex pattern of expressional regulation, suggesting that some diversification may have resulted from changes in their expression after duplication. In this study, the evolution of nsLTP genes within the Poaceae family was characterized via a survey of the pseudogenes and unigenes encoding the nsLTP in rice pseudomolecules and the NCBI unigene database. nsLTP-rich regions were detected in the distal portions of rice chromosomes 11 and 12; these may have resulted from the most recent large segmental duplication in the rice genome. Two independent tandem duplications were shown to occur within the nsLTP-rich regions of rice. The genomic distribution of the nsLTP genes in the rice genome differs from that in wheat. This may be attributed to gene migration, chromosomal rearrangement, and/or differential gene loss. The genomic distribution pattern of nsLTP genes in the Poaceae family points to the existence of some differences among cereal nsLTP genes, all of which diverged from an ancient gene. The unigenes encoding nsLTPs in each cereal species are clustered into five groups. The somewhat different distribution of nsLTP-encoding EST clones between the groups across cereal species imply that independent duplication(s) followed by subfunctionalization (and/or neofunctionalization) of the nsLTP gene family in each species occurred during speciation. [Abstract/Link to Full Text]

Lu C, Chen D, Zhang Z, Fang F, Wu Y, Luo L, Yin Z
Heat Shock Protein 90 regulates the stability of c-Jun in HEK293 Cells.
Mol Cells. 2007 Oct 31;24(2):210-4.
The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxy-carbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells. [Abstract/Link to Full Text]

Kim SY, Kim JH, Lee HS, Noh SM, Song KS, Cho JS, Jeong HY, Kim WH, Yeom YI, Kim NS, Kim S, Yoo HS, Kim YS
Meta- and gene set analysis of stomach cancer gene expression data.
Mol Cells. 2007 Oct 31;24(2):200-9.
We generated gene expression data from the tissues of 50 gastric cancer patients, and applied meta-analysis and gene set analysis to this data and three other stomach cancer gene expression data sets to define the gene expression changes in gastric tumors. By meta-analysis we identified genes consistently changed in gastric carcinomas, while gene set analysis revealed consistently changed biological themes. Genes and gene sets involved in digestion, fatty acid metabolism, and ion transport were consistently down-regulated in gastric carcinomas, while those involved in cellular proliferation, cell cycle, and DNA replication were consistently up-regulated. We also found significant differences between the genes and gene sets expressed in diffuse and intestinal type gastric carcinoma. By gene set analysis of cytogenetic bands, we identified many chromosomal regions with possible gross chromosomal changes (amplifications or deletions). Similar analysis of transcription factor binding sites (TFBSs), revealed transcription factors that may have caused the observed gene expression changes in gastric carcinomas, and we confirmed the overexpression of one of these, E2F1, in many gastric carcinomas by tissue array and immunohistochemistry. We have incorporated the results of our meta- and gene set analyses into a web accessible database ( [Abstract/Link to Full Text]

Seong KM, Baek JH, Ahn BY, Yu MH, Kim J
Rpn10p is a receptor for ubiquitinated Gcn4p in proteasomal proteolysis.
Mol Cells. 2007 Oct 31;24(2):194-9.
GCN4 is a typical eukaryotic transcriptional activator that is implicated in the expression of many genes involved in amino acids and purine biosyntheses under stress conditions. It is degraded by 26S proteasomes following ubiquitination. However, the immediate receptor for ubiquitinated Gcn4p has not yet been identified. We investigated whether ubiquitinated Gcn4p binds directly to Rpn10p as the ubiquitinated substrate receptor of the 26S proteasome. We found that the level of Gcn4p increased in cells deleted for Rpn10p but not in cells deleted for RAD23 and DSK2, the other ubiquitinated substrate receptors and, unlike Rpn10p, neither of these proteins recognized ubiquitinated Gcn4p. These results suggest that Rpn10p is the receptor that binds the polyubiquitin chain during ubiquitin-dependent proteolysis of Gcn4p. [Abstract/Link to Full Text]

Seo HS, Li J, Lee SY, Yu JW, Kim KH, Lee SH, Lee IJ, Paek NC
The Hypernodulating nts mutation induces jasmonate synthetic pathway in soybean leaves.
Mol Cells. 2007 Oct 31;24(2):185-93.
Symbiotic nitrogen fixation with nitrogen-fixing bacteria in the root nodules is a distinctly beneficial metabolic process in legume plants. Legumes control the nodule number and nodulation zone through a systemic negative regulatory system between shoot and root. Mutation in the soybean NTS gene encoding GmNARK, a CLAVATA1-like serine/threonine receptor-like kinase, causes excessive nodule development called hypernodulation. To examine the effect of nts mutation on the gene expression profile in the leaves, suppression subtractive hybridization was performed with the trifoliate leaves of nts mutant 'SS2-2' and the wild-type (WT) parent Sinpaldalkong2, and 75 EST clones that were highly expressed in the leaves of the SS2-2 mutant were identified. Interestingly, the expression of jasmonate (JA)-responsive genes such as vspA, vspB, and Lox2 were upregulated, whereas that of a salicylate-responsive gene PR1a was suppressed in the SS2-2 mutant. In addition, the level of JA was about two-fold higher in the leaves of the SS2-2 mutant than in those of the WT under natural growth conditions. Moreover, the JA-responsive gene expression persists in the leaves of SS2-2 mutant without rhizobia infection in the roots. Taken together, our results suggest that the nts mutation increases JA synthesis in mature leaves and consequently leads to constitutive expression of JA-responsive genes which is irrelevant to hypernodulation in the root. [Abstract/Link to Full Text]

Hartmann C
Skeletal development--Wnts are in control.
Mol Cells. 2007 Oct 31;24(2):177-84.
Approximately 200 individual skeletal elements, which differ in shape and size, are the building blocks of the vertebrate skeleton. Various features of the individual skeletal elements, such as their location, shape, growth and differentiation rate, are being determined during embryonic development. A few skeletal elements, such as the lateral halves of the clavicle and parts of the skull are formed by a process called intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts, while the majority of skeletal elements are formed via endochondral ossification. The latter process starts with the formation of a cartilaginous template, which eventually is being replaced by bone. This requires co-regulation of differentiation of the cell-types specific for cartilage and bone, chondrocytes and osteoblasts, respectively. In recent years it has been demonstrated that Wnt family members and their respective intracellular pathways, such as non-canonical and the canonical Wnt/beta-catenin pathway, play important and diverse roles during different steps of vertebrate skeletal development. Based on the recent discoveries modulation of the canonical Wnt-signaling pathway could be an interesting approach to direct stem cells into certain skeletal lineages. [Abstract/Link to Full Text]

Thompson EA
PPARgamma physiology and pathology in gastrointestinal epithelial cells.
Mol Cells. 2007 Oct 31;24(2):167-76.
Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed at very high levels in the gastrointestinal epithelium. Many of the functions of PPARgamma in gastrointestinal epithelial cells have been elucidated in recent years, and a pattern is emerging which suggests that this receptor plays an important role in gastrointestinal physiology. There is also strong evidence that PPARgamma is a colon cancer suppressor in pre-clinical rodent models of sporadic colon cancer, and there is considerable interest in exploitation of PPARgamma agonists as prophylactic or chemopreventive agents in colon cancer. Studies in mice and in human colon cancer cell lines suggest several mechanisms that might account for the tumor suppressive effects of PPARgamma agonists, although it is not in all cases clear whether these effects are altogether mediated by PPARgamma. Conversely, several reports suggest that PPARgamma agonists may promote colon cancer under certain circumstances. This possibility warrants considerable attention since several million individuals with type II diabetes are currently taking PPARgamma agonists. This review will focus on recent data related to four critical questions: what is the physiological function of PPARgamma in gastrointestinal epithelial cells; how does PPARgamma suppress colon carcinogenesis; is PPARgamma a tumor promoter; and what is the future of PPARgamma in colon cancer prevention? [Abstract/Link to Full Text]

Bartlett AH, Hayashida K, Park PW
Molecular and cellular mechanisms of syndecans in tissue injury and inflammation.
Mol Cells. 2007 Oct 31;24(2):153-66.
The syndecan family of heparan sulfate proteoglycans is expressed on the surface of all adherent cells. Syndecans interact with a wide variety of molecules, including growth factors, cytokines, proteinases, adhesion receptors and extracellular matrix components, through their heparan sulfate chains. Recent studies indicate that these interactions not only regulate key events in development and homeostasis, but also key mechanisms of the host inflammatory response. This review will focus on the molecular and cellular aspects of how syndecans modulate tissue injury and inflammation, and how syndecans affect the outcome of inflammatory diseases in vivo. [Abstract/Link to Full Text]

Ha HS, Huh JW, Kim DS, Kang DW, Cho BW, Kim HS
Promoter activity of the long terminal repeats of porcine endogenous retroviruses of the Korean domestic pig.
Mol Cells. 2007 Aug 31;24(1):148-51.
Porcine endogenous retroviruses (PERVs) in the pig genome represent a potential risk of infection in pig-to-human transplantation and are transmitted vertically. The solitary long terminal repeat (LTR) elements of the PERVs affect the replication properties of the individual viruses via their repeat sequences and by encoding a set of specific transcription factors. We examined the promoter activities of solitary LTR elements belonging to the PERV-A and -B families of the Korean domestic pig (KDP) using luciferase reporters. Three of the LTR structures (of PERV-A5-KDP, PERV-A7-KDP, PERV-A8-KDP) had different promoter activities in human HCT116 cells and monkey Cos7 cells, and potential negatively and positively acting regions affecting transcription were identified by deletion analysis. These data suggest that specific sequences in the U3 region of a given LTR element can affect the activities of promoter or enhancer elements in the PERV. [Abstract/Link to Full Text]

Jin EJ, Choi YA, Sonn JK, Kang SS
Suppression of ADAM 10-induced Delta-1 shedding inhibits cell proliferation during the chondro-inhibitory action of TGF-beta3.
Mol Cells. 2007 Aug 31;24(1):139-47.
Although transforming growth factors (TGFs) are implicated in the process of endochondral ossification, which is initiated by the differentiation of mesenchymal cells into chondrocytes, it is not clear how TGF-beta 3 regulates the chondrogenic differentiation of limb bud mesenchymal cells. Here, differential display polymerase chain reaction (DD-PCR) screening and RT-PCR analysis revealed that transcripts of A Disintegrin And Metalloprotease 10 (ADAM 10) decreased during the chondro-inhibitory action of TGF-beta 3 on cultured chick leg bud mesenchymal cells. Electroporation of ADAM 10 morpholino antisense oligonucleotides inhibited the ectodomain shedding of delta-1, and cell proliferation and subsequent precartilage condensation, in a manner similar to that caused by TGF-beta3. The suppression of mesenchymal cell proliferation induced by TGF-beta 3 and ADAM 10 morpholino antisense oligonucleotides was reversed by activation of ADAM 10 with phorbol 12-myristate 13-acetate (PMA) or knockdown of Notch-1 with siRNA. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta 3 downregulates ADAM 10 and inhibits cell proliferation and subsequent precartilage condensation by inhibiting the ectodomain shedding of delta-1, and that this results in the activation of Notch signaling. [Abstract/Link to Full Text]

Ju SA, Park SM, Lee SC, Kwon BS, Kim BS
Marked expansion of CD11c+CD8+ T-cells in melanoma-bearing mice induced by anti-4-1BB monoclonal antibody.
Mol Cells. 2007 Aug 31;24(1):132-8.
4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is expressed on activated T-cells, and 4-1BB signaling due to interaction with 4-1BB ligand or ligation with anti-4-1BB monoclonal antibody (mAb) costimulates T cells. It has been shown that administration of anti-4-1BB mAb induces anti-tumor immunity in mice, but the nature of the cellular subsets responsible for this immunity is uncertain. In this study we found that anti-4-1BB mAb administration to B16F10 melanoma-bearing mice induced marked expansion of CD11c+CD8+ T-cells in parallel with suppression of pulmonary tumors. The mAb-treated mice produced higher levels of IFN- in their tumor tissues, spleen and lymph nodes than mice exposed to control antibody. When the CD11c+CD8+ T-cells were purified and re-stimulated in vitro, they produced high levels of the Th1 cytokines, IFN- and IL-2, but low levels of the Th2 cytokines, IL-4 and IL-10. Furthermore, they expressed high levels of 4-1BB and CD107a, a marker of activated cytotoxic T-lymphocytes. Our results suggest that CD11c+CD8+ T-cells play a role in the anti-tumor immunity induced by anti-4-1BB mAb. [Abstract/Link to Full Text]

Park SW, Choi SY
Long-term expression of von Willebrand Factor by a VSV-G pseudotyped lentivirus enhances the functional activity of secreted B-Domain-deleted Coagulation Factor VIII.
Mol Cells. 2007 Aug 31;24(1):125-31.
von Willebrand factor (vWF) is a multimeric glycoprotein which functions within the coagulation system. It colocalizes with factor VIII (FVIII) by non-covalent interaction and alters its intracellular trafficking. vWF is also instrumental in maintaining the stability of secreted FVIII. The principal objective of this study was to generate a lentivirus-based vWF expression vector for gene therapy of hemophilia A. We inserted a vWF of 8.8 Kb into a lentiviral vector thereby producing VSV-G-pseudotyped vEx52. However, its titer was quite low, presumably because the length of vWF gene exceeds the size limit of the lentiviral vector. In order to overcome the low-titer, we concentrated the vEx52 and thus increased the efficiency of transduction approximately 6-fold with 1/100th of the volume. However, as concentration requires an additional laborious step, we attempted to enhance the transduction efficiency by deleting exons 24-46 and 29-46 in pRex52 to construct pRex23 and pRex28, and in pvEx52, yielding pvEx23 and pvEx28, respectively. The transfected pRex52 had a profound effect on the activity of secreted FVIII, and this activity declined as domains of vWF were deleted. However, when the domain-deleted vWF-lentiviruses were transduced into K562 cells, the vEx28 increased the activity of the secreted FVIII compared to what was observed with vEx52. This result is probably due to higher efficiencies of transduction and expression while retaining the essential domains required for proper interaction with FVIII. [Abstract/Link to Full Text]

Shin HJ, Lee H, Park JD, Hyun HC, Sohn HO, Lee DW, Kim YS
Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins.
Mol Cells. 2007 Aug 31;24(1):119-24.
TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants (KD) of LPS for immobilized CD14 and MD-2 were 8.7 microM, and 2.3 microM, respectively. The association rate constant (Kon) of LPS for MD-2 was 5.61 x 10(3) M-1S-1, and the dissociation rate constant (Koff) was 1.28 10 2 S 1, revealing slow association and fast dissociation with an affinity constant KD of 2.33 x 10-6 M at 25 degreesC. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex. [Abstract/Link to Full Text]

Choi SJ, Kim MJ, Heo HJ, Hong B, Cho HY, Kim YJ, Kim HK, Lim ST, Jun WJ, Kim EK, Shin DH
Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit.
Mol Cells. 2007 Aug 31;24(1):113-8.
The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide (Abeta). Abeta is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxi-dation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on Abeta-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2,7 -dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of Abeta in PC 12 cells, possibly by reducing oxidative stress. [Abstract/Link to Full Text]

Do JH, Kim IS, Park TK, Choi DK
Genome-wide examination of chromosomal aberrations in neuroblastoma SH-SY5Y cells by array-based comparative genomic hybridization.
Mol Cells. 2007 Aug 31;24(1):105-12.
Most neuroblastoma cells have chromosomal aberrations such as gains, losses, amplifications and deletions of DNA. Conventional approaches like fluorescence in situ hybridization (FISH) or metaphase comparative genomic hybridization (CGH) can detect chromosomal aberrations, but their resolution is low. In this study we used array-based comparative genomic hybridization to identify the chromosomal aberrations in human neuroblastoma SH-SY5Y cells. The DNA microarray consisting of 4000 bacterial artificial chromosome (BAC) clones was able to detect chromosomal regions with aberrations. The SH-SY5Y cells showed chromosomal gains in 1q12 approximately q44 (Chr1:142188905-246084832), 7 (over the whole chromosome), 2p25.3 approximately p16.3 (Chr2:18179-47899074), and 17q 21.32 approximately q25.3 (Chr17:42153031-78607159), while chromosomal losses detected were the distal deletion of 1p36.33 (Chr1:552910-563807), 14q21.1 approximately q21.3 (Chr14:37666271- 47282550), and 22q13.1 approximately q13.2 (Chr22:36885764-4190 7123). Except for the gain in 17q21 and the loss in 1p36, the other regions of gain or loss in SH-SY5Y cells were newly identified. [Abstract/Link to Full Text]

Shim HY, Park JH, Paik HD, Nah SY, Kim DS, Han YS
Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation.
Mol Cells. 2007 Aug 31;24(1):95-104.
The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation. [Abstract/Link to Full Text]

Yoo SC, Cho SH, Zhang H, Paik HC, Lee CH, Li J, Yoo JH, Lee BW, Koh HJ, Seo HS, Paek NC
Quantitative trait loci associated with functional stay-green SNU-SG1 in rice.
Mol Cells. 2007 Aug 31;24(1):83-94.
During monocarpic senescence in higher plants, functional stay-green delays leaf yellowing, maintaining photosynthetic competence, whereas nonfunctional stay-green retains leaf greenness without sustaining photosynthetic activity. Thus, functional stay-green is considered a beneficial trait that can increase grain yield in cereal crops. A stay-green japonica rice 'SNU-SG1' had a good seed-setting rate and grain yield, indicating the presence of a functional stay-green genotype. SNU-SG1 was crossed with two regular cultivars to determine the inheritance mode and identify major QTLs conferring stay-green in SNU-SG1. For QTL analysis, linkage maps with 100 and 116 DNA marker loci were constructed using selective genotyping with F2 and RIL (recombinant inbred line) populations, respectively. Molecular marker-based QTL analyses with both populations revealed that the functional stay-green phenotype of SNU-SG1 is regulated by several major QTLs accounting for a large portion of the genetic variation. Three main-effect QTLs located on chromosomes 7 and 9 were detected in both populations and a number of epistatic-effect QTLs were also found. The amount of variation explained by several digenic interactions was larger than that explained by main-effect QTLs. Two main-effect QTLs on chromosome 9 can be considered the target loci that most influence the functional stay-green in SNU-SG1. The functional stay-green QTLs may help develop low-input high-yielding rice cultivars by QTL-marker-assisted breeding with SNU-SG1. [Abstract/Link to Full Text]

Moon IS, Cho SJ, Jin I, Walikonis R
A simple method for combined fluorescence in situ hybridization and immunocytochemistry.
Mol Cells. 2007 Aug 31;24(1):76-82.
By combining in situ hybridization (ISH) and immunocytochemistry (IC), microscopic topological localization of mRNAs and proteins can be determined. Although this technique can be applied to a variety of tissues, it is particularly important for use on neuronal cells which are morphologically complex and in which specific mRNAs and proteins are located in distinct subcellular domains such as dendrites and dendritic spines. One common technical problem for combined ISH and IC is that the signal for immunocytochemical localization of proteins often becomes much weaker after conducting ISH. In this manuscript, we report a simplified but robust protocol that allows immunocytochemical localization of proteins after ISH. In this protocol, we fix cultured cortical or hippocampal neurons with 4% paraformaldehyde (PFA), rinse briefly in PBS, and then further fix the cells with C methanol. Our method has several major advantages over previously described ones in that (1) it is simple, as it is just consecutive routine fixation procedures, (2) it does not require any special alteration to the fixation procedures such as changes in salt concentration, and (3) it can be used with antibodies that are compatible with either methanol (MeOH-) or PFA-fixed target proteins. To our best knowledge, we are the first to employ this fixation method for fluorescence ISH + IC. [Abstract/Link to Full Text]

Recent Articles in Cell Research

Fong PM, Tian L, Chen ZJ
Arabidopsis thaliana histone deacetylase 1 (AtHD1) is localized in euchromatic regions and demonstrates histone deacetylase activity in vitro.
Cell Res. 2006 May;16(5):479-88.
Arabidopsis thaliana histone deacetylase 1 (AtHD1 or AtHDA19), a homolog of yeast RPD3, is a global regulator of many physiological and developmental processes in plants. In spite of the genetic evidence for a role of AtHD1 in plant gene regulation and development, the biochemical and cellular properties of AtHD1 are poorly understood. Here we report cellular localization patterns of AtHD1 in vivo and histone deacetylase activity in vitro. The transient and stable expression of a green fluorescent protein (GFP)-tagged AtHD1 in onion cells and in roots, seeds and leaves of the transgenic Arabidopsis, respectively, revealed that AtHD1 is localized in the nucleus presumably in the euchromatic regions and excluded from the nucleolus. The localization patterns of AtHD1 are different from those of AtHD2 and AtHDA6 that are involved in nucleolus formation and silencing of transgenes and repeated DNA elements, respectively. In addition, a histone deacetylase activity assay showed that the recombinant AtHD1 produced in bacteria demonstrated a specific histone deacetylase activity in vitro. The data suggest that AtHD1 is a nuclear protein and possesses histone deacetylase activities responsible for global transcriptional regulation important to plant growth and development. [Abstract/Link to Full Text]

Zhang BC, Chu QS
MSM and HIV/AIDS in China.
Cell Res. 2005 Nov-Dec;15(11-12):858-64.
This article profiles current status of spread and control of HIV/AIDS in China. China has a significant population of MSM (men who have sex with men) and they have been becoming very much alive in many ways since 1990s due to recent social changes. Some surveys indicate that great many of MSM are engaged in high-risk behaviors. In addition, majority of MSM have also experienced sexual encounters with women sometimes in their lives, which possibly contribute to spread of HIV to women. Some reports documented that HIV is becoming rampant among MSM since more than 1% of them are now infected. Political, cultural and custom elements could hinder intervention activities against HIV spread among MSM. Fortunately, many cities in China have seen that MSM were in cooperation with responsible institutions carrying out certain intervention measures. The general situation is promising. The authors forecast that the fast HIV spread among MSM of China could possibly be halted within several years when the authorities become more sensible to this issue, health service institutions offer unswerving efforts toward the MSM community and those who involve in MSM undertakes necessary responsibilities. [Abstract/Link to Full Text]

Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW
Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation.
Cell Res. 2005 Feb;15(2):92-6.
Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. KLF4 and KLF5 are two closely related members of the KLF family that have a similar tissue distribution in embryos and adults. However, the two KLFs often exhibit opposite effects on regulation of gene transcription, despite binding to similar, if not identical, cis-acting DNA sequences. In addition, KLF4 and 5 exert contrasting effects on cell proliferation in many instances; while KLF4 is an inhibitor of cell growth, KLF5 stimulates proliferation. Here we review the biological properties and biochemical mechanisms of action of the two KLFs in the context of growth regulation. [Abstract/Link to Full Text]

Zhang SB, Qian RL
The interaction between the human beta-globin locus control region and nuclear matrix.
Cell Res. 2002 Dec;12(5-6):411-6.
Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human beta-globin gene. However the molecular mechanisms by which the expression of beta-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681 approximately -10971 bp, HS3 core sequence -14991 approximately -14716 bp and HS4 core sequence -18586 approximately -18306 bp) of DNase I hypersensitive sites in the human beta-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human beta-like globin genes through their interaction with HSs (HS2, HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of beta-globin gene and to promote its transcription. [Abstract/Link to Full Text]

Hu ZH, Liu Q, Shang Q, Zheng M, Yang J, Zhang YL
Identification and characterization of a new member of serpin family- HongrES1 in rat epididymis.
Cell Res. 2002 Dec;12(5-6):407-10.
A full length cDNA named HongrES1 was isolated and cloned by screening rat epididymis cDNA library using a mouse EST as a probe and 5'RACE followed. It contained 1590bp nucleotides and its predicted protein had 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain. Tissue distribution pattern showed it was specifically expressed in adult rat epididymis; moreover, in situ hybridyzation indicated this gene was expressed in a limited region of the cauda epididymis near vas deference. Such kind of expression pattern sugested that HongrES1 had potential function in male reproduction. [Abstract/Link to Full Text]

Liu B, Jin GL, Zhao SH, Yu M, Xiong TA, Peng ZZ, Li K
Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping.
Cell Res. 2002 Dec;12(5-6):401-5.
Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs. [Abstract/Link to Full Text]

Gong M, Ni JH, Jia HT
Increased exchange rate of histone H1 on chromatin by exogenous myogenin expression.
Cell Res. 2002 Dec;12(5-6):395-400.
To explore the molecular mechanism of chromatin remodeling involved in the regulation of transcriptional activation of specific genes by a myogenic regulatory factor Myogenin, we used NIH3T3 fibroblasts with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching (FRAP) in living cells. The observation from FRAP experiments with myogenin transfected fibroblasts showed that the exchange rate of histone H1 in chromatin was obviously increased, indicating that forced expression of exogenous Myogenin can induce chromatin remodeling. The hyperacetylation of histones H3 and H4 from myogenin transfected fibroblasts was detected by triton-acid-urea (TAU)/SDS (2-D) electrophoresis and Western blot with specific antibodies against acetylated N-termini of histones H3 and H4. RT-PCR analysis indicated that the nAChR alpha-subunit gene was expressed in the transfected fibroblasts. These results suggest that the expression of exogenous Myogenin can induce chromatin remodeling and activate the transcription of Myogenin-targeted gene in non-muscle cells. [Abstract/Link to Full Text]

Song GL, Tang M, Liu CJ, Luo HY, Liang HM, Hu XW, Xi JY, Gao LL, Fleischmann B, Hescheler J
Developmental changes in functional expression and beta-adrenergic regulation of I(f) in the heart of mouse embryo.
Cell Res. 2002 Dec;12(5-6):385-94.
The hyperpolarization-activated current (I(f)) plays an important role in determining the spontaneous rate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart, therefore we studied developmental changes in functional expression and beta-adrenergic regulation of I(f) in embryonic mouse heart. The expression of I(f) is high in early developmental stage (EDS) (10.5 d after coitus) ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytes and even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating that these cells of the EDS embryonic heart have some properties of pacemaker cells. Beta-adrenergic agonist isoproterenol (ISO) stimulates I(f) in LDS but not in EDS cardiomyocytes, indicating that the beta-adrenergic regulation of I(f) is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of I(f) in EDS cells, indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore, the results demonstrate that I(f) is modulated by phosphorylation via cAMP dependent PKA both in EDS and LDS cells. [Abstract/Link to Full Text]

Lu DP, Tian L, O'Neill C, King NJ
Regulation of cellular adhesion molecule expression in murine oocytes, peri-implantation and post-implantation embryos.
Cell Res. 2002 Dec;12(5-6):373-83.
Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, alpha chain), and CD11a (LFA-1, alpha chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophectoderm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophectoderm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CD11a was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo. [Abstract/Link to Full Text]

Zhang XM, Xu YH
The associated regulators and signal pathway in rIL-16/CD4 mediated growth regulation in Jurkat cells.
Cell Res. 2002 Dec;12(5-6):363-72.
IL-16 is a ligand and chemotactic factor for CD4+ T cells. IL-16 inhibits the CD3 mediated lymphocyte activation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, the presence of co-activators etc. To understand the regulation function and mechanism of IL-16 on target cells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cells in vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10(-9)M), but inhibited the growth of the cells at higher concentration (10(-5)M). Results showed that 10(-5) M of rIL-16 treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, but up-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitor markedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively. The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells at a dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but, associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation of the ERK signal pathway in Jurkat cells. [Abstract/Link to Full Text]

Yan J, Ying H, Gu F, He J, Li YL, Liu HM, Xu YH
Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration.
Cell Res. 2002 Dec;12(5-6):353-61.
Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-1/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-1/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-1/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice. mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression of mfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistry assessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liver regeneration. These data suggested that MFREP-1 might play an important role in liver regeneration and be involved in the regulation of cell growth. [Abstract/Link to Full Text]

Guo JY, Xu J, Mao Q, Fu LL, Gu JR, De Zhu J
The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene.
Cell Res. 2002 Dec;12(5-6):339-52.
The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a region displaying a high score of loss of heterozygosity within chromosome 17p13.3 in human hepatocellular carcinoma in China. To unveil the underlying mechanisms for the transcription regulation of this gene and understand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by both bioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 gene was evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its transcription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragment containing the transcription start, extending from -112 to -55, represents the minimal promoter; 4, The consensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of the minimal promoter; 5, The bioinformatic analysis suggests that the C17orf25 gene may encode a protein in the family of the glyoxalase. Our data has provided some deep insight into both function and regulation of the C17orf25 gene in the context of the normal liver and hepatocellular carcinoma. [Abstract/Link to Full Text]

Feng WG, Wang YB, Zhang JS, Wang XY, Li CL, Chang ZL
cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in murine peritoneal macrophages.
Cell Res. 2002 Dec;12(5-6):331-7.
cAMP mediated signaling may play a suppressive role in immune response. We previously found that the cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increased the transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The present study examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40 expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNA expression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed that cAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-kappaB binding to IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12 production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMP signaling pathways. [Abstract/Link to Full Text]

Pan GJ, Chang ZY, Schöler HR, Pei D
Stem cell pluripotency and transcription factor Oct4.
Cell Res. 2002 Dec;12(5-6):321-9.
Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells. [Abstract/Link to Full Text]

Xu W, Liu LZ, Loizidou M, Ahmed M, Charles IG
The role of nitric oxide in cancer.
Cell Res. 2002 Dec;12(5-6):311-20.
Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelia (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties. Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis by upregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53, poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. [Abstract/Link to Full Text]

Jin XP, Peng JB, Huang F, Zhu YN, Fei J, Guo LH
A mRNA molecule encoding truncated excitatory amino acid carrier 1 (EAAC1) protein (EAAC2) is transcribed from an independent promoter but not an alternative splicing event.
Cell Res. 2002 Sep;12(3-4):257-62.
Glutamate transporter EAAC1 removes excitatory neurotransmitter in central nervous system, and also absorbs glutamate in epithelia of intestine, kidney, liver and heart for normal cell growth. When a mouse cDNA was screened using EAAC1 cDNA fragment as probe in our lab, a transcript (GenBank U75214) encoding an EAAC1 protein with 148 residues truncated at N-terminal was cloned and named as EAAC2. Sequence analysis shows that EAAC2 has it's own start code and unique 5'UTR that is different from that of EAAC1. A mouse genomic library was screened and a positive clone including EAAC1 CDS was sequenced (GenBank AF 322393) and indicates that normal EAAC1 transcript (GenBank U73521) is transcribed from 10 exons in terms of exon I, II, III, IV, V, VI, VII, VIII, IX, X, and EAAC2 transcript is consisted by exons from IV to IX as same as that of EAAC1 and with its unique exon beta upstream to exon IV and exon delta downstream to IX. EAAC2 transcript has a cluster of transcriptional start sites not overlapping with the transcriptional start sites of EAAC1. These results indicate that EAAC2 is transcribed from an independent promoter but not an alternative splicing event. [Abstract/Link to Full Text]

Ni WM, Chen XY, Xu ZH, Xue HW
A pin gene families encoding components of auxin efflux carriers in Brassica juncea.
Cell Res. 2002 Sep;12(3-4):247-55.
Based on the sequence information of Arabidopsis PIN1, two cDNAs encoding PIN homologues from Brassica juncea, Bjpin2 and Bjpin3, were isolated through cDNA library screening. Bjpin2 and Bjpin3 encoded proteins containing 640 and 635 amino acid residues, respectively, which shared 97.5% identities with each other and were highly homologous to Arabidopsis PIN1, PIN2 and other putative PIN proteins. BjPIN2 and BjPIN3 had similar structures as AtPIN proteins. Northern blot analysis indicated that Bjpin2 was expressed in stem, leaf and floral tissues, while Bjpin3 was expressed predominantly in stem and hypocotyls. Two promoter fragments of pin genes, Bjpin-X and Bjpin-Z, were isolated by 'genome walking' technique using primers at 5'-end of pin cDNA. Promoter-gus fusion studies revealed the GUS activities driven by Bjpin-X were at internal side of xylem and petal; while those driven by Bjpin-Z were detected at leaf vein, epidermal cell and cortex of stem, vascular tissues and anther. Results of the pin genes with different expression patterns in B. juncea suggested the presence of a gene family. [Abstract/Link to Full Text]

Ni WM, Chen XY, Xu ZH, Xue HW
Isolation and functional analysis of a Brassica juncea gene encoding a component of auxin efflux carrier.
Cell Res. 2002 Sep;12(3-4):235-45.
Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (Gälweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpinl), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPIN1 and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPIN1 and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpinl was expressed in most of the tissues tested, with a relatively higher level of transcript in flowers and a lower level in root tissues. Promoter-reporter gene fusion studies further revealed the expression of Bjpinl in the mature pollen grains, young seeds, root tip, leaf vascular tissue and trace bundle, stem epidermis, cortex and vascular cells. BjPIN1 was localized on the plasma membrane as demonstrated through fusion expression of green fluorescent protein (GFP). Auxin efflux carrier activity was elevated in transgenic Arabidopsis expressing BjPIN1. [Abstract/Link to Full Text]

Chen X, Zhang W, Gao YF, Su XQ, Zhai ZH
Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line.
Cell Res. 2002 Sep;12(3-4):229-33.
P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells. [Abstract/Link to Full Text]

Peng WM, Yu LL, Bao CY, Liao F, Li XS, Zuo MX
Transplanted neuronal precursors migrate and differentiate in the developing mouse brain.
Cell Res. 2002 Sep;12(3-4):223-8.
The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation, which is likely to be induced by some environmental factors. [Abstract/Link to Full Text]

Li B, Zhang YL
Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization.
Cell Res. 2002 Sep;12(3-4):215-21.
In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression subtractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88 genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes were firstly associated with UL. Three genes with notable difference were selected for Northern confirmation. Our results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate, testis, liver, heart and skeletal muscle. [Abstract/Link to Full Text]

Chhabra D, Bao S, dos Remedios CG
The distribution of cofilin and DNase I in vivo.
Cell Res. 2002 Sep;12(3-4):207-14.
Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassembled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one or more actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however, we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length. Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in their distributions. Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division. [Abstract/Link to Full Text]

Sun ZG, Kong WH, Zhang YJ, Yan S, Lu JN, Gu Z, Lin F, Tso JK
A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation.
Cell Res. 2002 Sep;12(3-4):199-206.
p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13(suc1)-agarose affinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accession number: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% identities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functional domains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant protein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis, a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paper reveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation. [Abstract/Link to Full Text]

Xu J, De Zhu J, Ni M, Wan F, Gu JR
The ATF/CREB site is the key element for transcription of the human RNA methyltransferase like 1(RNMTL1) gene, a newly discovered 17p13.3 gene.
Cell Res. 2002 Sep;12(3-4):177-97.
The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17p13.3 that suffers from a high frequent loss of heterozygosity in human hepatocellular carcinoma in China[1-5]. To understand the molecular mechanisms underlying transcription control of the RNMTL1 gene in human cancers, we decline using of the conventional approach where the cis-elements bound by the known transcription factors are primary targets, and carried out the systematic analyses to dissect the promoter structure and identify/characterize the key cis-elements that are responsible for its strong expression in cell. The molecular approaches applied included 1, the primer extension for mapping of the transcription starts; 2, the transient transfection/reporter assays on a large number of deletion and site-specific mutants of the promoter segment for defining the minimal promoter and the crucial elements within; and 3, the electrophoresis mobility shift assay with specific antibodies for reconfirming the nature of the transcription factors and their cognate cis-elements. We have shown that the interaction of an ATF/CREB element (-38 to -31) and its cognate transcription factors play a predominant role in the promoter activity of the RNMTL1 gene. The secondary DNA structures of the ATF/CREB element play a more vital role in the protein-DNA interaction. Finally, we reported a novel mechanism underlying the YY1 mediated transcription repression, namely, the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding. [Abstract/Link to Full Text]

Sarras MP, Yan L, Leontovich A, Zhang JS
Structure, expression, and developmental function of early divergent forms of metalloproteinases in hydra.
Cell Res. 2002 Sep;12(3-4):163-76.
Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra. [Abstract/Link to Full Text]

Chen D, Payne LG
Targeting epidermal Langerhans cells by epidermal powder immunization.
Cell Res. 2002 Jun;12(2):97-104.
Immune reactions to foreign or self-antigens lead to protective immunity and, sometimes, immune disorders such as allergies and autoimmune diseases. Antigen presenting cells (APC) including epidermal Langerhans cells (LCs) play an important role in the course and outcome of the immune reactions. Epidermal powder immunization (EPI) is a technology that offers a tool to manipulate the LCs and the potential to harness the immune reactions towards prevention and treatment of infectious diseases and immune disorders. [Abstract/Link to Full Text]

Shiokawa K, Kajita E, Hara H, Yatsuki H, Hori K
A developmental biological study of aldolase gene expression in Xenopus laevis.
Cell Res. 2002 Jun;12(2):85-96.
We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source. aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism. We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb) contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity. [Abstract/Link to Full Text]

Hu YX, Guo JY, Shen L, Chen Y, Zhang ZC, Zhang YL
Get effective polyclonal antisera in one month.
Cell Res. 2002 Jun;12(2):157-60.
According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks. In general, high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4 months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis. It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells. [Abstract/Link to Full Text]

Li MS, Li PF, Yang FY, He SP, Du GG, Li G
The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells.
Cell Res. 2002 Jun;12(2):151-6.
AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene. [Abstract/Link to Full Text]

Ji SJ, Liu F, Li EQ, Zhu YX
Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells.
Cell Res. 2002 Jun;12(2):143-50.
The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells. [Abstract/Link to Full Text]

Recent Articles in Cellular & Molecular Biology Letters

Abstracts of the 4th International Conference of Inhibitors of Protein Kinases and the Workshop on Modelling of Specific Molecular Recognition Processes, June 25-29, 2005, Warsaw, Poland.
Cell Mol Biol Lett. 2005;10 Suppl 29-149. [Abstract/Link to Full Text]

Abstracts of W. Mejbaum-Katzenellenbogen's Molecular Biology Seminars, 11. Amphiphiles and their Aggregates in Basic and Applied Science, May 15-19, 2005, Wroclaw/Kliczow, Poland.
Cell Mol Biol Lett. 2005;10 Suppl10-112. [Abstract/Link to Full Text]

Mozafari MR
Commentary: amphiphiles and their aggregates in basic and applied science. A post-conference thought on nomenclature.
Cell Mol Biol Lett. 2005;10(4):733-4. [Abstract/Link to Full Text]

Malakhova L, Bezlepkin VG, Antipova V, Ushakova T, Fomenko L, Sirota N, Gaziev AI
The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice.
Cell Mol Biol Lett. 2005;10(4):721-32.
Changes in the number of mitochondrial DNA (mtDNA) copies in the brain and spleen tissues of gamma-irradiated (3 Gy) mice were studied by comparative analysis of the long-extension PCR products of mtDNA (15.9 kb) and a fragment of the cluster nuclear beta-globin gene (8.7 kb) amplified simultaneously in one and the same test-tube within total DNA. The analysis showed that, compared to the nuclear beta-globin gene, an increase in mtDNA copy number (polyploidization) took place in the brain and spleen cells of mice exposed to gamma-radiation. This data led to the suggestion that the major mechanism for maintenance of the mitochondrial genome, which is constantly damaged by endogenous ROS and easily affected by ionizing radiation or other exogenous factors, is the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates because the repair systems in the mitochondria function at a low level of efficiency. [Abstract/Link to Full Text]

Mozafari MR
Liposomes: an overview of manufacturing techniques.
Cell Mol Biol Lett. 2005;10(4):711-9.
During the last few decades liposomes have attracted great interest as ideal models for biological membranes as well as efficient carriers for drugs, diagnostics, vaccines, nutrients and other bioactive agents. The extensive and ever increasing literature covering the field of liposomology written by researchers with diverse backgrounds is an indication of increasing interest in this field. Many techniques and methodologies have evolved for the manufacture of liposomes, on small and large scales, since their introduction to the scientific community around 40 years ago. This article intends to provide an overview of the advantages and disadvantages of liposome preparation methods in general with particular emphasis on the heating method, developed in our laboratory, as a model technique for fast production of the lipid vesicles. [Abstract/Link to Full Text]

Tagashira N, Plader W, Filipecki M, Yin Z, Wi?niewska A, Gaj P, Szwacka M, Fiehn O, Hoshi Y, Kondo K, Malepszy S
The metabolic profiles of transgenic cucumber lines vary with different chromosomal locations of the transgene.
Cell Mol Biol Lett. 2005;10(4):697-710.
The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper. [Abstract/Link to Full Text]

Jonusiene V, Sasnauskiene S, Juodka B
The cloning and characterization of Tetrahymena pyriformis translation elongation factor 1b alpha and gamma subunits.
Cell Mol Biol Lett. 2005;10(4):689-96.
The multi-subunit eukaryotic translation elongation factor 1 (eEF1) consists of two functionally distinct parts: G-protein eEF1A and guanine nucleotide exchange factor eEF1B. Here, we report on the cloning of cDNAs of both the alpha and gamma subunits of the eEF1B from the ciliated protozoan Tetrahymena pyriformis. The open reading frame of the eEF1Bgamma cDNA encodes a 399-amino acid long polypeptide with a calculated molecular mass of 45.2 kDa. The eEF1Balpha cDNA contains an open reading frame encoding a polypeptide of 228 amino acids. The calculated molecular mass of this protein is 25.2 kDa. The overall deduced amino acid sequences of eEF1Balpha and eEF1Bgamma show a considerable homology with the families of alpha and gamma proteins from other eukaryotic organisms. We demonstrated that eEF1Bgamma is an RNA-binding protein which is able to bind to different RNAs. [Abstract/Link to Full Text]

Cembrzy?ska-Nowak M, Liebhart J, Bie?kowska-Haba M, Liebhart E, Kulczak A, Siemieniec I, Dobek R, Dor A, Barg W, Panaszek B
The overproduction of nitric oxide associated with neutrophilic predominance is relevant to airway mycotic infections in asthmatics undergoing prolonged glucocorticoid treatment.
Cell Mol Biol Lett. 2005;10(4):677-87.
The complex relationship between the local inflammatory response and the spread of airway mycosis during prolonged glucocorticoid therapy in bronchial asthma patients remains unclear. We assessed the ability of airway leukocytes to produce nitric oxide (NO) in relation to differential inflammatory cell counts, levels of asthma severity, and coexisting airway mycotic infections. The study was carried out on leukocytes from the induced sputa (IS) of 14 patients with asthma complicated by mycotic airway infections undergoing prolonged glucocorticoid therapy (group FcA). Three groups of subjects without airway fungal infections were also studied: 18 glucocorticoid-treated asthmatics (group cA), 11 steroid-free asthmatics (group A), and 13 healthy control subjects (group H). In group FcA, both the level of spontaneous production of NO and the percentages of neutrophils in the IS were significantly higher than in all the remaining groups. Additionally, a significant positive correlation was noticed between the NO levels and both the percentages of neutrophils in the IS and the symptom intensity scores. The results suggest a possible predominant role of neutrophils in the overproduction of NO related to asthma severity and coexisting fungal infections in glucocorticoid-treated patients. [Abstract/Link to Full Text]

Haider S, Shameem S, Ahmed SP, Perveen T, Haleem DJ
Repeated administration of lead decreases brain 5-HT metabolism and produces memory deficits in rats.
Cell Mol Biol Lett. 2005;10(4):669-76.
Long-term exposure to low levels of lead (Pb2+) has been shown to produce learning and memory deficits in rodents and humans. These deficits are thought to be associated with altered brain monoamine neurotransmission. Increased brain 5-HT (5-hydroxytryptamine; serotonin) activity is thought to be a prerequisite for maintaining control over the cognitive information process, and is said to have a role in learning and memory. This study was designed to investigate the effects of Pb2+ administration on brain 5-HT metabolism and memory function in rats. Rats were injected daily for three weeks with Pb2+-acetate at a dose of 100 mg/kg body weight. The assessment of memory was done using the Radial arm maze (RAM) and Passive avoidance tests. The results showed spatial working memory (SWM) deficits as well as decreased brain 5-HT metabolism. Increased serotonin activity is considered to be an indication of improved cognitive performance. The results are discussed in the context of lead-induced decreases in 5-HT metabolism playing a role in the impairment of memory. [Abstract/Link to Full Text]

Jiang Q, Yan YH, Hu GK, Zhang YZ
Molecular cloning and characterization of a peroxiredoxin from Phanerochaete chrysosporium.
Cell Mol Biol Lett. 2005;10(4):659-68.
Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Here, we report on the cloning and functional characterization of a cDNA designated PcPrx-1, encoding peroxiredoxin from the white-rot fungus Phanerochaete chrysosporium. The full-length PcPrx-1 cDNA (932 bp) contains an open reading frame of 200 amino acid residues with a molecular mass of 22.1 kDa. The deduced primary structure of PcPrx-1 polypeptide shows a high level of sequence identity to other recently identified 2-cys peroxiredoxins. The recombinant PcPrx-1 protein was expressed as a histidine fusion protein in Escherichia coli and purified with a Ni2+-column. The purified protein was shown to have a protective effect against plasmid DNA cleavage by reactive oxygen species. The PcPrx-1 protein displays the ability to remove H2O2 in a ferrithiocyanate system. The results of this study suggest that PcPrx-1 may play a protective role against oxidative stress in P. chrysosporium. [Abstract/Link to Full Text]

Courdavault V, Burlat V, St-Pierre B, Gantet P, Giglioli-Guivarc'h N
Isolation of a cDNA encoding the alpha-subunit of CAAX-prenyltransferases from Catharanthus roseus and the expression of the active recombinant protein farnesyltransferase.
Cell Mol Biol Lett. 2005;10(4):649-57.
Crfta/ggt_Ia (AF525030), a cDNA encoding the ?-subunit of the two types of CaaX-prenyltransferase (CaaX-PTase), i.e. protein farnesyltransferase (PFT) and type I protein geranylgeranyltransferase, was cloned from Catharanthus roseus via a PCR strategy. Crfta/ggt_Ia is 1381-bp long and bears a 999-bp open reading frame encoding a protein of 332 residues (FTA) that shares 66% identity with its Lycopersicon esculentum orthologue. Southern blot analysis revealed that FTA is encoded by a single gene copy per haploid genome. Co-expression of Crfta/ggt_Ia and Crftb encoding the beta-subunit of PFT yielded purified active recombinant PFT. This enzyme is able to prenylate proteins from C. roseus, and could be used as a potent tool for prenylated protein identification. [Abstract/Link to Full Text]

Turek-Plewa J, Jagodzi?ski PP
The role of mammalian DNA methyltransferases in the regulation of gene expression.
Cell Mol Biol Lett. 2005;10(4):631-47.
The term epigenetic modification denotes reversible traits of gene expression that do not include alterations to the DNA sequence. These epigenetic alterations are responsible for chromatin structure stability, genome integrity, modulation of tissue-specific gene expression, embryonic development, genomic imprinting and X-chromosome inactivation in females. Epigenetic changes include reversible DNA methylation and histone acetylation or methylation. The modification of mammalian genomic DNA includes the methylation at the 5-position of the cytosine (C) residue within cytosine-guanine dinucleotides (CpG), resulting in the formation of 5-methylcytosine (m5C). Regulatory DNA sequences in vertebrates often have little or no methylation. The methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs), which play a special role in the initiation of chromatin remodeling and gene expression regulation. The mammalian DNMTs are DNMT1, DNMT3A and DNMT3B, which together with accessory proteins, like DNMT3L, are responsible for methylation pattern acquisition during gametogenesis, embryogenesis and somatic tissue development. Reversible epigenetic alterations lead to selective utilization of genome information through the activation or inactivation of transcription of functional genes during gametogenesis, embryogenesis and cell differentiation. Recently, several disparate isoforms of DNMT1 were identified in human somatic and female and male germ cells. Recent advances in the investigation of DNMT function in epigenetic DNA changes have formed the basis of the understanding of various disorder etiopathogeneses, and as a result, have facilitated and enabled new therapies with respect to these diseases. [Abstract/Link to Full Text]

Rog T, Vattulainen I, Karttunen M
Modeling glycolipids: take one.
Cell Mol Biol Lett. 2005;10(4):625-30.
Molecular dynamics simulations of glycolipid bilayers consisting of 1,2-di-O-palmitoyl-3-O-beta-D-glucosyl-sn-glycerol were performed using five different force field parameterizations. Comparing the results with experimental data revealed that only the all-atom model correctly reproduces both the phase behavior and the surface area per lipid. Only one of the united atom models studied reproduces the correct phase behavior. [Abstract/Link to Full Text]

Maczy?ski M, Zimecki M, Drozd-Szczygie? E, Ryng S
The synthesis, physicochemical properties and immunological activity of 5-amino-3-methylisoxazolo[5,4-d]4-pyrimidinone derivatives.
Cell Mol Biol Lett. 2005;10(4):613-23.
A series of 5-amino-3-methylisoxazole[5,4-d]4-pyrimidinone derivatives were obtained by reacting substituted 5-amino-3-methylisoxazol-4-carboxylic acid hydrazide with ethyl ortho-formate. The compounds were tested using the models of in vivo cellular and humoral immune response in mice and pokeweed mitogen-induced (PWM-induced) polyclonal antibody production in a culture of human peripheral blood mononuclear cells (PBMC). The compounds exhibited differential inhibitory activities in the described models, depending on the character and location of the substituted groups. We suggest that the compounds affect the early stages of the immune response. [Abstract/Link to Full Text]

Robertsson J, Petzold K, Löfvenberg L, Backman L
Folding of spectrin's SH3 domain in the presence of spectrin repeats.
Cell Mol Biol Lett. 2005;10(4):595-612.
The multifunctional protein spectrin contains several different structural motifs, such as spectrin repeats and a SH3 domain. Both triple-helix spectrin repeats and the SH3 domain are believed to form independent structural entities. In alpha-spectrins the SH3 domain is localized to repeat 9, where it is positioned between helix B and helix C in the repeat unit. The presence of SH3 in repeat 9 decreases the thermal stability considerably of this repeat unit while another insert in helix C does not seem to affect the stability. Addition of one or two adjacent repeat units increases the thermal stability from ca 25 degrees C to 41 and 48 degrees C, respectively. Despite the differences in thermal stability, the folding properties of peptides comprising the SH3 domain only or together with one or more repeats are more or less the same. [Abstract/Link to Full Text]

Towpik J
Regulation of mitochondrial translation in yeast.
Cell Mol Biol Lett. 2005;10(4):571-94.
This review provides an overview of the current state of knowledge regarding the control of very unusual mechanism of mitochondrial gene expression and the structure of mitochondrial ribosomes, with emphasis on the potential of the yeast Saccharomyces cerevisiae as a model organism. [Abstract/Link to Full Text]

Stimson LM, Vattulainen I, Róg T, Karttunen M
Exploring the effect of xenon on biomembranes.
Cell Mol Biol Lett. 2005;10(4):563-9.
We report the initial findings of 100 ns molecular dynamics simulations of the role of cellular membranes in general anaesthesia. The effect of xenon on hydrated dipalmitoylphosphatidylcholine bilayers is described. The xenon atoms were found to prefer the interfacial and central regions of the bilayer. The presence of xenon was observed to lead to a small increase in the surface area, membrane thickness, and order of the acyl chains. [Abstract/Link to Full Text]

Manju V, Balasubramaniyan V, Nalini N
Rat colonic lipid peroxidation and antioxidant status: the effects of dietary luteolin on 1,2-dimethylhydrazine challenge.
Cell Mol Biol Lett. 2005;10(3):535-51.
Colon cancer is the third most common cancer and second leading cause of cancer-related death in the United States. A number of recent articles demonstrate the importance of natural products as cancer chemopreventive agents. In this study, we evaluated the chemopreventive efficacy of luteolin, a flavonoid, on tissue lipid peroxidation and antioxidant status, which are used as biomarkers in DMH-induced experimental colon carcinogenesis. Rats were given a weekly subcutaneous injection of DMH at a dose of 20 mg/kg body weight for 15 weeks. Luteolin (0.2 mg/kg body weight/everyday p.o.) was given to the DMH-treated rats at the initiation and post-initiation stages of carcinogenesis. The animals were killed after 30 weeks. After a total experimental period of 32 weeks (including 2 weeks of acclimatization), tumor incidence was 100% in DMH-treated rats. In those DMH-treated rats that had received luteolin during the initiation or post-initiation stages of colon carcinogenesis, the incidence of cancer and the colon tumor size was significantly reduced as compared to that for DMH-treated rats not receiving luteolin. In the presence of DMH, relative to the results for the control rats, there were decreased levels of lipid peroxidation, as denoted by thiobarbituric acid reactive substances (TBARS), conjugated dienes and lipid hydroperoxides, decreased activities of the enzymic antioxidants superoxide dismutase (SOD) and catalase (CAT), and elevated levels of glutathione and the glutathione-dependent enzymes reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR), and of the non-enzymic antioxidants vitamin C and vitamin E. Our study shows that intragastric administration of luteolin inhibits colon carcinogenesis, not only by modulating lipid peroxidation and antioxidant status, but also by preventing DMH-induced histopathological changes. Our results thus indicate that luteolin could act as a potent chemopreventive agent for colon carcinogenesis. [Abstract/Link to Full Text]

Zimmermann P, Zentgraf U
The correlation between oxidative stress and leaf senescence during plant development.
Cell Mol Biol Lett. 2005;10(3):515-34.
In plants, besides being the final step leading to the death of the whole organism, senescence has a developmental function involving the coordinated degradation of macromolecules and the mobilization of nutrients out of senescing tissues into developing parts of the plant. Free radicals are thought to play an essential role in senescence, especially those derived from oxygen. Since these molecules are extremely toxic, the levels of the different reactive oxygen species have to be tightly regulated. However, at low concentrations, hydrogen peroxide may also serve as a signalling molecule. Therefore, a coordinated regulation of the free radical scavenging system, which comprises enzymatic components such as catalase, superoxide dismutase and ascorbate peroxidase, and non-enzymatic molecules such as ascorbate and glutathione is essential. The increased radical levels displayed during senescence are not only caused by the elevated production of radicals but also by a loss in antioxidant capacity. [Abstract/Link to Full Text]

Sándor Z, Varga A, Horváth P, Nagy B, Szolcsányi J
Construction of a stable cell line uniformly expressing the rat TRPV1 receptor.
Cell Mol Biol Lett. 2005;10(3):499-514.
We constructed and analyzed a new cell line called HT5-1, which stably expresses an enhanced green fluorescent protein-tagged version of the rat vanilloid receptor 1 (VR1/TRPV1). The fluorescent receptor allowed easy measurement of receptor expression and expression level-based purification of cells via fluorescence-activated cell sorting. The HT5-1 cell line was compared to cells transiently transfected with the fluorescent receptor, to cells expressing the native rat vanilloid receptor, and to isolated capsaicin-sensitive rat trigeminal sensory neurons. Fura-2 microfluorimetry measurements of the calcium influx upon capsaicin induction showed that, by contrast to transiently transfected cells, HT5-1 cells respond uniformly to the stimulation, due to the similar level of receptor expression in individual cells. HT5-1 cells showed similar behaviour to isolated trigeminal root ganglion neurons, including marked tachyphylaxis upon repeated capsaicin induction, and a lack of calcium ion release from intracellular storage sites. [Abstract/Link to Full Text]

Janus A, Robak T, Smolewski P
The mammalian target of the rapamycin (mTOR) kinase pathway: its role in tumourigenesis and targeted antitumour therapy.
Cell Mol Biol Lett. 2005;10(3):479-98.
The mammalian target of rapamycin (mTOR) is a kinase responsible for mitogen-induced cell proliferation/survival signaling. Its activation in response to mitogens leads to a cell-cycle progression from G1 to S phase. mTOR controls the activation of ribosomal protein translation and the initiation of cap-dependent translation. A role of mTOR signaling pathway dysregulation in tumourigenesis is postulated. mTOR and pathways upstream of this kinase were found to be frequently upregulated in neoplastic diseases. Therefore, it is also an attractive target for antitumour therapy. Several mTOR inhibitors were developed, including rapamycin and its analogues: CCI-779, RAD001 and AP23573. After promising phase I studies, their potential clinical significance is currently under evaluation in several phase II-III trials on patients with solid tumours and some hematological malignancies. [Abstract/Link to Full Text]

Krokosz A, Szweda-Lewandowska Z
Changes in the activity of acetylcholinesterase and Na,K-ATPase in human erythrocytes irradiated with X-rays.
Cell Mol Biol Lett. 2005;10(3):471-8.
The response of human erythrocytes to X-rays in the dose range from 40 Gy to 600 Gy was determined on the basis of changes in the activities of AChE and ATPase. The Na,K-ATPase activity increased above the control value at doses below 200 Gy, while at the doses higher than 200 Gy, it decreased, reaching 96% of the control value at a dose of 600 Gy. In the range of doses up to 200 Gy, the AChE activity, expressed as Vmax, did not change. At higher doses, it fell drastically, reaching 33% of the control value at a dose of 600 Gy. Simultaneously, the enzyme substrate affinity decreased at 200 Gy, and then started to increase at lower values of Vmax. The obtained results suggest that under appropriate conditions, low doses of radiation may have the opposite effects to high doses. [Abstract/Link to Full Text]

Konopka K, Fallah B, Monzon-Duller J, Overlid N, Düzgünes N
Serum-resistant gene transfer to oral cancer cells by Metafectene and GeneJammer: application to HSV-tk/ganciclovir-mediated cytotoxicity.
Cell Mol Biol Lett. 2005;10(3):455-70.
Cationic lipids and polyamines have been used as non-viral gene transfer reagents, both in vitro and in vivo. One of the limitations to their use in vivo is the inhibition of gene delivery by serum. We showed previously that, in the absence of serum, relatively high cytotoxicity in oral cancer cell lines could be achieved via transfection of the Herpes Simplex Virus thymidine kinase (HSV-tk) gene followed by treatment with ganciclovir (GCV), despite the low efficiency of transfection (Konopka et al., Gene Ther. Mol. Biol. 8 (2004) 307-318). In this study we evaluated the effect of high concentrations (20-60%) of fetal bovine serum (FBS) on the transfection efficiency of two novel reagents, the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in HSC-3 and H357 human oral squamous cell carcinoma (OSCC) cells. We also examined whether the HSV-tk gene delivered in the presence of FBS (up to 60%, could induce cell death following treatment with GCV. Transfection was optimized using a luciferase-expressing plasmid. Both Metafectene- and GeneJammer-mediated luciferase gene expression in HSC-3 cells was reduced by 40-50% when transfection was performed in the presence of 20-60% FBS. The delivery of the HSV-tk gene by Metafectene in the absence and the presence of 60% FBS, followed by GCV treatment for 9 days, resulted in 95% and 70% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 90% and 40% cytotoxicity, respectively, after 9 days. In contrast, very low transfection activity and a much higher inhibitory effect of serum were observed in H357 cells. Nevertheless, about 35% GCV-mediated cytotoxicity was observed with H357 cells at both 0% and 60% FBS, using GeneJamer. Thus, Metafectene and GeneJammer can be used in the delivery of genes in biological milieu and in the gene therapy of OSCC in animal models. [Abstract/Link to Full Text]

Wesierska-Gadek J, Schmid G
The subcellular distribution of the p53 tumour suppressor, and organismal ageing.
Cell Mol Biol Lett. 2005;10(3):439-53.
The p53 protein, the product of a tumour suppressor gene, is a key regulator of cell growth, differentiation and apoptosis. It is able to induce a transient cell cycle arrest and terminal senescence. Most of its functions are exerted by the transcriptional activation of genes involved in cell cycle control, DNA repair and apoptosis. The activation of p53 is primarily mediated by post-translational modifications that affect its conformation and capacity to bind to several proteins, resulting in its stabilization and enhanced DNA-binding potential. Another way to regulate the biological function of p53 involves changes in its intracellular distribution. This paper presents an overview of the role of p53 in cellular senescence and the regulation of p53 activity by its intracellular distribution. [Abstract/Link to Full Text]

Song BK, Nadarajah K, Romanov MN, Ratnam W
Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.
Cell Mol Biol Lett. 2005;10(3):425-37.
The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing. [Abstract/Link to Full Text]

Chetty CS, Vemuri MC, Campbell K, Suresh C
Lead-induced cell death of human neuroblastoma cells involves GSH deprivation.
Cell Mol Biol Lett. 2005;10(3):413-23.
Lead (Pb2+) is a toxic heavy metal that has adverse effects on the health of humans and other animals. The developing central nervous system is especially sensitive and vulnerable to Pb2+ toxicity. In this study, the effects of low levels of Pb2+ exposure on human SH-SY5Y neuroblastoma cell cultures were assessed. The cells were exposed to Pb2+ (0.01 microM-10 microM) for 48 hrs, and the level of cell proliferation was determined. Pb2+ significantly inhibited the proliferation of neuroblastoma cells in a concentration-dependent manner. A 50% inhibition (IC50) in cellular proliferation was observed with 5 microM Pb2+. A significant decrease in the levels of glutathione (GSH), a critical intracellular antioxidant, was observed at all the lead concentrations. There was also a multifold increase in the activity of caspase-3, a key executioner of apoptosis, and in the levels of prostaglandin E2 (PGE2). Our results suggest that the neurotoxic effects of Pb may be mediated by apoptosis and PGE2 release, which could be potentially detrimental to neuronal survival. [Abstract/Link to Full Text]

Ming F, Mi GH, Lu Q, Yin S, Zhang SS, Guo B, Shen DL
Cloning and characterization of cDNA for the Oryza sativa phosphate transporter.
Cell Mol Biol Lett. 2005;10(3):401-11.
A putative high-affinity phosphate (Pi) transporter gene in rice (Oryza sativa), OsLPT1, was isolated by RT-PCR from the leaves of the plants. The 1635-bp nucleotide sequence of OsLPT1 spans an open reading frame encoding a polypeptide of 535 amino acids with sequence similarity to phosphate transporters from other plant species. Southern blot analysis showed that the OsLPT1 gene might be present in three transcripts in the rice genome. RT-PCR analysis demonstrated the expression of OsLPT1 in both leaves and roots. The expression of OsLPT1 in the roots was enhanced by Pi deprivation. In situ hybridization revealed OsLPT1 expression in mesophyll cells, xylem parenchyma and phloem cells in the leaves, and in the epidermis, exodermis, and in the vasculature surrounding metaxylem vessels in the roots. The data suggests that the OsLPT1 protein may be involved in enhancing phosphate uptake under conditions of Pi starvation, and in the translocation of Pi among cells in shoots to increase the efficiency of internal Pi use. [Abstract/Link to Full Text]

Modzelewska B, Kostrzewska A
The influence of methylene blue on the spontaneous contractity of the non-pregnant human myometrium and on the myometrial response to DEA/NO.
Cell Mol Biol Lett. 2005;10(3):389-400.
Nitric oxide (NO) is a potent inhibitor of spontaneous contractions of the human non-pregnant myometrium; however, the precise mechanism by which NO causes the myometrial smooth muscles to relax remains unclear. The aim of this study was to determine the influence of methylene blue (MB) on myometrial contractions and the response of the myometrium to DEA/NO in vitro. Concentration-response curves to DEA/NO were constructed in the absence and presence of MB (5x10(-6), 10(-4) and 10(-2) mol/l) and 5x10(-3) mol/l cystamine. Cystamine did not counteract the DEA/NO-induced relaxation of the myometrial strips. MB itself, excluding the lowest concentration, caused noticeable changes in spontaneous activity. The changes involved a concentration-dependent increase in the frequency of contractions, and a decrease in their amplitude. In conclusion, our results confirm that NO relaxes the human myometrium via a cGMP-independent mechanism. The results obtained in the presence of MB may be misleading because of its complex influence on myometrial contractile activity. [Abstract/Link to Full Text]

Abe F, Horikoshi K
Enhanced production of isoamyl alcohol and isoamyl acetate by ubiquitination-deficient Saccharomyces cerevisiae mutants.
Cell Mol Biol Lett. 2005;10(3):383-8.
Aromatic compounds are an important element in the flavor of yeast-fermented alcohol. We isolated mutants of Saccharomyces cerevisiae capable of growth at high levels of hydrostatic pressure. Among them, the HPG1 mutants, with a defect in their Rsp5 ubiquitin ligase, were found to produce high amounts of aromatics due to enhanced leucine uptake, with isoamyl alcohol production 2- to 3-fold and isoamyl acetate production 4- to 8-fold that of the wild-type strain. The result suggests that the HPG1/RSP5 mutant alleles could be new resources for producing these flavoring compounds for yeast-fermented alcoholic beverages. [Abstract/Link to Full Text]

Kuliszkiewicz-Janus M, Tuz MA, Baczy?ski S, Prajs I, Ja?wiec B
31P MRS analysis of the phospholipid composition of normal human peripheral blood mononuclear cells (PBMC).
Cell Mol Biol Lett. 2005;10(3):373-82.
The aim of this investigation was to characterize the phospholipid composition of normal human blood mononuclear cells using 31P NMR spectroscopy. Mononuclear cells of peripheral blood were obtained from 10 volunteers. Phospholipid extracts were prepared from 60x10(6) cells according to modified Folch's method. An AMX 300 Bruker spectrometer 7.05 T was used. The 31P spectrum of phospholipid extracts from normal human PBMC consisted of 9 peaks, with one each for phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL), and another one due to the external reference substance, methylenediphosphonic acid (MDPA). The concentrations of these phospholipids (PL), based on the integral intensities, were as follows: 0.398 +/- 0.078 mmole/l for PC; 0.033 +/- 0.019 mmole/l for CPLAS; 0.155 +/- 0.043 mmole/l for SM; 0.266 +/- 0.104 mmole/l for PI+PE; 0.101 +/- 0.040 mmole/l for PS, and 0.026 +/- 0.033 mmole/l for CL. The results of this study confirmed that 31P MRS is a convenient tool for measuring the phospholipid concentrations of biological samples. [Abstract/Link to Full Text]

Recent Articles in BMC Cell Biology

Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Yamamoto M, Sugimoto T
Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.
BMC Cell Biol. 2007 Nov 29;8(1):51.
ABSTRACT: BACKGROUND: Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results: Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01--0.5mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01-1.0 mg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusions: Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. [Abstract/Link to Full Text]

Cho GW, Shin SM, Kim HK, Ha SA, Kim S, Yoon JH, Hur SY, Kim TE, Kim JW
HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis.
BMC Cell Biol. 2007 Nov 28;8(1):50.
ABSTRACT: BACKGROUND: The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. RESULTS: To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. CONCLUSIONS: Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation. [Abstract/Link to Full Text]

Aijaz S, Sanchez-Heras E, Balda MS, Matter K
Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2.
BMC Cell Biol. 2007 Nov 20;8(1):49.
ABSTRACT: BACKGROUND: Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. RESULTS: We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. CONCLUSIONS: Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation. [Abstract/Link to Full Text]

Annesley SJ, Bandala-Sanchez E, Ahmed AU, Fisher PR
Filamin repeat segments required for photosensory signalling in Dictyostelium discoideum.
BMC Cell Biol. 2007 Nov 12;8(1):48.
ABSTRACT: BACKGROUND: Filamin is an actin binding protein which is ubiquitous in eukaryotes and its basic structure is well conserved - an N-terminal actin binding domain followed by a series of repeated segments which vary in number in different organisms. D. discoideum is a well established model organism for the study of signalling pathways and the actin cytoskeleton and as such makes an excellent organism in which to study filamin. Ddfilamin plays a putative role as a scaffolding protein in a photosensory signalling pathway and this role is thought to be mediated by the unusual repeat segments in the rod domain. RESULTS: To study the role of filamin in phototaxis, a filamin null mutant, HG1264, was transformed with constructs each of which expressed wild type filamin or a mutant filamin with a deletion of one of the repeat segments. Transformants expressing the full length filamin to wild type levels completely rescued the phototaxis defect in HG1264, however if filamin was expressed at lower than wild type levels the phototaxis defect was not restored. The transformants lacking any one of the repeat segments 2-6 retained defective phototaxis and thermotaxis phenotypes, whereas transformants expressing filaminDelta1 exhibited a range of partial complementation of the phototaxis phenotype which was related to expression levels. Immunofluorescence microscopy showed that filamin lacking any of the repeat segments still localised to the same actin rich areas as wild type filamin. Ddfilamin interacts with RasD and IP experiments demonstrated that this interaction did not rely upon any single repeat segment or the actin binding domain. CONCLUSIONS: This paper demonstrates that wild type levels of filamin expression are essential for the formation of functional photosensory signalling complexes and that each of the repeat segments 2-6 are essential for filamins role in phototaxis. By contrast, repeat segment 1 is not essential provided the mutated filamin lacking repeat segment 1 is expressed at a high enough level. The defects in photo/thermosensory signal transduction caused by the absence of the repeats are due neither to mislocalisation of filamin nor to the loss of RasD recruitment to the previously described photosensory signalling complex. [Abstract/Link to Full Text]

Hattier T, Andrulis E, Tartakoff AM
Immobility, inheritance and plasticity of shape of the yeast nucleus.
BMC Cell Biol. 2007 Nov 9;8(1):47.
ABSTRACT: BACKGROUND: Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR. RESULTS: Upon increased Esc1p expression, cell division continues and dramatic elaborations of the nuclear envelope extend into the cytoplasm. These "escapades" include nuclear pores and associate with the nucleolus, but exclude chromatin. Escapades are not inherited by daughter nuclei. This exclusion reflects their relative immobility, which we document in studies of prezygotes. Moreover, excess Esc1p affects the levels of multiple transcripts, not all of which originate at telomere-proximal loci. Unlike Esc1p and the colocalizing protein, Mlp1p, overexpression of selected proteins of the inner nuclear membrane is toxic. CONCLUSIONS: Esc1p is the first non-membrane protein of the nuclear periphery which - like proteins of the nuclear lamina of higher eukaryotes - can modify the shape of the yeast nucleus. The elaborations of the nuclear envelope ("escapades") which appear upon induction of excess Esc1p are not inherited during mitotic growth. The lack of inheritance of such components could help sustain cell growth when parental nuclei have acquired potentially deleterious characteristics. [Abstract/Link to Full Text]

Thomas SG, Calaminus SD, Auger JM, Watson SP, Machesky LM
Studies on the actin-binding protein HS1 in platelets.
BMC Cell Biol. 2007 Nov 9;8(1):46.
ABSTRACT: BACKGROUND: The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice. RESULTS: The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay. CONCLUSIONS: This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin. [Abstract/Link to Full Text]

Schutze N, Schenk R, Fiedler J, Mattes T, Jakob F, Brenner RE
CYR61/CCN1 and WISP3/CCN6 are chemoattractive ligands for human multipotent mesenchymal stroma cells.
BMC Cell Biol. 2007 Oct 31;8(1):45.
ABSTRACT: BACKGROUND: CCN proteins are known to be involved in development, homeostasis and repair of mesenchymal tissues. Since these processes implicate recruitment of cells with the potential to be committed to various phenotypes, we studied the effect of CYR61/CCN1 and WISP3/CCN6 on migration of human bone marrow derived mesenchymal stroma cells (MSCs) in comparison to those of in vitro osteogenic differentiated MSCs using a modified Boyden chamber assay. RESULTS: CYR61 and WISP3 were purified as fusion proteins with a C-terminal Fc-tag from baculovirus infected SF21 cells using protein G sepharose columns. CYR61 and WISP3 stimulated cell migration of undifferentiated MSCs in a dose-dependent manner. CYR61 and WISP3 had similar effects on committed osteogenic precursor cells. Checkerboard analysis revealed that CYR61 and WISP3 stimulated true directed cell migration (chemotaxis) of MSCs and committed osteogenic precursors. In MSCs the chemotactic activity of WISP3 but not CYR61 was mediated through integrin alphavbeta5. CONCLUSIONS: Our results indicate that CYR61 and WISP3 can function as soluble ligands transmitting chemotactic signals to human MSCs but differ in the involvement of integrin alphavbeta5. This may be relevant for their possible role in connective tissue repair. [Abstract/Link to Full Text]

Blackwell E, Kim HJ, Stone DE
The pheromone-induced nuclear accumulation of the Fus3 MAPK in yeast depends on its phosphorylation state and on Dig1 and Dig2.
BMC Cell Biol. 2007 Oct 26;8(1):44.
ABSTRACT: BACKGROUND: Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers. RESULTS: Dig1, Dig2, and Ste12 are nuclear proteins that interact with Fus3. We found that the pheromone-induced nuclear accumulation of a Fus3 GFP reporter is reduced in cells lacking Dig1 or Dig2, whereas Fus3T180A Y182A GFP localization was unaffected by the absence of these proteins. This suggests that Dig1 and Dig2 contribute to the retention of phosphorylated Fus3 in the nucleus. Moreover, overexpression of Ste12 caused the hyper accumulation of Fus3 GFP (but not Fus3T180A Y182A GFP) in the nuclei of pheromone-treated cells, suggesting that Ste12 also plays a role in the nuclear retention of phosphorylated Fus3, either by directly interacting with it or by transcribing genes whose protein products are Fus3 tethers. We have previously reported that overexpression of the Msg5 phosphatase inhibits the nuclear localization of Fus3. Here we show that this effect depends on the phosphatase activity of Msg5, and provide evidence that both nuclear and cytoplasmic Msg5 can affect the localization of Fus3. CONCLUSIONS: Our data are consistent with a model in which the pheromone-induced phosphorylation of Fus3 increases its affinity for nuclear tethers, which contributes to its nuclear accumulation and is antagonized by Msg5. [Abstract/Link to Full Text]

Bhadriraju K, Elliott JT, Nguyen M, Plant AL
Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy.
BMC Cell Biol. 2007 Oct 17;8(1):43.
ABSTRACT: BACKGROUND: In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells. RESULTS: Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a reproducible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC50 for myosin light chain phosphorylation using Y27632 was determined to be 2.1+/-0.6 micromolar. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods. CONCLUSIONS: Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development. [Abstract/Link to Full Text]

Easwaran HP, Leonhardt H, Cardoso MC
Distribution of DNA replication proteins in Drosophila cells.
BMC Cell Biol. 2007;842.
BACKGROUND: DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. RESULTS: Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. CONCLUSION: We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very transient interactions at the RF. [Abstract/Link to Full Text]

Zilberberg L, ten Dijke P, Sakai LY, Rifkin DB
A rapid and sensitive bioassay to measure bone morphogenetic protein activity.
BMC Cell Biol. 2007;841.
BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and were originally identified as proteins that induce ectopic bone formation. BMPs were shown subsequently to be involved in several biological processes during development and in adult tissues through the regulation of the growth, differentiation and apoptosis of various cell types. An alkaline phosphatase (ALP)-based assay is the most widely used assay to evaluate BMP activity. However, the ALP assay is not rapid and not sensitive enough to measure BMP activity at physiological concentrations. In this paper, we describe a highly sensitive, rapid, and specific cell-based assay for the quantification of BMP activity. RESULTS: Two cells lines, C2C12 and HepG2 were stably transfected with a reporter plasmid consisting of BMP-responsive elements from the Id1 promoter fused to a luciferase reporter gene. Exposure of cells containing this construct to BMPs induces the expression of luciferase, which can be quantified with a luminometer. The bioassay is specific for BMPs and can detect BMP-4 activity at a concentration as low as 3 pM. Related family members, such as TGF-beta1, TGF-beta2 and TGF-beta3, do not induce the reporter gene. CONCLUSION: The assay is rapid (less than 24 hours) and can be used, as described in this paper, to measure BMP activity in complex solutions and in cell culture in a simple and efficient way. [Abstract/Link to Full Text]

Li G, Liu T, Tarokh A, Nie J, Guo L, Mara A, Holley S, Wong ST
3D cell nuclei segmentation based on gradient flow tracking.
BMC Cell Biol. 2007;840.
BACKGROUND: Reliable segmentation of cell nuclei from three dimensional (3D) microscopic images is an important task in many biological studies. We present a novel, fully automated method for the segmentation of cell nuclei from 3D microscopic images. It was designed specifically to segment nuclei in images where the nuclei are closely juxtaposed or touching each other. The segmentation approach has three stages: 1) a gradient diffusion procedure, 2) gradient flow tracking and grouping, and 3) local adaptive thresholding. RESULTS: Both qualitative and quantitative results on synthesized and original 3D images are provided to demonstrate the performance and generality of the proposed method. Both the over-segmentation and under-segmentation percentages of the proposed method are around 5%. The volume overlap, compared to expert manual segmentation, is consistently over 90%. CONCLUSION: The proposed algorithm is able to segment closely juxtaposed or touching cell nuclei obtained from 3D microscopy imaging with reasonable accuracy. [Abstract/Link to Full Text]

Schneider D, Fuhrmann E, Scholz I, Hess WR, Graumann PL
Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes.
BMC Cell Biol. 2007;839.
BACKGROUND: In spite of their abundance and importance, little is known about cyanobacterial cell biology and their cell cycle. During each cell cycle, chromosomes must be separated into future daughter cells, i.e. into both cell halves, which in many bacteria is achieved by an active machinery that operates during DNA replication. Many cyanobacteria contain multiple identical copies of the chromosome, but it is unknown how chromosomes are segregated into future daughter cells, and if an active or passive mechanism is operative. In addition to an outer and an inner cell membrane, cyanobacteria contain internal thylakoid membranes that carry the active photosynthetic machinery. It is unclear whether thylakoid membranes are invaginations of the inner cell membrane, or an independent membrane system. RESULTS: We have used different fluorescent dyes to study the organization of chromosomes and of cell and thylakoid membranes in live cyanobacterial cells. FM1-43 stained the outer and inner cytoplasmic membranes but did not enter the interior of the cell. In contrast, thylakoid membranes in unicellular Synechocystis cells became visible through a membrane-permeable stain only. Furthermore, continuous supply of the fluorescent dye FM1-43 resulted in the formation of one to four intracellular fluorescent structures in Synechocystis cells, within occurred within 30 to 60 minutes, and may represent membrane vesicles. Using fluorescent DNA stains, we found that Synechocystis genomic DNA is compacted in the cell centre that is devoid of thylakoid membranes. Nucleoids segregated very late in the cell cycle, just before complete closing of the division septum. In striking contrast to Bacillus subtilis, which possesses an active chromosome segregation machinery, fluorescence intensity of stained nucleoids differed considerably between the two Synechocystis daughter cells soon after cell division. CONCLUSION: Our experiments strongly support the idea that the cytoplasmic and thylakoid membranes are not directly connected, but separate entities, in unicellular cyanobacteria. Our findings suggest that a transport system may exist between the cytoplasmic membrane and thylakoids, which could mediate the extension of thylakoid membranes and possibly also protein transport from the cytoplasmic membrane to thylakoid membranes. The cell cycle studies in Synechocystis sp. PCC 6803 show that the multiple chromosome copies per cell segregate very late in the cell cycle and in a much less stringent manner than in B. subtilis cells, indicating that chromosomes may become segregated randomly and in a passive fashion, possibly through constriction of the division septum. [Abstract/Link to Full Text]

Iida K, Li Y, McGrath BC, Frank A, Cavener DR
PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice.
BMC Cell Biol. 2007;838.
BACKGROUND: Deficiency of the PERK eIF2 alpha kinase in humans and mice results in postnatal exocrine pancreatic atrophy as well as severe growth and metabolic anomalies in other organs and tissues. To determine if the exocrine pancreatic atrophy is due to a cell-autonomous defect, the Perk gene was specifically ablated in acinar cells of the exocrine pancreas in mice. RESULTS: We show that expression of PERK in the acinar cells is required to maintain their viability but is not required for normal protein synthesis and secretion. Exocrine pancreatic atrophy in PERK-deficient mice was previously attributed to uncontrolled ER-stress followed by apoptotic cell death based on studies in cultured fibroblasts. However, we have found no evidence for perturbations in the endoplasmic reticulum or ER-stress and show that acinar cells succumb to a non-apoptotic form of cell death, oncosis, which is associated with a pronounced inflammatory response and induction of the pancreatitis stress response genes. We also show that mice carrying a knockout mutation of PERK's downstream target, ATF4, exhibit pancreatic deficiency caused by developmental defects and that mice ablated for ATF4's transcriptional target CHOP have a normal exocrine pancreas. CONCLUSION: We conclude that PERK modulates secretory capacity of the exocrine pancreas by regulating cell viability of acinar cells. [Abstract/Link to Full Text]

Li X, Leder P
Identifying genes preferentially expressed in undifferentiated embryonic stem cells.
BMC Cell Biol. 2007;837.
BACKGROUND: The mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood. RESULTS: To address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-J kappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene. CONCLUSION: Taken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells. [Abstract/Link to Full Text]

Rao AR, Cecchi GA, Magnasco M
High performance computing environment for multidimensional image analysis.
BMC Cell Biol. 2007;8 Suppl 1S9.
BACKGROUND: The processing of images acquired through microscopy is a challenging task due to the large size of datasets (several gigabytes) and the fast turnaround time required. If the throughput of the image processing stage is significantly increased, it can have a major impact in microscopy applications. RESULTS: We present a high performance computing (HPC) solution to this problem. This involves decomposing the spatial 3D image into segments that are assigned to unique processors, and matched to the 3D torus architecture of the IBM Blue Gene/L machine. Communication between segments is restricted to the nearest neighbors. When running on a 2 Ghz Intel CPU, the task of 3D median filtering on a typical 256 megabyte dataset takes two and a half hours, whereas by using 1024 nodes of Blue Gene, this task can be performed in 18.8 seconds, a 478x speedup. CONCLUSION: Our parallel solution dramatically improves the performance of image processing, feature extraction and 3D reconstruction tasks. This increased throughput permits biologists to conduct unprecedented large scale experiments with massive datasets. [Abstract/Link to Full Text]

Boucheron LE, Bi Z, Harvey NR, Manjunath B, Rimm DL
Utility of multispectral imaging for nuclear classification of routine clinical histopathology imagery.
BMC Cell Biol. 2007;8 Suppl 1S8.
BACKGROUND: We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420-700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis. RESULTS: For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains. CONCLUSION: Our results indicate that multispectral imagery for routine H&E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery. [Abstract/Link to Full Text]

Peng H, Long F, Zhou J, Leung G, Eisen MB, Myers EW
Automatic image analysis for gene expression patterns of fly embryos.
BMC Cell Biol. 2007;8 Suppl 1S7.
BACKGROUND: Staining the mRNA of a gene via in situ hybridization (ISH) during the development of a D. melanogaster embryo delivers the detailed spatio-temporal pattern of expression of the gene. Many biological problems such as the detection of co-expressed genes, co-regulated genes, and transcription factor binding motifs rely heavily on the analyses of these image patterns. The increasing availability of ISH image data motivates the development of automated computational approaches to the analysis of gene expression patterns. RESULTS: We have developed algorithms and associated software that extracts a feature representation of a gene expression pattern from an ISH image, that clusters genes sharing the same spatio-temporal pattern of expression, that suggests transcription factor binding (TFB) site motifs for genes that appear to be co-regulated (based on the clustering), and that automatically identifies the anatomical regions that express a gene given a training set of annotations. In fact, we developed three different feature representations, based on Gaussian Mixture Models (GMM), Principal Component Analysis (PCA), and wavelet functions, each having different merits with respect to the tasks above. For clustering image patterns, we developed a minimum spanning tree method (MSTCUT), and for proposing TFB sites we used standard motif finders on clustered/co-expressed genes with the added twist of requiring conservation across the genomes of 8 related fly species. Lastly, we trained a suite of binary-classifiers, one for each anatomical annotation term in a controlled vocabulary or ontology that operate on the wavelet feature representation. We report the results of applying these methods to the Berkeley Drosophila Genome Project (BDGP) gene expression database. CONCLUSION: Our automatic image analysis methods recapitulate known co-regulated genes and give correct developmental-stage classifications with 99+% accuracy, despite variations in morphology, orientation, and focal plane suggesting that these techniques form a set of useful tools for the large-scale computational analysis of fly embryonic gene expression patterns. [Abstract/Link to Full Text]

Singh R
Surface similarity-based molecular query-retrieval.
BMC Cell Biol. 2007;8 Suppl 1S6.
BACKGROUND: Discerning the similarity between molecules is a challenging problem in drug discovery as well as in molecular biology. The importance of this problem is due to the fact that the biochemical characteristics of a molecule are closely related to its structure. Therefore molecular similarity is a key notion in investigations targeting exploration of molecular structural space, query-retrieval in molecular databases, and structure-activity modelling. Determining molecular similarity is related to the choice of molecular representation. Currently, representations with high descriptive power and physical relevance like 3D surface-based descriptors are available. Information from such representations is both surface-based and volumetric. However, most techniques for determining molecular similarity tend to focus on idealized 2D graph-based descriptors due to the complexity that accompanies reasoning with more elaborate representations. RESULTS: This paper addresses the problem of determining similarity when molecules are described using complex surface-based representations. It proposes an intrinsic, spherical representation that systematically maps points on a molecular surface to points on a standard coordinate system (a sphere). Molecular surface properties such as shape, field strengths, and effects due to field super-positioning can then be captured as distributions on the surface of the sphere. Surface-based molecular similarity is subsequently determined by computing the similarity of the surface-property distributions using a novel formulation of histogram-intersection. The similarity formulation is not only sensitive to the 3D distribution of the surface properties, but is also highly efficient to compute. CONCLUSION: The proposed method obviates the computationally expensive step of molecular pose-optimisation, can incorporate conformational variations, and facilitates highly efficient determination of similarity by directly comparing molecular surfaces and surface-based properties. Retrieval performance, applications in structure-activity modeling of complex biological properties, and comparisons with existing research and commercial methods demonstrate the validity and effectiveness of the approach. [Abstract/Link to Full Text]

Cecchi GA, Rao AR, Centeno MV, Baliki M, Apkarian AV, Chialvo DR
Identifying directed links in large scale functional networks: application to brain fMRI.
BMC Cell Biol. 2007;8 Suppl 1S5.
BACKGROUND: Biological experiments increasingly yield data representing large ensembles of interacting variables, making the application of advanced analytical tools a forbidding task. We present a method to extract networks of correlated activity, specifically from functional MRI data, such that: (a) network nodes represent voxels, and (b) the network links can be directed or undirected, representing temporal relationships between the nodes. The method provides a snapshot of the ongoing dynamics of the brain without sacrificing resolution, as the analysis is tractable even for very large numbers of voxels. RESULTS: We find that, based on topological properties of the networks, the method provides enough information about the dynamics to discriminate between subtly different brain states. Moreover, the statistical regularities previously reported are qualitatively preserved, i.e. the resulting networks display scale-free and small-world topologies. CONCLUSION: Our method expands previous approaches to render large scale functional networks, and creates the basis for an extensive and -due to the presence of mixtures of directed and undirected links- richer motif analysis of functional relationships. [Abstract/Link to Full Text]

Altinok A, Kiris E, Peck AJ, Feinstein SC, Wilson L, Manjunath BS, Rose K
Model based dynamics analysis in live cell microtubule images.
BMC Cell Biol. 2007;8 Suppl 1S4.
BACKGROUND: The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data. RESULTS: In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior. CONCLUSION: Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior. [Abstract/Link to Full Text]

Long F, Peng H, Sudar D, Leličvre SA, Knowles DW
Phenotype clustering of breast epithelial cells in confocal images based on nuclear protein distribution analysis.
BMC Cell Biol. 2007;8 Suppl 1S3.
BACKGROUND: The distribution of chromatin-associated proteins plays a key role in directing nuclear function. Previously, we developed an image-based method to quantify the nuclear distributions of proteins and showed that these distributions depended on the phenotype of human mammary epithelial cells. Here we describe a method that creates a hierarchical tree of the given cell phenotypes and calculates the statistical significance between them, based on the clustering analysis of nuclear protein distributions. RESULTS: Nuclear distributions of nuclear mitotic apparatus protein were previously obtained for non-neoplastic S1 and malignant T4-2 human mammary epithelial cells cultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 and the number of days in cultured. A probabilistic ensemble approach was used to define a set of consensus clusters from the results of multiple traditional cluster analysis techniques applied to the nuclear distribution data. Cluster histograms were constructed to show how cells in any one phenotype were distributed across the consensus clusters. Grouping various phenotypes allowed us to build phenotype trees and calculate the statistical difference between each group. The results showed that non-neoplastic S1 cells could be distinguished from malignant T4-2 cells with 94.19% accuracy; that proliferating S1 cells could be distinguished from differentiated S1 cells with 92.86% accuracy; and showed no significant difference between the various phenotypes of T4-2 cells corresponding to increasing tumor sizes. CONCLUSION: This work presents a cluster analysis method that can identify significant cell phenotypes, based on the nuclear distribution of specific proteins, with high accuracy. [Abstract/Link to Full Text]

Marée R, Geurts P, Wehenkel L
Random subwindows and extremely randomized trees for image classification in cell biology.
BMC Cell Biol. 2007;8 Suppl 1S2.
BACKGROUND: With the improvements in biosensors and high-throughput image acquisition technologies, life science laboratories are able to perform an increasing number of experiments that involve the generation of a large amount of images at different imaging modalities/scales. It stresses the need for computer vision methods that automate image classification tasks. RESULTS: We illustrate the potential of our image classification method in cell biology by evaluating it on four datasets of images related to protein distributions or subcellular localizations, and red-blood cell shapes. Accuracy results are quite good without any specific pre-processing neither domain knowledge incorporation. The method is implemented in Java and available upon request for evaluation and research purpose. CONCLUSION: Our method is directly applicable to any image classification problems. We foresee the use of this automatic approach as a baseline method and first try on various biological image classification problems. [Abstract/Link to Full Text]

Staadt OG, Natarajan V, Weber GH, Wiley DF, Hamann B
Interactive processing and visualization of image data for biomedical and life science applications.
BMC Cell Biol. 2007;8 Suppl 1S10.
BACKGROUND: Applications in biomedical science and life science produce large data sets using increasingly powerful imaging devices and computer simulations. It is becoming increasingly difficult for scientists to explore and analyze these data using traditional tools. Interactive data processing and visualization tools can support scientists to overcome these limitations. RESULTS: We show that new data processing tools and visualization systems can be used successfully in biomedical and life science applications. We present an adaptive high-resolution display system suitable for biomedical image data, algorithms for analyzing and visualization protein surfaces and retinal optical coherence tomography data, and visualization tools for 3D gene expression data. CONCLUSION: We demonstrated that interactive processing and visualization methods and systems can support scientists in a variety of biomedical and life science application areas concerned with massive data analysis. [Abstract/Link to Full Text]

Proceedings of the 2006 International Workshop on Multiscale Biological Imaging, Data Mining and Informatics, Santa Barbara, USA (BII06).
BMC Cell Biol. 2007;8 Suppl 1S1-10.
The 2006 International Workshop on Multiscale Biological Imaging, Data Mining and Informatics was held at Santa Barbara, on Sept 7-8, 2006. Based on the presentations at the workshop, we selected and compiled this collection of research articles related to novel algorithms and enabling techniques for bio- and biomedical image analysis, mining, visualization, and biology applications. [Abstract/Link to Full Text]

Wu Y, Laughlin RC, Henry DC, Krueger DE, Hudson JS, Kuan CY, He J, Reppert J, Tomkins JP
Naturally occurring and stress induced tubular structures from mammalian cells, a survival mechanism.
BMC Cell Biol. 2007;836.
BACKGROUND: Tubular shaped mammalian cells in response to dehydration have not been previously reported. This may be due to the invisibility of these cells in aqueous solution, and because sugars and salts added to the cell culture for manipulation of the osmotic conditions inhibit transformation of normal cells into tubular shaped structures. RESULTS: We report the transformation of normal spherical mammalian cells into tubular shaped structures in response to stress. We have termed these transformed structures 'straw cells' which we have associated with a variety of human tissue types, including fresh, post mortem and frozen lung, liver, skin, and heart. We have also documented the presence of straw cells in bovine brain and prostate tissues of mice. The number of straw cells in heart, lung tissues, and collapsed straw cells in urine increases with the age of the mammal. Straw cells were also reproduced in vitro from human cancer cells (THP1, CACO2, and MCF7) and mouse stem cells (D1 and adipose D1) by dehydrating cultured cells. The tubular center of the straw cells is much smaller than the original cell; houses condensed organelles and have filamentous extensions that are covered with microscopic hair-like structures and circular openings. When rehydrated, the filaments uptake water rapidly. The straw cell walls, have a range of 120 nm to 200 nm and are composed of sulfated-glucose polymers and glycosylated acidic proteins. The transformation from normal cell to straw cells takes 5 to 8 hr in open-air. This process is characterized by an increase in metabolic activity. When rehydrated, the straw cells regain their normal spherical shape and begin to divide in 10 to 15 days. Like various types of microbial spores, straw cells are resistant to harsh environmental conditions such as UV-C radiation. CONCLUSION: Straw cells are specialized cellular structures and not artifacts from spontaneous polymerization, which are generated in response to stress conditions, like dehydration. The disintegrative, mobile, disruptive and ubiquitous nature of straw cells makes this a possible physiological process that may be involved in human health, longevity, and various types of diseases such as cancer. [Abstract/Link to Full Text]

Piergentili R
Evolutionary conservation of lampbrush-like loops in drosophilids.
BMC Cell Biol. 2007;835.
BACKGROUND: Loopin-1 is an abundant, male germ line specific protein of Drosophila melanogaster. The polyclonal antibody T53-F1 specifically recognizes Loopin-1 and enables its visualization on the Y-chromosome lampbrush-like loop named kl-3 during primary spermatocyte development, as well as on sperm tails. In order to test lampbrush-like loop evolutionary conservation, extensive phase-contrast microscopy and immunostaining with T53-F1 antibody was performed in other drosophilids scattered along their genealogical tree. RESULTS: In the male germ line of all species tested there are cells showing giant nuclei and intranuclear structures similar to those of Drosophila melanogaster primary spermatocytes. Moreover, the antibody T53-F1 recognizes intranuclear structures in primary spermatocytes of all drosophilids analyzed. Interestingly, the extent and conformation of the staining pattern is species-specific. In addition, the intense staining of sperm tails in all species suggests that the terminal localization of Loopin-1 and its orthologues is conserved. A comparison of these cytological data and the data coming from the literature about sperm length, amount of sperm tail entering the egg during fertilization, shape and extent of both loops and primary spermatocyte nuclei, seems to exclude direct relationships among these parameters. CONCLUSION: Taken together, the data reported strongly suggest that lampbrush-like loops are a conserved feature of primary spermatocyte nuclei in many, if not all, drosophilids. Moreover, the conserved pattern of the T53-F1 immunostaining indicates that a Loopin-1-like protein is present in all the species analyzed, whose localization on lampbrush-like loops and sperm tails during spermatogenesis is evolutionary conserved. [Abstract/Link to Full Text]

Tighe A, Ray-Sinha A, Staples OD, Taylor SS
GSK-3 inhibitors induce chromosome instability.
BMC Cell Biol. 2007;834.
BACKGROUND: Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC) may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 - a protein kinase, which in concert with APC, targets beta-catenin for proteolysis - and ask whether GSK-3 is required for accurate chromosome segregation. RESULTS: To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3beta. Cells deficient for GSK-3beta exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3beta repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation. CONCLUSION: Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability. [Abstract/Link to Full Text]

Stehle IM, Postberg J, Rupprecht S, Cremer T, Jackson DA, Lipps HJ
Establishment and mitotic stability of an extra-chromosomal mammalian replicon.
BMC Cell Biol. 2007;833.
BACKGROUND: Basic functions of the eukaryotic nucleus, like transcription and replication, are regulated in a hierarchic fashion. It is assumed that epigenetic factors influence the efficiency and precision of these processes. In order to uncouple local and long-range epigenetic features we used an extra-chromosomal replicon to study the requirements for replication and segregation and compared its behavior to that of its integrated counterpart. RESULTS: The autonomous replicon replicates in all eukaryotic cells and is stably maintained in the absence of selection but, as other extra-chromosomal replicons, its establishment is very inefficient. We now show that following establishment the vector is stably associated with nuclear compartments involved in gene expression and chromosomal domains that replicate at the onset of S-phase. While the vector stays autonomous, its association with these compartments ensures the efficiency of replication and mitotic segregation in proliferating cells. CONCLUSION: Using this novel minimal model system we demonstrate that relevant functions of the eukaryotic nucleus are strongly influenced by higher nuclear architecture. Furthermore our findings have relevance for the rational design of episomal vectors to be used for genetic modification of cells: in order to improve such constructs with respect to efficiency elements have to be identified which ensure that such constructs reach regions of the nucleus favorable for replication and transcription. [Abstract/Link to Full Text]

Howard RA, Sharma P, Hajjar C, Caldwell KA, Caldwell GA, du Breuil R, Moore R, Boyd L
Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation.
BMC Cell Biol. 2007;832.
BACKGROUND: Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease and Parkinson's disease. Proteins containing long, homopolymeric stretches of glutamine are especially prone to form aggregates. It has long been known that the small protein modifier, ubiquitin, localizes to these aggregates. In this report, nematode and cell culture models for polyglutamine aggregation are used to investigate the role of the ubiquitin pathway in protein aggregation. RESULTS: Ubiquitin conjugating enzymes (Ubc's) were identified that affect polyglutamine aggregates in C. elegans. Specifically, RNAi knockdown of ubc-2 or ubc-22 causes a significant increase in the size of aggregates as well as a reduction in aggregate number. In contrast, RNAi of ubc-1, ubc-13, or uev-1 leads to a reduction of aggregate size and eliminates ubiquitin and proteasome localization to aggregates. In cultured human cells, shRNA knockdown of human homologs of these Ubc's (Ube2A, UbcH5b, and E2-25K) causes similar effects indicating a conserved role for ubiquitination in polyglutamine protein aggregation. CONCLUSION: Results of knockdown of different Ubc enzymes indicate that at least two different and opposing ubiquitination events occur during polyglutamine aggregation. The loss of ubiquitin localization after ubc-1, ubc-13, or uev-1 knockdown suggests that these enzymes might be directly involved in ubiquitination of aggregating proteins. [Abstract/Link to Full Text]

Recent Articles in Cell Communication and Signaling

Perbal B
International CCN society and intercellular signaling.
Cell Commun Signal. 2007;51. [Abstract/Link to Full Text]

Abou Zeid N, Vallés AM, Boyer B
Serine phosphorylation regulates paxillin turnover during cell migration.
Cell Commun Signal. 2006;48.
BACKGROUND: Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines 188 and 190. However, the function of this phosphorylation event remains unknown. The purpose of this work was to determine the role of paxillin phosphorylation on residues S188 and S190 in the regulation of cell migration. RESULTS: We used NBT-II epithelial cells that can be induced to migrate when plated on collagen. To examine the role of paxillin serines 188/190 in cell migration, we constructed an EGFP-tagged paxillin mutant in which S188/S190 were mutated into unphosphorylatable alanine residues. We provide evidence that paxillin is regulated by proteasomal degradation following polyubiquitylation of the protein. During active cell migration on collagen, paxillin is protected from proteasome-dependent degradation. We demonstrate that phosphorylation of serines 188/190 is necessary for the protective effect of collagen. In an effort to understand the physiological relevance of paxillin protection from degradation, we show that cells expressing the paxillin S188/190A interfering mutant spread less, have reduced protrusive activity but migrate more actively. CONCLUSION: Our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility. [Abstract/Link to Full Text]

Planque N
Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers.
Cell Commun Signal. 2006;47.
Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy. [Abstract/Link to Full Text]

Perbal B
New insight into CCN3 interactions--nuclear CCN3 : fact or fantasy?
Cell Commun Signal. 2006;46.
The identification of potential partners for CCN3(NOV) sheds new light on the biological activity of this signaling protein. In particular, the physical interaction of CCN3 with the IL33 cytokine combined with previous data indicating that CCN3 expression was regulated by TNFalpha and IL1 cytokines, point to CCN3 as a potent player in a variety of inflammatory responses, including neurodegenerative disease, and arthritis. Nuclear proteins that are involved in the regulation of RNA processing and chromatin remodeling were also found to interact with CCN3. These observations reinforce the concept that routing of CCN3 to the cell nucleus where it acts as a transcription regulator, might constitute a key element in the balance between the anti- and pro-proliferative activities of CCN3 proteins. [Abstract/Link to Full Text]

Wilsbacher JL, Moores SL, Brugge JS
An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand.
Cell Commun Signal. 2006;45.
BACKGROUND: Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity. RESULTS: We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation. CONCLUSION: Our results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1. [Abstract/Link to Full Text]

Bhuiyan MB, Murad F, Fant ME
The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide.
Cell Commun Signal. 2006;44.
BACKGROUND: The human placenta, a non-neuronal tissue, contains an active cholinergic system comprised of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and high affinity muscarinic receptors. The cell(s) of origin of placental ACh and its role in trophoblast function has not been defined. These studies were performed to define the cellular location of ACh synthesis (ChAT) in the human placenta and to begin studying its functional role. RESULTS: Using immunohistochemical techniques, ChAT was observed primarily within the cytotrophoblasts of preterm placentae as well as some mesenchymal elements. Similar intense immunostaining of the cytotrophoblast was observed for endothelium-derived nitric oxide synthase (eNOS) suggesting that ACh may interact with nitric oxide (NO)-dependent signaling pathways. The ability of carbamylcholine (CCh), an ACh analogue, to stimulate a rise in intracellular Ca++ and NO production in trophoblasts was therefore tested using the BeWob30 choriocarcinoma cell as a model system. First, CCh significantly increased intracellular calcium as assessed by fluorescence microscopy. We then examined the ability of CCh to stimulate NO production by measuring total nitrite/nitrate production in conditioned media using chemiluminescence-based analysis. CCh, alone, had no effect on NO production. However, CCh increased measurable NO approximately 100% in the presence of 10 nM estradiol. This stimulatory effect was inhibited by 1 (micro)M scopolamine suggesting mediation via muscarinic receptors. Estradiol, alone, had no effect on total NO or eNOS protein or mRNA. CONCLUSION: These data demonstrate that placental ChAT localizes to the cytotrophoblast and some mesenchymal cells in human placenta. It further suggests that ACh acts via muscarinic receptors on the trophoblast cell membrane to modulate NO in an estrogen-dependent manner. [Abstract/Link to Full Text]

Perbal B
NOV story: the way to CCN3.
Cell Commun Signal. 2006;43.
The principal aim of this historical review- the first in a new series- is to present the basic concepts that led to the discovery of NOV and to show how our ideas evolved regarding the role and functions of this new class of proteins. It should prove particularly useful to the new comers and to students who are engaged in this exciting field. It is also a good opportunity to acknowledge the input of those who participated in the development of this scientific endeavour. [Abstract/Link to Full Text]

Caldwell KK, Sosa M, Buckley CT
Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize phosphatidylinositols and inositol phosphates.
Cell Commun Signal. 2006 Jan 30;4(1):2.
ABSTRACT: BACKGROUND: Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs). RESULTS: We found that several, but not all, of these enzymes contain identifiable D-domain sequences. Further, we found a high degree of conservation of these sequences and their location in human and mouse proteins; notable exceptions were PI 3-kinase C2-gamma, PI 4-kinase type IIbeta, and inositol polyphosphate 1-phosphatase. CONCLUSIONS: The results indicate that there may be extensive crosstalk between MAPK signaling and signaling pathways that are regulated by cellular levels of PIs or IPs. [Abstract/Link to Full Text]

Li CL, Coullin P, Bernheim A, Joliot V, Auffray C, Zoroob R, Perbal B
Integration of Myeloblastosis Associated Virus proviral sequences occurs in the vicinity of genes encoding signaling proteins and regulators of cell proliferation.
Cell Commun Signal. 2006;41.
AIMS: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8-10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS: Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. RESULTS: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. CONCLUSION: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors. [Abstract/Link to Full Text]

Gao R, Brigstock DR
Activation of nuclear factor kappa B (NF-kappaB) by connective tissue growth factor (CCN2) is involved in sustaining the survival of primary rat hepatic stellate cells.
Cell Commun Signal. 2005 Nov 22;314.
BACKGROUND/AIMS: Connective tissue growth factor (CCN2) is a matricellular protein that plays a role in hepatic stellate cell (HSC)-mediated fibrogenesis. The aim of this study was to investigate the regulation by CCN2 of cell survival pathways in primary HSC. METHODS: Primary HSC were obtained by in situ enzymatic perfusion of rat liver. NF-kappaB activation was assessed by immunoblotting for IkappaBalpha phosphorylation and degradation and by NF-kappaB p50 or p65 nuclear accumulation. NF-kappaB DNA-binding activity was determined by gel mobility shift assay while NF-kappaB response gene expression was evaluated using a luciferase reporter. Cell viability was assessed by Trypan blue staining or ATP luminescent assay while apoptosis was evaluated by caspase-3 activity. RESULTS: CCN2 induced IkappaBalpha phosphorylation and degradation as well as nuclear accumulation of NF-kappaB. Activated NF-kappaB comprised three dimers, p65/p65, p65/p50 and p50/p50, that individually bound to DNA-binding sites and subsequently triggered transcriptional activity. This was confirmed by showing that CCN2 promoted activity of a NF-kappaB luciferase reporter. CCN2 promoted survival of serum-starved HSC and protected the cells from death induced by blocking the NF-kappaB signaling pathway using Bay-11-7082, a specific inhibitor of IkappaBalpha phosphorylation. CONCLUSION: CCN2 contributes to the survival of primary HSC through the NF-kappaB pathway. [Abstract/Link to Full Text]

Marcus DC, Liu J, Lee JH, Scherer EQ, Scofield MA, Wangemann P
Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium.
Cell Commun Signal. 2005 Nov 2;313.
BACKGROUND: It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. RESULTS: The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 microM) did not inhibit the effect of UTP (1 microM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. CONCLUSION: The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2. [Abstract/Link to Full Text]

Swain RK, Katoh M, Medina A, Steinbeisser H
Xenopus frizzled-4S, a splicing variant of Xfz4 is a context-dependent activator and inhibitor of Wnt/beta-catenin signaling.
Cell Commun Signal. 2005 Oct 19;312.
BACKGROUND: Secreted Frizzled related proteins (SFRPs) are extracellular regulators of Wnt signaling. These proteins contain an N-terminal cysteine rich domain (CRD) highly similar to the CRDs of the Frizzled family of seven-transmembrane proteins that act as Wnt receptors. SFRPs can bind to Wnts and prevent their interaction with the Frizzled receptor. Recently it has been reported that a splice variant of human Frizzled-4 (FZD4S) lacking the transmembrane and the cytoplasmic domains of Frizzled-4 can activate rather than inhibit Wnt-8 activity in Xenopus embryos. This indicates that secreted CRD containing proteins such as Frizzled ecto-domains and SFRPs may not always act as Wnt inhibitors. It is not known how FZD4S can activate Wnt/beta-catenin signaling and what biological role this molecule plays in vivo. RESULTS: Here we report that the Xenopus frizzled-4 is alternatively spliced to give rise to a putative secreted protein that lacks the seven-transmembrane and the cytoplasmic domains. We performed functional experiments in Xenopus embryos to investigate how this novel splicing variant, Xfz4S, can modulate the Wnt/beta-catenin pathway. We show that Xfz4S as well as the extracellular domain of Xfz8 (ECD8) can act as both activators and inhibitors of Wnt/beta-catenin signaling dependent on the Wnt ligand presented. The positive regulation of Wnt/beta-catenin signaling by the extracellular domains of Frizzled receptors is mediated by the members of low density lipoprotein receptor-related protein (LRP-5/6) that act as Wnt coreceptors. CONCLUSION: This work provides evidence that the secreted extracellular domains of Frizzled receptors may act as both inhibitors and activators of Wnt signaling dependent on the Wnt ligand presented. [Abstract/Link to Full Text]

Asano M, Kubota S, Nakanishi T, Nishida T, Yamaai T, Yosimichi G, Ohyama K, Sugimoto T, Murayama Y, Takigawa M
Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells.
Cell Commun Signal. 2005 Oct 5;311.
BACKGROUND: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro. RESULTS: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-beta. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis. CONCLUSION: These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament. [Abstract/Link to Full Text]

Harmon EB, Porter JM, Porter JE
Beta-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA-independent mechanisms.
Cell Commun Signal. 2005 Aug 9;310.
BACKGROUND: Interstitial cystitis (IC) is a debilitating disease characterized by chronic inflammation of the urinary bladder, yet specific cellular mechanisms of inflammation in IC are largely unknown. Multiple lines of evidence suggest that beta-adrenergic receptor (AR) signaling is increased in the inflamed urothelium, however the precise effects of these urothelial cell signals have not been studied. In order to better elucidate the AR signaling mechanisms of inflammation associated with IC, we have examined the effects of beta-AR stimulation in an immortalized human urothelial cell line (UROtsa). For these studies, UROtsa cells were treated with effective concentrations of the selective beta-AR agonist isoproterenol, in the absence or presence of selective inhibitors of protein kinase A (PKA). Cell lysates were analyzed by radioimmunoassay for generation of cAMP or by Western blotting for induction of protein products associated with inflammatory responses. RESULTS: Radioligand binding demonstrated the presence of beta-ARs on human urothelial UROtsa cell membranes. Stimulating UROtsa cells with isoproterenol led to concentration-dependent increases of cAMP production that could be inhibited by pretreatment with a blocking concentration of the selective beta-AR antagonist propranolol. In addition, isoproterenol activation of these same cells led to significant increases in the amount of phosphorylated extracellular signal-regulated kinase (pERK), inducible nitric oxide synthase (iNOS) and the induced form of cyclooxygenase (COX-2) when compared to control. Moreover, preincubation of UROtsa cells with the selective PKA inhibitors H-89 or Rp-cAMPs did not diminish this isoproterenol mediated phosphorylation of ERK or production of iNOS and COX-2. CONCLUSION: Functional beta-ARs expressed on human urothelial UROtsa cell membranes increase the generation of cAMP and production of protein products associated with inflammation when activated by the selective beta-AR agonist isoproterenol. However, the increased production of iNOS and COX-2 by isoproterenol is not blocked when UROtsa cells are preincubated with inhibitors of PKA. Therefore, UROtsa cell beta-AR activation significantly increases the amount of iNOS and COX-2 produced by a PKA-independent mechanism. Consequently, this immortalized human urothelial cell line can be useful in characterizing potential AR signaling mechanisms associated with chronic inflammatory diseases of the bladder. [Abstract/Link to Full Text]

Wary KK, Humtsoe JO
Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF- and VEGF-induced capillary morphogenesis of endothelial cells.
Cell Commun Signal. 2005 Aug 2;39.
BACKGROUND: Angiogenesis, or the remodeling of existing vasculature serves as a lifeline to nourish developing embryos and starved tissues, and to accelerate wound healing, diabetic retinopathy, and tumor progression. Recent studies indicate that angiogenesis requires growth factor activity as well as cell adhesion events mediated by alpha5beta1 and alphavbeta3 integrins. We previously demonstrated that human lipid phosphate phosphohydrolase-3 (LPP3) acts as a cell-associated ligand for alpha5beta1 and alphavbeta3 integrins. Here, we test the hypothesis that an anti-LPP3 antibody can inhibit basic fibroblast growth factor (bFGF)-and vascular endothelial growth factor (VEGF)-induced capillary morphogenesis of endothelial cells (ECs). RESULTS: We report that bFGF and VEGF up-regulate LPP3 protein expression in ECs. Immunoprecipitation analyses show that LPP3 is a cell surface protein and undergoes N-glycosylation. Fluorescent activated cell sorting (FACS) data suggest that anti-LPP3-RGD detects native neoepitope on the surface of activated ECs. Moreover, we demonstrate LPP3 protein expression in tumor endothelium alongside VEGF. The embedding of ECs into three-dimensional type I collagen in the presence of bFGF and VEGF induce capillary formation. Importantly, we show that the addition of an anti-LPP3 antibody specifically and significantly blocks bFGF- and VEGF-induced capillary morphogenesis of ECs. CONCLUSION: These data suggest that activated ECs as well as tumor endothelium express LPP3 protein. In an in vitro assay, the anti-LPP3-RGD specifically blocks bFGF and VEGF induced capillary morphogenesis of ECs. Our results, therefore, suggest a role for LPP3 in angiogenesis. [Abstract/Link to Full Text]

Ralt D
Intercellular communication, NO and the biology of Chinese medicine.
Cell Commun Signal. 2005 May 18;3(1):8.
New multiple categories of health disciplines have become popular in the west and integration between the medicinal approaches has become essential. The hypothesis presented here suggests a novel integrative view that combines Western biochemistry with the Chinese medicinal concept of qi. The core for this hypothesis is that transmission of qi along the meridians is based on informational molecules that travel via an intercellular communication system. Acupuncture at specific points enhances the flow of the signaling molecules through this communication system. Nitric oxide is suggested as a prime candidate for such a signaling molecule in the meridian system. The biochemistry of nitric oxide can shed light on the biology underlying Chinese medicine while Chinese medicinal data can provide a clue to the sought after framework for nitric oxide. [Abstract/Link to Full Text]

Wary KK
Recognizing scientific excellence in the biology of cell adhesion.
Cell Commun Signal. 2005 Apr 18;3(1):7.
The prestigious 2005 Japan Prize for Cell Biology has been awarded to Dr. Masatoshi Takeichi, Director of RIKEN Developmental Biology, Kobe, Japan, and Dr. Erkki Ruoslahti, Distinguished Professor, The Burnham Institute, La Jolla, USA for their "Fundamental contribution in elucidating the molecular mechanisms of cell adhesion". The award is scheduled to be presented to the scientists in ceremonies in Tokyo on April 20, 2005 as part of a week-long celebration of "Japan Prize Week". [Abstract/Link to Full Text]

Moritani NH, Kubota S, Sugahara T, Takigawa M
Comparable response of ccn1 with ccn2 genes upon arthritis: An in vitro evaluation with a human chondrocytic cell line stimulated by a set of cytokines.
Cell Commun Signal. 2005 Apr 15;3(1):6.
BACKGROUND: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. RESULTS: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-alpha. To induce pro-inflammatory or reparative responses, TGF-beta was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-alpha stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-beta and a glucocorticoid. CONCLUSION: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis. [Abstract/Link to Full Text]

Schutze N, Noth U, Schneidereit J, Hendrich C, Jakob F
Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.
Cell Commun Signal. 2005 Mar 17;3(1):5.
BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways. [Abstract/Link to Full Text]

Hemavathy KC, Suppiah K, Hashmi G, Novetsky AD, Wang JC
TPO/Mpl Studies in Agnogenic Myeloid Metaplasia.
Cell Commun Signal. 2005 Feb 3;3(1):4.
BACKGROUND: Agnogenic myeloid metaplasia (AMM) is one of the Philadelphia chromosome negative myeloproliferative disorder and is diagnosed by hyperplasia of atypical megakaryocytes, hepatosplenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Fibrosis is considered to be a secondary consequence of enhanced levels of fibrogenic growth factors such as TGF beta1, bFGF and PDGF produced by enhanced numbers of megakaryocytes, while the primary cause is considered to be the enhanced proliferation of a defective stem cell. We have previously reported that thrombopoietin (TPO) is elevated in patients with AMM. Others have reported that Mpl protein is decreased in these patients. Since TPO is essential for the development of megakaryocytes, and Mpl protein is the receptor for TPO, we extended the study of TPO/Mpl to in vitro and in vivo cell culture systems to better understand the mechanism that leads to reduced Mpl protein in AMM patients. RESULTS: Plasma TPO levels were significantly elevated and Mpl protein levels were significantly reduced in AMM patients in concordance with previous studies. Platelet Mpl transcripts in AMM were however similar to those in controls. We also cloned Mpl cDNA from AMM patients and tested for their ability to make functional proteins in vitro and in the in vivo system of 293 T human embryonic kidney cells. Their expression including the glycosylated forms was similar to those from the controls. We also measured the level of translation initiation factor, eIF4E and found it to be increased in patients with AMM demonstrating that the reduced Mpl protein may not be due to translation defects. CONCLUSIONS: Our studies using the in vitro and in vivo systems further confirm that reduced Mpl protein levels are not due to defects in its transcription/translation. Reduced Mpl protein could be due to its increased internalisation owing to enhanced plasma TPO or in vivo intrinsic defects in patients with AMM. [Abstract/Link to Full Text]

Reilly GC, Golden EB, Grasso-Knight G, Leboy PS
Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes.
Cell Commun Signal. 2005 Feb 3;3(1):3.
BACKGROUND: During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes. RESULTS: Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition. CONCLUSION: Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate. [Abstract/Link to Full Text]

de Bovis B, Derouet D, Gauchat JF, Elson G, Gascan H, Delapeyričre O
clc is co-expressed with clf or cntfr in developing mouse muscles.
Cell Commun Signal. 2005 Jan 31;3(1):1.
BACKGROUND: The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor alpha component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR). RESULTS: Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. CONCLUSION: This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development. [Abstract/Link to Full Text]

Kingsley K, Plopper GE
Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways.
Cell Commun Signal. 2005 Jan 31;3(1):2.
BACKGROUND: A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. RESULTS: Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-beta1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-beta1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-beta1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. CONCLUSION: These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis. [Abstract/Link to Full Text]

Kyurkchiev S, Yeger H, Bleau AM, Perbal B
Potential cellular conformations of the CCN3(NOV) protein.
Cell Commun Signal. 2004 Sep 10;2(1):9.
AIM: To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R. RESULTS: Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R (adrenocortical carcinoma) cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled. CONCLUSIONS: The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein. [Abstract/Link to Full Text]

Dudley AC, Thomas D, Best J, Jenkins A
The STATs in cell stress-type responses.
Cell Commun Signal. 2004 Aug 6;2(1):8.
In the early 1990's, a new cell signaling pathway was described. This new paradigm, now known as the JAK/STAT pathway, has been extensively investigated in immune-type cells in response to interferons and interleukins. However, recent evidence suggests that the JAK/STAT pathway also mediates diverse cellular responses to various forms of biological stress including hypoxia/reperfusion, endotoxin, ultraviolet light, and hyperosmolarity. The current literature describing the JAK/STAT pathway's role in cellular stress responses has been reviewed herein, but it is clear that our knowledge in this area is far from complete. [Abstract/Link to Full Text]

Perbal B
APC: the toll road to continued high quality communication.
Cell Commun Signal. 2004 Jul 8;2(1):7.
In this article we briefly review the reasons and advantages that underly our publisher's decision to introduce article-processing charges (APC) for manuscripts submitted to Cell Communication and Signaling. The charge is an attempt to develop a new business model for distributing biomedical information and has been accepted in a number of other journals. APCs will enable BioMed Central to continue to provide their excellent service and will help to establish our journal. [Abstract/Link to Full Text]

Marino F, Cosentino M, Ferrari M, Cattaneo S, Frigo G, Fietta AM, Lecchini S, Frigo GM
Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers.
Cell Commun Signal. 2004 Jun 30;2(1):6.
BACKGROUND: The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. RESULTS: The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or omega-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the alpha1 subunits of T-type Ca++ channels (namely, alpha1G, alpha1H, and alpha1I). CONCLUSIONS: In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels. [Abstract/Link to Full Text]

Maroni PD, Koul S, Meacham RB, Koul HK
Mitogen Activated Protein kinase signal transduction pathways in the prostate.
Cell Commun Signal. 2004 Jun 25;2(1):5.
The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. [Abstract/Link to Full Text]

Thirkill TL, Hendren SR, Soghomonians A, Mariano NF, Barakat AI, Douglas GC
Regulation of trophoblast beta1-integrin expression by contact with endothelial cells.
Cell Commun Signal. 2004 Jun 9;2(1):4.
BACKGROUND: In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of beta1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells. RESULTS: When cultured alone, trophoblasts expressed low levels of beta1 integrin as determined by quantitative immunofluorescence microscopy. When trophoblasts were cultured on top of endothelial cells for 24 h, the expression of trophoblast beta1 integrin was significantly increased as determined by image analysis. beta1 Integrin expression was not increased when trophoblasts were cultured with endothelial cell-conditioned medium, suggesting that upregulation requires direct contact between trophoblasts and endothelial cells. To identify endothelial cell surface molecules responsible for induction of trophoblast integrin expression, trophoblasts were cultured in dishes coated with recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), or alphaVbeta3 integrin. Trophoblast beta1 integrin expression (assessed by immunofluorescence microscopy and Western blotting) was increased when PECAM-1 or alphaVbeta3 integrin, but not ICAM-1, was used as substrate. CONCLUSIONS: Direct contact between trophoblasts and endothelial cells increases the expression of trophoblast beta1 integrin. [Abstract/Link to Full Text]

Rafiee P, Heidemann J, Ogawa H, Johnson NA, Fisher PJ, Li MS, Otterson MF, Johnson CP, Binion DG
Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling.
Cell Commun Signal. 2004 Jun 2;2(1):3.
The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection. [Abstract/Link to Full Text]

Recent Articles in Eukaryotic Cell

Fujioka T, Mizutani O, Furukawa K, Sato N, Yoshimi A, Yamagata Y, Nakajima T, Abe K
MpkA-Dependent and -independent cell wall integrity signaling in Aspergillus nidulans.
Eukaryot Cell. 2007 Aug;6(8):1497-510.
Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Delta and mpk1Delta mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Delta and swi6Delta mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmADelta, Answi4Delta Answi6Delta, and mpkADelta strains) and analyzed mpkA and CWG transcripts after treatment with a beta-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The beta-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkADelta strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except alpha-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae. [Abstract/Link to Full Text]

McFadden DC, Fries BC, Wang F, Casadevall A
Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans.
Eukaryot Cell. 2007 Aug;6(8):1464-73.
Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions. [Abstract/Link to Full Text]

Oide S, Krasnoff SB, Gibson DM, Turgeon BG
Intracellular siderophores are essential for ascomycete sexual development in heterothallic Cochliobolus heterostrophus and homothallic Gibberella zeae.
Eukaryot Cell. 2007 Aug;6(8):1339-53.
Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1 to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Delta nps2) was defective in ascus/ascospore development in homozygous Delta nps2 crosses. Homozygous crosses of the remaining 11 Delta nps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae Delta nps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae Delta nps2 strains, demonstrating that abnormal sexual development of Delta nps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae Delta nps2 strains restored WT fertility. NPS1, a G. zeae NPS gene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophus and the homothallic ascomycete G. zeae. [Abstract/Link to Full Text]

Ding C, Butler G
Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation.
Eukaryot Cell. 2007 Aug;6(8):1310-9.
Candida parapsilosis is an important cause of candidiasis, yet few molecular tools are available. We adapted a recyclable nourseothricin resistance marker gene (SAT1) originally developed for use with C. albicans in order to generate gene knockouts from C. parapsilosis. We first replaced the promoters driving expression of the FLP recombinase and the SAT1 genes with the equivalent sequences from C. parapsilosis. We then used the cassette to generate a homozygous knockout of C. parapsilosis URA3. The ura3 knockouts have altered colony morphologies. We also knocked out both alleles of an ortholog of BCR1, a gene encoding a transcription factor known to be required for biofilm development in C. albicans. We show that C. parapsilosis BCR1 is necessary for biofilm development in C. parapsilosis and for expression of the cell wall protein encoded by RBT1. Our results suggest that there are significant similarities in the regulation of biofilms between the two species, despite the fact that C. parapsilosis does not generate true hyphae and that BCR1 regulates the expression of many hypha-specific adhesins in C. albicans. [Abstract/Link to Full Text]

Saveria T, Halbach A, Erdmann R, Volkmer-Engert R, Landgraf C, Rottensteiner H, Parsons M
Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.
Eukaryot Cell. 2007 Aug;6(8):1439-49.
Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism. [Abstract/Link to Full Text]

Rexer CH, Chalker DL
Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila.
Eukaryot Cell. 2007 Aug;6(8):1320-9.
Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (DeltaLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome. [Abstract/Link to Full Text]

Kragl C, Schrettl M, Abt B, Sarg B, Lindner HH, Haas H
EstB-mediated hydrolysis of the siderophore triacetylfusarinine C optimizes iron uptake of Aspergillus fumigatus.
Eukaryot Cell. 2007 Aug;6(8):1278-85.
Aspergillus fumigatus excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three N(5)-cis-anhydromevalonyl-N(5)-hydroxy-N(2)-acetyl-l-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene, estB, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of estB in A. fumigatus eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo. Escherichia coli-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three N(2)-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in A. fumigatus. [Abstract/Link to Full Text]

Yamamoto A, Ueda J, Yamamoto N, Hashikawa N, Sakurai H
Role of heat shock transcription factor in Saccharomyces cerevisiae oxidative stress response.
Eukaryot Cell. 2007 Aug;6(8):1373-9.
The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO(2) and the thiol oxidants diamide and 1-chloro-2,4-dinitrobenzene (CDNB). Similar to heat shock, the oxidizing reagents are potent inducers of both efficient HSE binding and extensive phosphorylation of Hsf1. The inducible phosphorylation of Hsf1 is regulated by the intramolecular domain-domain interactions and affects HSE structure-specific transcription. Unlike the heat shock, diamide, or CDNB response, menadione or KO(2) activation of Hsf1 is inhibited by cyclic-AMP-dependent protein kinase (PKA) activity, which negatively regulates the activator functions of other transcriptional regulators implicated in the oxidative stress response. These results demonstrate that Hsf1 is a member of the oxidative stress-responsive activators and that PKA is a general negative regulator in the superoxide anion response. [Abstract/Link to Full Text]

Nimrichter L, Frases S, Cinelli LP, Viana NB, Nakouzi A, Travassos LR, Casadevall A, Rodrigues ML
Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations.
Eukaryot Cell. 2007 Aug;6(8):1400-10.
The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules. [Abstract/Link to Full Text]

Ren M, Santhanam A, Lee P, Caplan A, Garrett S
Alteration of the protein kinase binding domain enhances function of the Saccharomyces cerevisiae molecular chaperone Cdc37.
Eukaryot Cell. 2007 Aug;6(8):1363-72.
Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative "protein kinase binding domain" of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases. [Abstract/Link to Full Text]

Periz G, Dharia D, Miller SH, Keller LR
Flagellar elongation and gene expression in Chlamydomonas reinhardtii.
Eukaryot Cell. 2007 Aug;6(8):1411-20.
Lithium (Li(+)) affects the physiology of cells from a broad range of organisms including plants and both vertebrate and invertebrate animals. Although its effects result presumably from changes in gene expression elicited by its interaction with intracellular signal transduction pathways, the molecular mechanisms of Li(+) action are not well understood. The biflagellate green alga Chlamydomonas reinhardtii is an ideal genetic model for the integration of the effects on Li(+) on signal transduction, gene expression, and aspects of flagellar biogenesis. Li(+) causes C. reinhardtii flagella to elongate to approximately 1.4 times their normal length and blocks flagellar motility (S. Nakamura, H. Tabino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). We report here that Li(+) treatment increases the abundance of several flagellar mRNAs, including alpha- and beta-tubulin and pcf3-21. Li(+)-induced flagellar gene expression occurs in cells pretreated with cycloheximide, suggesting that the abundance change is a response that does not require new protein synthesis. Deletion analysis of the flagellar alpha1-tubulin gene promoter showed that sequences necessary for Li(+)-induced expression differed from those for acid shock induction and contain a consensus binding site for CREB/ATF and AP-1 transcription factors. These studies suggest potential promoter elements, candidate factors, and signal transduction pathways that may coordinate the C. reinhardtii cellular response to Li(+). [Abstract/Link to Full Text]

Koehler RN, Rachfall N, Rolfes RJ
Activation of the ADE genes requires the chromatin remodeling complexes SAGA and SWI/SNF.
Eukaryot Cell. 2007 Aug;6(8):1474-85.
The activation of the ADE regulon genes requires the pair of transcription factors Bas1 and Pho2. In a genome-wide screen for additional regulators of the pathway, strains with mutations in multiple subunits of the chromatin remodeling complexes SAGA and SWI/SNF were uncovered. These mutants exhibited decreased expression of an ADE5,7-lacZ reporter and native ADE compared to the wild-type strains, but the expression of the BAS1 and PHO2 genes was not substantially decreased. An unregulated Bas1-Pho2 fusion protein depended upon SAGA and SWI/SNF activity to promote transcription of a reporter. A significant but low-level association of Gcn5-myc and Snf2-myc with the ADE5,7 promoter was independent of adenine growth conditions and independent of the presence of the activator proteins Bas1 and Pho2. However, the increase in occupancy of Bas1 and Pho2 at ADE5,7 depended on both SAGA and SWI/SNF. The loss of catalytic activity of both SAGA and SWI/SNF complexes in the gcn5Delta snf2Delta double mutant was severely detrimental to ADE-lacZ reporter expression and native ADE gene expression, indicating complementary roles for these complexes. We conclude that Bas1 and Pho2 do not recruit the SAGA and SWI/SNF complexes to the ADE5,7 promoter but that the remodeling complexes are necessary to increase the binding of Bas1 and Pho2 in response to the adenine regulatory signal. Our data support the model that the SAGA and SWI/SNF complexes engage in global surveillance that is necessary for the specific response by Bas1 and Pho2. [Abstract/Link to Full Text]

Dolezal P, Dancis A, Lesuisse E, Sutak R, Hrdý I, Embley TM, Tachezy J
Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis.
Eukaryot Cell. 2007 Aug;6(8):1431-8.
Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes. [Abstract/Link to Full Text]

Moroney JV, Ynalvez RA
Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii.
Eukaryot Cell. 2007 Aug;6(8):1251-9. [Abstract/Link to Full Text]

Saxena AK, Wu Y, Garboczi DN
Plasmodium p25 and p28 surface proteins: potential transmission-blocking vaccines.
Eukaryot Cell. 2007 Aug;6(8):1260-5. [Abstract/Link to Full Text]

Deng F, Allen TD, Hillman BI, Nuss DL
Comparative analysis of alterations in host phenotype and transcript accumulation following hypovirus and mycoreovirus infections of the chestnut blight fungus Cryphonectria parasitica.
Eukaryot Cell. 2007 Aug;6(8):1286-98.
Infection of the chestnut blight fungus, Cryphonectria parasitica, by hypovirus CHV1-EP713 or by reovirus MyRV1-Cp9B21 or MyRV2-CpC18 results in reduced fungal virulence (hypovirulence). However, additional phenotypic changes caused by the two groups of mycoviruses are quite different. We now report that the loss of female fertility and the resulting absence of virus transmission through sexual spores observed after hypovirus infection was not observed for reovirus-infected C. parasitica. Consistent with this result, expression of two genes involved in sexual reproduction, the pheromone precursor gene, Mf2/1, and the yeast STE12-like transcriptional factor gene, cpst12, was less reduced in reovirus-infected strains than in the hypovirus CHV1-EP713-infected strain. Analysis with a custom microarray cDNA chip containing expressed sequence tag clones representing approximately 2,200 unique C. parasitica genes identified 140 and 128 host genes that were responsive to MyRV1-Cp9B21 or MyRV2-CpC18 infection, respectively. Comparison of these virus-responsive genes revealed an overlap of 85 genes, even though the nucleotide sequence identity for the two reoviruses is less than 50%. Significantly, 84 of the 85 genes were altered in the same direction. Further comparison revealed that 51% and 48% of the MyRV1-Cp9B21- and MyRV2-CpC18-responsive genes were also responsive to CHV1-EP713 infection. Finally, similar to results reported for CHV1-EP713 infection, a high percentage (59% and 66%) of the reovirus-responsive genes were also differentially expressed following disruption of the cellular G-protein signal transduction pathway. These data support the hypothesis that hypovirus and reovirus infections perturb common and specific C. parasitica regulatory pathways to cause hypovirulence and distinct sets of phenotypic changes. [Abstract/Link to Full Text]

Southern TR, Jolly CE, Lester ME, Hayman JR
EnP1, a microsporidian spore wall protein that enables spores to adhere to and infect host cells in vitro.
Eukaryot Cell. 2007 Aug;6(8):1354-62.
Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection. [Abstract/Link to Full Text]

Turnock DC, Ferguson MA
Sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major.
Eukaryot Cell. 2007 Aug;6(8):1450-63.
The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. Five sugar nucleotides, GDP-alpha-d-mannose, UDP-alpha-d-N-acetylglucosamine, UDP-alpha-d-glucose, UDP-alpha-galactopyranose, and GDP-beta-l-fucose, were common to all three species; one, UDP-alpha-d-galactofuranose, was common to T. cruzi and L. major; three, UDP-beta-l-rhamnopyranose, UDP-alpha-d-xylose, and UDP-alpha-d-glucuronic acid, were found only in T. cruzi; and one, GDP-alpha-d-arabinopyranose, was found only in L. major. The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes. [Abstract/Link to Full Text]

Balajee SA, Tay ST, Lasker BA, Hurst SF, Rooney AP
Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America.
Eukaryot Cell. 2007 Aug;6(8):1392-9.
Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study. [Abstract/Link to Full Text]

Teodorovic S, Braverman JM, Elmendorf HG
Unusually low levels of genetic variation among Giardia lamblia isolates.
Eukaryot Cell. 2007 Aug;6(8):1421-30.
Giardia lamblia, an intestinal pathogen of mammals, including humans, is a significant cause of diarrheal disease around the world. Additionally, the parasite is found on a lineage which separated early from the main branch in eukaryotic evolution. The extent of genetic diversity among G. lamblia isolates is insufficiently understood, but this knowledge is a prerequisite to better understand the role of parasite variation in disease etiology and to examine the evolution of mechanisms of genetic exchange among eukaryotes. Intraisolate genetic variation in G. lamblia has never been estimated, and previous studies on interisolate genetic variation have included a limited sample of loci. Here we report a population genetics study of intra- and interisolate genetic diversity based on six coding and four noncoding regions from nine G. lamblia isolates. Our results indicate exceedingly low levels of genetic variation in two out of three G. lamblia groups that infect humans; this variation is sufficient to allow identification of isolate-specific markers. Low genetic diversity at both coding and noncoding regions, with an overall bias towards synonymous substitutions, was discovered. Surprisingly, we found a dichotomous haplotype structure in the third, more variable G. lamblia group, represented by a haplotype shared with one of the homogenous groups and an additional group-specific haplotype. We propose that the distinct patterns of genetic-variation distribution among lineages are a consequence of the presence of genetic exchange. More broadly, our findings have implications for the regulation of gene expression, as well as the mode of reproduction in the parasite. [Abstract/Link to Full Text]

Levdansky E, Romano J, Shadkchan Y, Sharon H, Verstrepen KJ, Fink GR, Osherov N
Coding tandem repeats generate diversity in Aspergillus fumigatus genes.
Eukaryot Cell. 2007 Aug;6(8):1380-91.
Genes containing multiple coding mini- and microsatellite repeats are highly dynamic components of genomes. Frequent recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the amino acid sequence of the corresponding protein. In bacteria and yeasts, the expansion of such coding repeats in cell wall proteins is associated with alterations in immunogenicity, adhesion, and pathogenesis. We hypothesized that identification of repeat-containing putative cell wall proteins in the human pathogen Aspergillus fumigatus may reveal novel pathogenesis-related elements. Here, we report that the genome of A. fumigatus contains as many as 292 genes with internal repeats. Fourteen of 30 selected genes showed size variation of their repeat-containing regions among 11 clinical A. fumigatus isolates. Four of these genes, Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090, encode putative cell wall proteins containing a leader sequence and a glycosylphosphatidylinositol anchor motif. All four genes are expressed and produce variable-size mRNA encoding a discrete number of repeat amino acid units. Their expression was altered during development and in response to cell wall-disrupting agents. Deletion of one of these genes, Afu3g08990, resulted in a phenotype characterized by rapid conidial germination and reduced adherence to extracellular matrix suggestive of an alteration in cell wall characteristics. The Afu3g08990 protein was localized to the cell walls of dormant and germinating conidia. Our findings suggest that a subset of the A. fumigatus cell surface proteins may be hypervariable due to recombination events in their internal tandem repeats. This variation may provide the functional diversity in cell surface antigens which allows rapid adaptation to the environment and/or elusion of the host immune system. [Abstract/Link to Full Text]

Jong A, Wu CH, Chen HM, Luo F, Kwon-Chung KJ, Chang YC, Lamunyon CW, Plaas A, Huang SH
Identification and characterization of CPS1 as a hyaluronic acid synthase contributing to the pathogenesis of Cryptococcus neoformans infection.
Eukaryot Cell. 2007 Aug;6(8):1486-96.
Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Delta strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast. [Abstract/Link to Full Text]

Hsu M, Yu EY, Singh SM, Lue NF
Mutual dependence of Candida albicans Est1p and Est3p in telomerase assembly and activation.
Eukaryot Cell. 2007 Aug;6(8):1330-8.
Telomerase is an RNA-protein complex responsible for extending one strand of the telomere terminal repeats. Analysis of the telomerase complex in budding yeasts has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The TERT subunit is essential for telomerase catalysis, while the functions of Est1p and Est3p have not been precisely elucidated. In an earlier study, we showed that telomerase derived from a Candida est1-null mutant is defective in primer utilization in vitro; it exhibits reduced initiation and processivity on primers that terminate in two regions of the telomere repeat. Here we show that telomerase derived from a Candida est3-null mutant has nearly identical defects in primer utilization and processivity. Further analysis revealed an unexpected mutual dependence of Est1p and Est3p in their assembly into the full telomerase complex, which accounts for the similarity between the mutant enzymes. We also developed an affinity isolation and an in vitro reconstitution protocol for the telomerase complex that will facilitate future mechanistic studies. [Abstract/Link to Full Text]

Simm C, Lahner B, Salt D, LeFurgey A, Ingram P, Yandell B, Eide DJ
Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution.
Eukaryot Cell. 2007 Jul;6(7):1166-77.
Previous studies of the yeast Saccharomyces cerevisiae indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these methods, we determined that zinc levels in the vacuole vary with zinc status and can rise to almost 100 mM zinc (i.e., 7 x 10(8) atoms of vacuolar zinc per cell). Moreover, this zinc can be mobilized effectively to supply the needs of as many as eight generations of progeny cells under zinc starvation conditions. While the Zrc1 and Cot1 zinc transporters are essential for zinc uptake into the vacuole under steady-state growth conditions, additional transporters help mediate zinc uptake into the vacuole during "zinc shock," when zinc-limited cells are resupplied with zinc. In addition, we found that other compartments of the cell do not provide significant stores of zinc. In particular, zinc accumulation in mitochondria is low and is homeostatically regulated independently of vacuolar zinc storage. Finally, we observed a strong correlation between zinc status and the levels of magnesium and phosphorus accumulated in cells. Our results implicate zinc as a major determinant of the ability of the cell to store these other important nutrients. [Abstract/Link to Full Text]

Souza RT, Santos MR, Lima FM, El-Sayed NM, Myler PJ, Ruiz JC, da Silveira JF
New Trypanosoma cruzi repeated element that shows site specificity for insertion.
Eukaryot Cell. 2007 Jul;6(7):1228-38.
A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other T. cruzi sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5' end of the element and a sequence domain of approximately 500 bp without a well-defined borderline at the 3' end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of approximately 0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be approximately 173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site. [Abstract/Link to Full Text]

Griffiths S, Portman N, Taylor PR, Gordon S, Ginger ML, Gull K
RNA interference mutant induction in vivo demonstrates the essential nature of trypanosome flagellar function during mammalian infection.
Eukaryot Cell. 2007 Jul;6(7):1248-50.
We demonstrate that trypanosomes compromised in flagellar function are rapidly cleared from infected mice. Analysis of the PFR2 bloodstream RNA interference mutant revealed that defective cell motility occurred prior to cytokinesis failure. This validation provides a paradigm for the flagellum as a target for future assays and interventions against this human pathogen. [Abstract/Link to Full Text]

Ueno K, Uno J, Nakayama H, Sasamoto K, Mikami Y, Chibana H
Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata.
Eukaryot Cell. 2007 Jul;6(7):1239-47.
In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C. glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, H(2)O(2), UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified. [Abstract/Link to Full Text]

Mandel MA, Barker BM, Kroken S, Rounsley SD, Orbach MJ
Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition.
Eukaryot Cell. 2007 Jul;6(7):1189-99.
Coccidioides species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of Coccidioides, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes. Coccidioides strains contain either a MAT1-1 or a MAT1-2 idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the MAT loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the MAT loci are transcribed. Two genes frequently found in common sequences flanking MAT idiomorphs, APN2 and COX13, are within the MAT loci in Coccidioides, but the MAT1-1 and MAT1-2 copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the MAT loci occurred prior to the separation of Coccidioides from Uncinocarpus reesii. An analysis of 436 Coccidioides isolates from patients and the environment indicates that in both Coccidioides immitis and C. posadasii, there is a 1:1 distribution of MAT loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment. [Abstract/Link to Full Text]

Parsons M, Karnataki A, Feagin JE, DeRocher A
Protein trafficking to the apicoplast: deciphering the apicomplexan solution to secondary endosymbiosis.
Eukaryot Cell. 2007 Jul;6(7):1081-8. [Abstract/Link to Full Text]

Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe K, Niimi M, Uehara Y, Cannon RD
Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Jul;6(7):1150-65.
The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, ADDelta. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K(m) value and the V(max) value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening. [Abstract/Link to Full Text]

Recent Articles in The Journal of Biological Chemistry

Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S
Monoubiquitylation of alpha -synuclein by SIAH promotes its aggregation in dopaminergic cells.
J Biol Chem. 2007 Dec 10; .
a-Synuclein plays a major role in familial and sporadic Parkinson's disease. Unraveling the mechanisms that promote a-synuclein aggregation is essential to understand the formation of Lewy bodies and possibly the mechanisms involved in dopaminergic cell death. a-Synuclein is known to be ubiquitylated in Lewy bodies, but the role of a-synuclein ubiquitylation has been mysterious. We now report that the E3 ubiquitin-ligase SIAH directly interacts with and monoubiquitylates a-synuclein, and promotes its aggregation both in vitro and in vivo. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates a-synuclein at lysines 12, 21 and 23, which were previously shown to be ubiquitylated in Lewy bodies. Additionally, SIAH ubiquitylates lysines 10, 34, 43 and 96 as well. Suppression of SIAH expression by shRNA to SIAH-1 and SIAH-2 abolished a-synuclein monoubiquitylation in human dopaminergic cells, indicating that endogenous SIAH ubiquitylates a-synuclein. Moreover, SIAH co-immunoprecipitated with a-synuclein from brain extracts, indicating a physiological interaction. Inhibition of proteasomal, lysosomal and autophagic pathways, as well as overexpression of an ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of a-synuclein by SIAH, suggesting that different proteolytic pathways and deubiquitinases play a role in regulating a-synuclein monoubiquitylation. Additionally, monoubiquitylation increased the aggregation of a-synuclein in vitro as determined by Western blot and electron microscopy assays. At the electron microscopy level, monoubiquitylated a-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased a-synuclein aggregation in vivo as observed by the increased formation of a-synuclein inclusion bodies within dopaminergic cells. The formation of a-synuclein inclusions was prevented when endogenous SIAH expression was suppressed by shRNAs to SIAH-1 and SIAH-2. Furthermore, inclusions formed by monoubiquitylated a-synuclein were not protective to SH-SY5Y cells. Instead, they were toxic to some extent. Our data suggest that monoubiquitylation represents a possible trigger event for a-synuclein aggregation and Lewy body formation. [Abstract/Link to Full Text]

Bäumer AT, Ten Freyhaus H, Sauer H, Wartenberg M, Kappert K, Schnabel P, Konkol C, Hescheler J, Vantler M, Rosenkranz S
Pi3 kinase-dependent membrane recruitment of rac-1 and p47phox is critical for alpha PDGF receptor-induced production of reactive oxygen species.
J Biol Chem. 2007 Dec 10;
PDGF plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (NOX)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependency of PDGF-AA-induced ROS production on the cytosolic NOX subunits rac-1 and p47phox, and systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed NOX as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47phox and rac-1 from the cytosol to the cell membrane. Experiments performed in p47phox(-/-) cells and inhibition of rac-1 or overexpression of dominant-negative rac revealed that these NOX subunits are required for PDGF-dependent NOX activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs which lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, PI3K, PLCgamma, SHP-2). Lack of PI3K signaling (but not Src, PLCgamma, or SHP-2) completely abolished PDGF-dependent p47phox and rac-1 translocation, increase of NOX activity and ROS production. Conversely, a mutant alphaPDGFR capable to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha(but not p110beta)was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, BrdU incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis, but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47phox and rac-1 to the cell membrane, thereby assembling the active NOX complex. ROS are required for PDGF-AA-dependent chemotaxis, but not proliferation. [Abstract/Link to Full Text]

Kweon SM, Cho YJ, Minoo P, Groffen J, Heisterkamp N
Activity of Bcr GTPase activating domain is regulated through direct protein-protein interaction with RhoGDI.
J Biol Chem. 2007 Dec 10;
The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by G-nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and G-nucleotide dissociation inhibitors (GDIs). Little is known how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert RacGTP to RacGDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to RacGTP and catalyzes the conversion of bound GTP to GDP on Rac. The presence of activated Rac diminished the RhoGDIalpha-Bcr interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of the RacGTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, which indicates that binding of RhoGDIalpha and RacGTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates the Bcr GAP activity. [Abstract/Link to Full Text]

Jarboe LR, Hyduke DR, Tran LM, Chou KJ, Liao JC
Determination of the Escherichia coli S-nitrosoglutathione response network using integrated biochemical and systems analysis.
J Biol Chem. 2007 Dec 10;
During infection or denitrification, bacteria encounter reactive nitrogen species. Though the molecular targets of and defensive response against nitric oxide (NO) in E. coli are well studied, the response elements specific to Snitrosothiols are less clear. Previously, we employed an integrated systems biology approach to unravel the E. coli NO-response network. Here we use a similar approach to confirm that S-nitrosoglutathione (GSNO) primarily impacts E. coli's metabolic and regulatory programs in minimal medium by reaction with homocysteine and cysteine and subsequent disruption of the methionine biosynthesis pathway. Targeting of homocysteine and cysteine results in altered regulatory activity of MetJ, MetR and CysB, activation of the stringent response and growth inhibition. Deletion of metJ or supplementation with methionine strongly attenuated the effect of GSNO on growth and gene expression. Furthermore, GSNO inhibited the ArcAB twocomponent system. Consistent with the underlying nitrosative and thiol-oxidative chemistry, growth inhibition and the majority of the regulatory perturbations were dependent upon GSNO internalization by the Dpp dipeptide transporter. Contrastingly, perturbation of NsrR appeared to be a result of the submicromolar levels of NO released from GSNO and did not require GSNO internalization. [Abstract/Link to Full Text]

Tartari CJ, Gunby RH, Coluccia AM, Sottocornola R, Cimbro B, Scapozza L, Donella-Deana A, Pinna LA, Gambacorti-Passerini C
Characterization of some molecular mechanisms governing autoactivation of catalytic domain of the anaplastic lymphoma kinase.
J Biol Chem. 2007 Dec 10;
NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of Anaplastic Large Cell Lymphoma (ALCL) cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Here, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Since kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Y F) three potential autophosphorylation sites contained in the 'Y-x-x-x-Y-Y' motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant), did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the 'Y-x-x-x-Y-Y' motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK. [Abstract/Link to Full Text]

Edwin F, Patel TB
A novel role of sprouty 2 in regulating cellular apoptosis.
J Biol Chem. 2007 Dec 10;
Sprouty (SPRY1) proteins modulate receptor tyrosine kinase signaling and, thereby, regulate cell migration and proliferation. Here, we have examined the role of endogenous human SPRY2 (hSPRY2) in the regulation of cellular apoptosis. Small inhibitory RNA (siRNA)- mediated silencing of hSPRY2 abolished the anti-apoptotic action of serum in adrenal cortex adenocarcinoma (SW13) cells. Silencing of hSPRY2 decreased serum- or epidermal growth factor (EGF)- elicited activation of AKT and ERK1/2 and reduced the levels of EGF receptor. Silencing of hSPRY2 also inhibited serum induced activation of p90RSK and decreased phosphorylation of pro-apoptotic protein BAD by p90RSK. Inhibiting both the ERK1/2 and AKT pathways abolished the ability of serum to protect against apoptosis mimicking the effects of silencing hSPRY2. Serum transactivated the EGFR and inhibition of the EGFR by a neutralizing antibody attenuated the anti-apoptotic actions of serum. Consistent with the role of EGFR and perhaps other growth factor receptors in the anti-apoptotic actions of serum, the tyrosine kinase binding domain of c-Cbl (Cbl-TKB) protected against downregulation of the growth factor receptors such as EGFR and preserved the anti-apoptotic actions of serum when hSpry2 was silenced. Additionally, silencing of Spry2 in c-Cbl null cells did not alter the ability of serum to promote cell survival. Moreover, reintroduction of WT hSPRY2, but not its mutants that do not bind c-Cbl or CIN85, into SW13 cells after endogenous hSPRY2 had been silenced restored the anti-apoptotic actions of serum. Overall, we conclude that endogenous hSPRY2- mediated regulation of apoptosis requires c-Cbl and is manifested by the ability of hSPRY2 to sequester c-Cbl and thereby augment signaling via growth factor receptors. [Abstract/Link to Full Text]

Kimball SR, Do AN, Kutzler L, Cavener DR, Jefferson LS
Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis.
J Biol Chem. 2007 Dec 10;
mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein due to its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity. [Abstract/Link to Full Text]

Badugu R, Garcia M, Bondada V, Joshi A, Geddes JW
N-terminal of calpain 1 is a mitochondrial targeting sequence.
J Biol Chem. 2007 Dec 10;
The ubiquitous m- and micro-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, micro-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of micro-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form micro-calpain, are present in the mitochondrial intermembrane space. The N-terminal of calpain 1 contains an amphipathic a-helical domain, and is distinct from the N-terminal of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N-terminal of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The localization of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of micro-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease. [Abstract/Link to Full Text]

Piao W, Song C, Chen H, Wahl LM, Fitzgerald KA, O'Neill LA, Medvedev AE
Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance.
J Biol Chem. 2007 Dec 10;
Toll-like receptor (TLR) 4 recognition of lipopolysaccharide (LPS) triggers signalosome assembly among TLR4, sorting (e.g., MyD88-adapter-like, Mal) and signaling (e.g., MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors and production of inflammatory mediators. In this study, we examined whether tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, IRAK-2, TRAF-6 and is important for signaling. Overexpression of wild-type (WT) Mal in human embryonic kidney (HEK) 293T cells induced its constitutive tyrosine phosphorylation and led to activation of p38, NF-kappaB, and IL-8 gene expression. Mutagenesis of Y86, Y106, and Y159 tyrosine residues within the Toll-IL-1R (TIR) domain impaired Mal tyrosine phosphorylation, interactions with Bruton tyrosine kinase (Btk), phosphorylation of p38, and NF-kappaB activation. LPS triggered tyrosine phosphorylation of endogenous Mal and initiated Mal-Btk interactions in 293/TLR4/MD-2 cells and human monocytes that were suppressed in endotoxin-tolerant cells. Compared to WT-Mal, Y86A-, Y106A-, and Y159A-Mal variants exhibited higher interactions with TLR4 and MyD88, while associations with IL-1R-associated kinase (IRAK)-2 and TNFR-associated factor (TRAF)-6 were not affected. Overexpression of Y86A and Y106A Mal in 293/TLR4/MD-2 cells exerted dominant-negative effects on TLR4-inducible p38 phosphorylation and NF-kappaB reporter activation to the extent comparable to P125H Mal-mediated suppression. In contrast, tyrosine-deficient Mal species did not affect NF-kappaB activation when signaling was initiated at the post-receptor level by overexpression of MyD88, IRAK-2 or TRAF-6. Thus, tyrosine phosphorylation of Mal is required for adapter signaling, regulates Mal interactions with TLR4 and receptor signaling, and is inhibited in endotoxin tolerance. [Abstract/Link to Full Text]

Madsen L, Pedersen LM, Liaset B, Ma T, Petersen RK, van den Berg S, Pan J, Müller-Decker K, Dülsner ED, Kleemann R, Kooistra T, Dřskeland SO, Kristiansen K
cAMP-depending signaling regulates the adipogenic effect of N-6 polyunsaturated fatty acids.
J Biol Chem. 2007 Dec 10;
The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial as fundamentally opposing results both in vivo and in vitro have been reported. Using in vitro cell culture models we show that the adipogenic action of the n-6 PUFA arachidonic acid was dependent on the intracellular levels of cAMP. In conditions with baseline intracellular levels of cAMP, n-6 PUFAs acted pro-adipogenic, whereas n-6 PUFAs acted anti-adipogenic when the intracellular levels of cAMP were elevated. The anti-adipogenic action of n-6 PUFAs was dependent on a PKA-mediated induction of cyclooxygenase (COX) expression and activity. In vivo the intracellular levels of cAMP are modulated in response to dietary intake of different classes of macronutrients. Accordingly, we show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice. The high protein diet increased the glucagon/insulin ratio, leading to elevated cAMP-dependent signaling and induction of COX-mediated prostaglandin synthesis. Mice fed the high protein diet had a markedly lower feed efficiency than mice fed the high carbohydrate diet. Yet, oxygen consumption and apparent heat production were similar. Mice on a high protein diet had increased hepatic expression of PGC-1a and genes involved in energy demanding processes like urea synthesis and gluconeogenesis. We conclude that cAMP signaling is pivotal in regulating the adipogenic effect of n-6 PUFAs, and that diet-induced differences in cAMP levels can explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity. [Abstract/Link to Full Text]

Datta K, von Hippel PH
Direct spectroscopic study of reconstituted transcription complexes reveals that intrinsic termination is driven primarily by thermodynamic destabilization of the nucleic acid framework.
J Biol Chem. 2007 Dec 10;
Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine (2-AP) residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, while the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-AP probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency and disrupt the upstream end of the hybrid, as well as interfering with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently 'lock' the polymerase to the nucleic acid scaffold and thus hold the RNA-DNA hybrid 'in frame'. At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, while hairpin-dependent pausing provides time for the process to go to completion. [Abstract/Link to Full Text]

Smith AE, Kim SH, Liu F, Jia W, Vinogradov E, Gyles CL, Bishop RE
PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7.
J Biol Chem. 2007 Dec 10;
The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the R3 core oligosaccharide's first outer core glucose unit. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation, while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagP5-14 allele, which mostly eliminates a periplasmic amphipathic a-helical domain, but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane, but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier. [Abstract/Link to Full Text]

Bonente G, Howes BD, Caffarri S, Smulevich G, Bassi R
Interactions between the photosystem II subunit psbs and xanthophylls studied in vivo and in vitro.
J Biol Chem. 2007 Dec 10;
The photosystem II subunit PsbS is essential for excess energy dissipation (qE), however both lutein and zeaxanthin are needed for its full activation. Based on previous work two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton sensing trigger which activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS binding sites, each activated by the protonation of a DCCD (dicychlohexylcarbodiimide) binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1 npq4 and lut2 npq4 mutants lacking, respectively, zeaxanthin and lutein. Double mutants PsbS(E122Q)npq1 and PsbS(E226Q)npq1 had no qE, while PsbS(E122Q)lut2, and PsbS(E226Q)lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a DCCD binding site and suggest that the dependence of NPQ on xanthophyll composition is not due to pigment binding to PsbS. In order to verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment binding proteins, probably of the Lhcb type. [Abstract/Link to Full Text]

Worby CA, Gentry MS, Dixon JE
Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG).
J Biol Chem. 2007 Dec 10;
Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual-specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PTG/R5), a scaffold protein that binds both glycogen and many of the enzymes involved in glycogen synthesis, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG-stimulated glycogen accumulation by 25%, and co-expression of malin and laforin abolished PTG-stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin-dependent manner, both in vivo and in vitro, and targets PTG for proteasome-dependent degradation. These results suggest an additional mechanism, involving laforin and malin, in regulating glycogen metabolism. [Abstract/Link to Full Text]

Zheng Y, Boeglin WE, Schneider C, Brash AR
A 49 kD Mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality.
J Biol Chem. 2007 Dec 10;
Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (~49 kD) at the C-terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and a-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereo-specifically labeled [11-3H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C16/C18:2-phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala215), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R-specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:36, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the ss-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the ss-barrel domain was then acquired independently in the animal and plant kingdoms. [Abstract/Link to Full Text]

Abdulhussein R, Koo DH, Vogel WF
Identification of disulfide-linked dimers of the receptor tyrosine kinase DDR1.
J Biol Chem. 2007 Dec 7;
Discoidin Domain Receptor 1 (DDR1) is a transmembrane receptor tyrosine kinase activated by triple-helical collagen. So far 6 different isoforms of DDR1 have been described. Aberrant expression and signaling of DDR1 has been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and lung fibrosis. Here we show that DDR1 exists as a disulfide-linked dimer in transfected as well as endogenously expressing cells. This dimer formation occurred irrespective of its kinase domain, as dimers were also found for the truncated DDR1d isoform. A deletion analysis of the extracellular domain showed that DDR1 mutants lacking the stalk region failed to form dimers while deletion of the discoidin domain did not prevent dimerization. Point mutagenesis within the stalk region suggested that cysteines 303 and 348 are necessary for dimerization, collagen-binding and activation of kinase function. The identification of DDR1 dimers provides new insights into the molecular structure of receptor tyrosine kinases and suggests distinct signaling mechanisms of each receptor subfamily. [Abstract/Link to Full Text]

Tan K, Duquette M, Liu JH, Shanmugasundaram K, Joachimiak A, Gallagher JT, Rigby AC, Wang JH, Lawler J
Heparin-induced cis- and trans- dimerization modes of the thrombospondin-1 N-terminal domain.
J Biol Chem. 2007 Dec 7;
Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by X-ray crystallography. We have found that (1) dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra, and (2) dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42 and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhance the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1. [Abstract/Link to Full Text]

Poree B, Kypriotou M, Chadjichristos C, Beauchef G, Renard E, Legendre F, Melin M, Gueret S, Hartmann DJ, Mallein-Gerin F, Pujol JP, Boumediene K, Galera P
Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of SP1/SP3 ratio and of the binding activity of both factors to the COL2A1 promoter.
J Biol Chem. 2007 Dec 7;
Type II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. IL-6 is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R or both, inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-35 sequence that binds Sp1/Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression, and their binding activity to the 63-bp promoter. In ChIP experiments, IL-6/sIL-6R induce increase of Sp3 recruitment to the detriment of Sp1. Knock-down of Sp1/Sp3 by siRNA and decoy strategies were found to prevent the IL-6 and/or sIL-6R induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene, and that a heterotypic Sp1/Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R alone or in combination, decrease both the Sp1/Sp3 ratio and DNA binding activities to inhibit COL2A1 transcription. [Abstract/Link to Full Text]

Miller WL, Matewish MJ, McNally DJ, Ishiyama N, Anderson EM, Brewer D, Brisson JR, Berghuis AM, Lam JS
Flagellin glycosylation in pseudomonas aeruginosa pak requires the O-antigen biosynthesis enzyme WbpO.
J Biol Chem. 2007 Dec 7;
Pseudomonas aeruginosa PAK (serotype O6) produces a single, polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen deficient mutants wbpL, wbpO and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild-type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared to PAK flagellin (pI 4.6). These differences were due to the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Since WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP coupled-enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetyl-hexuronic acid as a substrate. [Abstract/Link to Full Text]

Molina-Cruz A, Dejong RJ, Charles B, Gupta L, Kumar S, Jaramillo-Gutierrez G, Barillas-Mury C
Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and plasmodium.
J Biol Chem. 2007 Dec 6;
The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive better a bacterial challenge, while reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body following a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a P. berghei-infected meal, indicating that the oxidative stress following a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by dsRNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity, and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism. [Abstract/Link to Full Text]

Robertson PD, Warren EM, Zhang H, Friedman DB, Lary JW, Cole JL, Tutter AV, Walter JC, Fanning E, Eichman BF
Domain architecture and biochemical characterization of vertebrate MCM10.
J Biol Chem. 2007 Dec 6;
Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed amino-terminal (NTD), internal (ID), and carboxy-terminal (CTD) structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the NTD forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- (ss) and double-stranded (ds) DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the amino-terminal 323 residues of the p180 subunit of DNA polymerase a-primase (pol a). We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and pol a, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome. [Abstract/Link to Full Text]

Tian W, Wijewickrama GT, Kim JH, Das S, Tun MP, Gokhale N, Jung JW, Kim KP, Cho W
Mechanism of regulation of group IVA phospholipase A2 Activity by Ser727 phosphorylation.
J Biol Chem. 2007 Dec 7;
Although group IVA phospholipase A2 (cPLA2a) has been reported to be phosphorylated at multiple Ser residues, the mechanisms by which phosphorylation at different sites regulates cPLA2a activities are not fully understood. To explore the possibility that phosphorylation of Ser727 modulates cellular protein-protein interactions, we measured the effect of Ser727 mutations on the interaction of cPLA2a with a reported cPLA2a-binding protein, p11. In vitro activity assays and membrane binding measurements by surface plasmon resonance analysis showed that a heterotetramer (A2t) of p11 and annexin A2, but not p11 or annexin A2 alone, directly binds cPLA2a via Ser727, which keeps the enzyme from binding the membrane and catalyzing the phospholipid hydrolysis. Phosphorylation of Ser727 disrupts this inhibitory cPLA2a-A2t interaction, thereby activating cPLA2a. Subcellular translocation and activity measurements in HEK293 cells cotransfected with cPLA2a and p11 also showed that p11, in the form of A2t, inhibits cPLA2a by the same mechanism and that phosphorylation of Ser727 activates cPLA2a by interfering with the inhibitory cPLA2a-A2t interaction. Collectively, these studies provide new insight into the regulatory mechanism of cPLA2a through Ser727 phosphorylation. [Abstract/Link to Full Text]

Tu CL, Chang W, Xie Z, Bikle DD
Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.
J Biol Chem. 2007 Dec 7;
Extracellular Ca(2)(+) (Ca(2)(+)(o)) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca(2)(+) signaling during Ca(2)(+)(o) -induced differentiation. Inactivation of the endogenous CaR gene by adenoviral expression of a CaR antisense cDNA inhibited the Ca(2)(+)(o)-induced increase in intracellular free calcium (Ca(2)(+)(i)) and expression of terminal differentiation genes, while promoting apoptosis. Ca(2)(+)(o) also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca(2)(+)(o) concentration ([Ca(2)(+)](o)) from 0.03 to 2 mM rapidly induced the co-localization of a-, ss-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca(2)(+)(o)-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca(2)(+)(o)-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of E-cadherin/catenin/PI3K complex was sensitive to the pharmacologic inhibition of Src-family tyrosine kinases, but was not affected by inhibition of Ca(2)(+)(o)-induced rise in Ca(2)(+)(i). Inhibition of CaR expression blocked the Ca(2)(+)(o)-induced tyrosine phosphorylation of ss-, - and p120-catenin, PI3K, and the tyrosine kinase Fyn, and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca(2)(+)(o)-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src-family tyrosine kinase-mediated signaling. [Abstract/Link to Full Text]

Chen SJ, Lin G, Chang KJ, Yeh LS, Wang CC
Translational efficiency of a non-AUG initiation codon is significantly affected by its sequence context in yeast.
J Biol Chem. 2007 Dec 7;
Previous studies have shown that translation of mRNA for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at position -3 to -1 relative to the initiation codon. A/A/R (R represents A or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. While an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (-3 to -1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. These results suggest that sequence context is more important for translation initiation in yeast than previously appreciated. [Abstract/Link to Full Text]

Snyder M, Huang XY, Zhang JJ
Identification of novel direct STAT3 target genes for control of growth and differentiation.
J Biol Chem. 2007 Dec 7;
Stat3 (Signal Transducer and Activator of Transcription 3) is a key regulator of gene expression in response to signaling of the gp130 family cytokines including interleukin 6 (IL-6), oncostatin M (OSM) and leukemia inhibitory factor (LIF). Many efforts have been made to identify Stat3 target genes and to understand the mechanism of how Stat3 regulates gene expression. Using the microarray technique, hundreds of genes have been documented to be potential Stat3 target genes in different cell types. However, only a small fraction of these genes have been proven to be true direct Stat3 target genes. Here, we report the identification of novel direct Stat3 target genes using a genome-wide screening procedure based on the Chromatin Immunoprecipitation (ChIP) method. These novel Stat3 target genes are involved in a diverse array of biological processes such as oncogenesis, cell growth and differentiation. We show that Stat3 can act as both a repressor and activator on its direct target genes. We further show that most of the novel Stat3 direct target genes are dependent on Stat3 for their transcriptional regulation. In addition, using a physiological cell system, we demonstrate that Stat3 is required for the transcriptional regulation of two of the newly identified direct Stat3 target genes important for muscle differentiation. [Abstract/Link to Full Text]

Sadikovic B, Andrews J, Carter D, Robinson J, Rodenhiser DI
Genome-wide H3K9 histone acetylation profiles are altered in benzopyrene treated MCF7 breast cancer cells.
J Biol Chem. 2007 Dec 7;
Current toxicogenomic approaches generate transcriptional profiles that can be used to develop gene expression signatures of environmental toxicants and address their mechanisms of action. It has become evident however, that the intricate processes governing gene transcription are overlaid with a complex set of molecular instructions involving epigenetic modifications. These commands regulate both gene expression and chromatin organization through coordinated sets of histone modifications and heritable DNA methylation patterns. While the effects of specific environmental toxicants on gene expression are the subject of much study, the epigenetic effects of such compounds are poorly understood. Here we have used human promoter tiling arrays in conjunction with chromatin immunoprecipitation to identify changes in histone acetylation profiles due to chemical exposure. Chromatin from cells exposed to an industrial pollutant, the polyaromatic hydrocarbon benzo(a)pyrene, was immunoprecipitated with antibodies against acetylated histones. Affymetrix promoter tiling microarrays were probed to generate epigenomic profiles of hypo- and hyperacetylated chromatin localized to gene promoter regions. Statistical analyses, data mining and expression studies revealed that treated cells possessed differentially acetylated gene promoter regions and gene-specific expression changes. This ChIP-on-chip approach permits genome-wide profiling of histone acetylation patterns due to chemical exposures that can be used to identify chromatin-related signatures of environmental toxicants and potentially determine the molecular pathways these changes target. This approach also has potential applications for profiling histone modifications and DNA methylation changes during embryonic development, in cancer biology and in the development and assessment of cancer therapeutics. [Abstract/Link to Full Text]

Leclerc D, Rozen R
Endoplasmic reticulum stress increases the expression of methylenetetrahydrofolate reductase through the IRE1 transducer.
J Biol Chem. 2007 Dec 7;
Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-B, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well-characterized stress inducers (tunicamycin, thapsigargin and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Since IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 (JNK1) reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival. [Abstract/Link to Full Text]

Knierim B, Hofmann KP, Gärtner W, Hubbell WL, Ernst OP
Rhodopsin and 9-demethyl retinal analog: Effect of a partial agonist on displacement of transmembrane helix 6 in class A GPCRs.
J Biol Chem. 2007 Dec 6;
Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis-configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling (SDSL). Earlier SDSL studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of helix TM6 by ca. 6 A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm-pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm-pigments. When the proton concentration is further increased, a new state arises in 9-dm-pigments which is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state. [Abstract/Link to Full Text]

Pauff JM, Zhang J, Bell CE, Hille R
Substrate orientation in xanthine oxidase, crystal structure with 2-hydroxy-6-methylpurine.
J Biol Chem. 2007 Dec 6;
Xanthine oxidoreductase catalyzes the final two steps of purine catabolism, and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzyme's flavin site, but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3 angstroms. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine, and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C6 position opposite Arg 880, and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg 880 in stabilizing the MoV transition state. [Abstract/Link to Full Text]

Hochbaum D, Hong K, Barila G, Ribeiro-Neto F, Altschuler DL
Epac, in synergy with PKA, is required for cAMP-mediated mitogenesis.
J Biol Chem. 2007 Dec 6;
cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA, are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for TSH or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and TSH-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP. [Abstract/Link to Full Text]

Recent Articles in Bioscience, Biotechnology, and Biochemistry

Asatiani MD, Elisashvili VI, Wasser SP, Reznick AZ, Nevo E
Free-Radical Scavenging Activity of Submerged Mycelium Extracts from Higher Basidiomycetes Mushrooms.
Biosci Biotechnol Biochem. 2007 Dec 7; .
Twenty-four Basidiomycetes strains were evaluated to determine there free-radical scavenging capacity in submerged cultivation. The scavenging capacity of the extracts varied from 1 to 85% depending on the mushroom species, solvent used, and concentration. A calculation of EC(50) of extracts from several wood-rotting basidiomycetes showed high scavenging abilities at low effective concentration. [Abstract/Link to Full Text]

Sugahara T, Yamauchi S, Kondo A, Ohno F, Tominaga S, Nakashima Y, Kishida T, Akiyama K, Maruyama M
First Stereoselective Synthesis of meso-Secoisolariciresinol and Comparison of Its Biological Activity with (+) and (-)-Secoisolariciresinol.
Biosci Biotechnol Biochem. 2007 Dec 7;
The first stereoselective synthesis of meso-secoisolariciresinol is reported. A comparison of the cytotoxic and immunosuppressive activity between meso-secoisolariciresinol and optically active secoisolariciresinols was similarly performed for the first time. Both enantiomers of secoisolariciresinol accelerated IgM production, although meso-secoisolariciresinol did not affect IgM production. Only meso-secoisolariciresinol showed cytotoxic activity. [Abstract/Link to Full Text]

Yun JW, Kim YK, Lee BS, Kim CW, Hyun JS, Baik JH, Kim JJ, Kim BH
Effect of Dietary Epigallocatechin-3-gallate on Cytochrome P450 2E1-Dependent Alcoholic Liver Damage: Enhancement of Fatty Acid Oxidation.
Biosci Biotechnol Biochem. 2007 Dec 7;
This study was designed to determine whether dietary epigallocatechin-3-gallate (EGCG), the most abundant catechin polyphenol in green tea, can protect the liver from cytochrome P450 2E1 (CYP2E1)-dependent alcoholic liver damage. Compared with an ethanol group, when EGCG was present in the ethanol diet, the formation of a fatty liver was significantly reduced and the serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were much lower. Ethanol treatment significantly elevated hepatic CYP2E1 expression while simultaneously reducing hepatic phospho-acetyl CoA carboxylase (p-ACC) and carnitine palmitoyl-transferase 1 (CPT-1) levels. While EGCG markedly reversed the effect of ethanol on hepatic p-ACC and CPT-1 levels, it had no effect on the ethanol-induced elevation in CYP2E1 expression. EGCG prevents ethanol-induced hepatotoxicity and inhibits the development of a fatty liver. These effects were associated with improvements in p-ACC and CPT-1 levels. The use of EGCG might be useful in treating patients with an alcoholic fatty liver. [Abstract/Link to Full Text]

Nakaya M, Oguri H, Takahashi K, Fukushi E, Watanabe K, Oikawa H
Relative and Absolute Configuration of Antitumor Agent SW-163D.
Biosci Biotechnol Biochem. 2007 Dec 7;
Our interest on engineering non-ribosomal synthetase responsible for SW-163 biosynthesis prompted us to determine the relative and absolute configuration of antitumor cyclic depsipeptide SW-163s. We first isolated and identified SW-163 homologs D, F and G as known compounds UK-63598, UK-65662 and UK-63052, respectively. Both enantiomers of the unusual constitutive amino acid, N-methylnorcoromic acid, were synthesized in chiral forms starting from (R)- and (S)-1,2-propanediol. The hydrolyzate of SW-163D, a major constituent of this family, was converted with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA), and the resulting mixture of amino acid derivatives was subjected to an LC/MS analysis. Compared with authentic samples, the analytical data unambiguously show that SW-163D consisted of L-Ala, D-Ser and (1S, 2S)-N-methylnorcoronamic acid. The remaining stereochemistry of the N-methylcysteine moieties was determined from NOE data. [Abstract/Link to Full Text]

López-Mirabal HR, Winther JR, Kielland-Brandt MC
Genetic Interaction between the ero1-1 and leu2 Mutations in Saccharomyces cerevisiae.
Biosci Biotechnol Biochem. 2007 Dec 7;
The conditional ero1-1 mutant, deficient in the ER-localized PDI oxidase Ero1p, is blocked in disulfide bond formation under restrictive conditions, such as high temperature, lack of oxygen, or high concentrations of membrane-permeant thiols. Previous studies of the physiological consequences of the ero1-1 mutation were carried out in a leu2 mutant. The ero1-1 leu2 strain does not grow in standard synthetic complete medium at 30 degrees C, a defect that can be remedied by increasing the L-leucine concentration in the medium or by transforming the ero1-1 leu2 strain with the LEU2 wild-type allele. In addition, the LEU2 gene can partially complement the growth impairment at 37 degrees C of the ero1-1 leu2 mutant. The leucine transporter Bap2p exhibits a dramatic decrease in stability in an ero1-1 strain, which may account for the pronounced leucine demand observed in the ero1-1 leu2 mutant. [Abstract/Link to Full Text]

Hayashi Y, Onaka H, Itoh N, Seto H, Dairi T
Cloning of the Gene Cluster Responsible for Biosynthesis of KS-505a (Longestin), a Unique Tetraterpenoid.
Biosci Biotechnol Biochem. 2007 Dec 7;
KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid. [Abstract/Link to Full Text]

Mori H, Iwahashi H
Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation.
Biosci Biotechnol Biochem. 2007 Dec 7;
To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 muM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.). [Abstract/Link to Full Text]

Sawamura H, Fukuwatari T, Shibata K
Effects of Excess Biotin Administration on the Growth and Urinary Excretion of Water-Soluble Vitamins in Young Rats.
Biosci Biotechnol Biochem. 2007 Dec 7;
To determine the effects of excess biotin administration on growth and water-soluble vitamin metabolism, weaning rats were fed on a 20% casein diet containing 0.00002% biotin, or same diet with 0.04, 0.08, 0.10, 0.20, 0.50, 0.80 or 1.0% added biotin for 28 days. More than 0.08% biotin administration decreased the food intake and body weight gain compared with the levels in control rats. An accumulation of biotin in such tissues as the liver, brain and kidney increased in a dose-dependent manner, and the both bound and free biotin contents in the liver also increased in a dose-dependent manner. An excess administration of biotin did not affect the urinary excretion of other water-soluble vitamins, suggesting no effect on the metabolism of other water-soluble vitamins. The results of the food intake and body weight gain indicated that the lowest observed adverse effect level for young rats was 79.2 mg/kg body weight/day, while the no observed adverse effect level was 38.4 mg/kg/day. These results suggested immediately setting a tolerable upper intake level for biotin. [Abstract/Link to Full Text]

Lee SH, Kim YH, Yu HJ, Cho NS, Kim TH, Kim DC, Chung CB, Hwang YI, Kim KH
Enhanced Bioavailability Soy Isoflavones by Complexation with beta-Cyclodextrin in Rats.
Biosci Biotechnol Biochem. 2007 Dec 7;
In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 mug.min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 mug.min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE (p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD). [Abstract/Link to Full Text]

Kim EK, Kim EY, Moon PD, Um JY, Kim HM, Lee HS, Sohn Y, Park SK, Jung HS, Sohn NW
Lithospermi radix Extract Inhibits Histamine Release and Production of Inflammatory Cytokine in Mast Cells.
Biosci Biotechnol Biochem. 2007 Dec 7;
Lithospermi radix (LR, Borraginaceae, the root of Lithospermum erythrorhizon Siebold. et Zuccarinii) is used in herbal medicine to treat such conditions as eczema, skin burns and frostbite. This study investigates the effects of LR on the anti-allergy mechanism. LR inhibited the release of histamine from rat peritoneal mast cells by compound 48/80 in a dose-dependent manner. LR orally administered at 6.59 mg/100 g also inhibited the anti-DNP IgE-induced passive cutaneous anaphylaxis reaction. LR inhibited the PMA plus A23187-induced increase in IL-6, IL-8, and TNF-alpha expression in HMC-1 cells. In addition, LR also inhibited nuclear factor-kappa B (NF-kappaB) activation and IkappaB-alpha degradation. These results show that LR had an inhibitory effect on the atopic allergic reaction. Furthermore, the in vivo and in vitro anti-allergic effect of LR suggests possible therapeutic applications of this agent for inflammatory allergic diseases. [Abstract/Link to Full Text]

Kumada HO, Koizumi Y, Sekiya J
Purification and Characterization of Dipeptidase Hydrolyzing L-Cysteinylglycine from Radish Cotyledon.
Biosci Biotechnol Biochem. 2007 Dec 7;
Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.3 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants. [Abstract/Link to Full Text]

Kato S, Shimizu-Ibuka A, Mura K, Takeuchi A, Tokue C, Arai S
Molecular Cloning and Characterization of an alpha-Amylase from Pichia burtonii 15-1.
Biosci Biotechnol Biochem. 2007 Dec 7;
An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher. [Abstract/Link to Full Text]

Ichikawa S, Fujii R, Fujiwara D, Komiyama Y, Kaisho T, Sakaguchi M, Konishi Y
MyD88 but Not TLR2, 4 or 9 Is Essential for IL-12 Induction by Lactic Acid Bacteria.
Biosci Biotechnol Biochem. 2007 Dec 7;
Although lactic acid bacteria (LAB) affect the immune system, for example, having an anti-allergic effect, little is known about the actual mechanisms of immune modulation. Toll-like receptors (TLRs) recognize conserved microbial molecular patterns, and are presumed to be involved in the recognition of LAB. However, there are few detailed reports examining the relationships between TLR and LAB. We measured here production of IL-12, a cytokine considered to play an important role in anti-allergic effects, induced by Lactobacillus paracasei strain KW3110 and other typical LAB by cells from TLR2-, TLR4-, TLR9- and myeloid differentiation factor 88 (MyD88)-deficient mice. Unexpectedly, similar cytokine production from wild-type and TLR2-, 4- and 9-deficient mice was observed. In contrast, cells from MyD88-deficient mice failed to respond to stimulation with LAB. It is therefore concluded that although LAB, including strain KW3110, are not likely to be recognized by TLR2, 4 or 9, MyD88 is essential for the response to these bacteria. [Abstract/Link to Full Text]

Kubota T, Shimono J, Kanameda C, Izumi Y
The First Thermophilic alpha-Oxoamine Synthase Family Enzyme That Has Activities of 2-Amino-3-ketobutyrate CoA Ligase and 7-Keto-8-aminopelargonic Acid Synthase: Cloning and Overexpression of the Gene from an Extreme Thermophile, Thermus thermophilus, and Characterization of Its Gene Product.
Biosci Biotechnol Biochem. 2007 Dec 7;
The first thermophilic alpha-oxoamine synthase family enzyme was identified. The gene (ORF TTHA1582), which is annotated to code putative alpha-oxoamine synthase family enzymes, 7-keto-8-aminopelargonic acid (KAPA) synthase (BioF, 8-amino-7-oxononanoate synthase, EC and 2-amino-3-ketobutyrate CoA ligase (KBL, EC, in a genomic database, was cloned from an extreme thermophile, Thermus thermophilus, and overexpressed in Escherichia coli. The recombinant TTHA1582 protein was purified and characterized. It exhibited activity of BioF, which catalyzes the condensation of pimeloyl-CoA and L-alanine to produce a biotin intermediate KAPA, CoASH, and CO(2) with pyridoxal 5'-phosphate as a cofactor. The protein is a dimer with a subunit of 43 kDa that shows an amino acid sequence identity of 35% with E. coli BioF. The optimum temperature and pH were about 70 degrees C and about 6.0. The enzyme showed high thermostability at temperatures of up to 70 degrees C for 1 h, and a half-life of 1 h at 80 degrees C. Thus the TTHA1582 protein was found to have the highest optimum temperature and thermostablility of the alpha-oxoamine synthase family enzymes so far reported. Substrate specificity experiments revealed that it was also able to catalyze the KBL reaction, which used acetyl-CoA and glycine as substrates, and that enzyme activity was seen with the following combinations of substrates: acetyl-CoA and glycine, L-alanine, or L-serine; pimeloyl-CoA and L-alanine, glycine, or L-serine; palmitoyl-CoA and L-alanine. This suggests that the recombinant TTHA1582 protein has broad substrate specificity, unlike the reported mesophilic enzymes of the alpha-oxoamine synthase family. [Abstract/Link to Full Text]

Mizoguchi H, Tanaka-Masuda K, Mori H
A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome.
Biosci Biotechnol Biochem. 2007 Dec 7;
We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by lambda Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome. [Abstract/Link to Full Text]

Kim C, Lee IH, Lee K, Ryu SS, Lee SH, Lee KJ, Lee J, Kang JY, Kim TS
Multi-Well Chip for Forming a Uniform Embryoid Body in a Tiny Droplet with Mouse Embryonic Stem Cells.
Biosci Biotechnol Biochem. 2007 Dec 7;
A multi-well chip (MWC) is described by which mouse embryonic carcinoma (EC) stem cells form a comparatively more rapid and uniform embryoid body (EB) over the conventional hanging drop (HD) method. The newly developed MWC consists of an array of extruded through-holes, each of which holds a droplet of the cell suspension. The study found that the small curvature radius of the droplet in the MWC improved the EB formation rate of a hanging drop from 70% to 98%. Furthermore, the EBs formed by the MWC were uniformly round in shape regardless of the number of suspended cells ranging from 0.5x10(3) to 20x10(3). The ratio of beating colonies from the MWC was over 2-fold larger than that from HD. The experiments demonstrate that the MWC will be a valuable experimental tool for robust and reproducible EB-based differentiation of a defined number of ES cells. [Abstract/Link to Full Text]

Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Tamada Y
A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm.
Biosci Biotechnol Biochem. 2007 Dec 7;
We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm. [Abstract/Link to Full Text]

Sobajima H, Tani T, Chujo T, Okada K, Suzuki K, Mori S, Minami E, Nishiyama M, Nojiri H, Yamane H
Identification of a Jasmonic Acid-Responsive Region in the Promoter of the Rice 12-Oxophytodienoic Acid Reductase 1 Gene OsOPR1.
Biosci Biotechnol Biochem. 2007 Dec 7;
The rice 12-oxophytodienoic acid reductase 1 gene (OsOPR1), isolated as a jasmonic acid (JA)-responsive gene, has been suggested to be involved in defense responses in rice. We identified a 19-base pair region that is essential to the JA-responsiveness of OsOPR1 by deletion and mutation analysis of the promoter by dual luciferase assay. This region contains possible recognition sites for basic leucine zipper transcription factors. [Abstract/Link to Full Text]

Yunoki K, Hirose S, Ohnishi M
Ethyl Esterification of Long-Chain Unsaturated Fatty Acids Derived from Grape Must by Yeast during Alcoholic Fermentation.
Biosci Biotechnol Biochem. 2007 Dec 7;
The composition of total fatty acid ethyl ester (FAEE) in yeast cells and the liquid phase separated from grape must during alcoholic fermentation at different temperatures was investigated by using the solid-phase extraction method. Thirteen FAEE from butyric to linolenic acids were detected during fermentation. Significant amounts of long-chain unsaturated FAEE, including linoleic and linolenic acids derived from grape material, had already accumulated in the yeast cells by day 3 during fermentation. [Abstract/Link to Full Text]

Takeda M, Miyanoiri Y, Nogami T, Oda K, Saito T, Kato K, Koizumi JI, Katahira M
Structural Analysis of the Fundamental Polymer of the Sheath Constructed by Sphaerotilus natans.
Biosci Biotechnol Biochem. 2007 Dec 7;
The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing beta-D-GlcA, beta-D-Glc, alpha-D-GalN, and beta-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine. [Abstract/Link to Full Text]

Harada A, Yagi H, Saito A, Azakami H, Kato A
Relationship between the Stability of Hen Egg-White Lysozymes Mutated at Sites Designed to Interact with alpha-Helix Dipoles and Their Secretion Amounts in Yeast.
Biosci Biotechnol Biochem. 2007 Dec 7;
The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change DeltaG of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and DeltaG of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability. [Abstract/Link to Full Text]

Dejima K, Ohshima A, Yanai T, Yamamoto R, Takata R, Yoshikawa T
Effects of Polysaccharide Derived from Black Currant on Relieving Clinical Symptoms of Japanese Cedar Pollinosis: A Randomized Double-Blind, Placebo-Controlled Trial.
Biosci Biotechnol Biochem. 2007 Dec 7;
We investigated the efficacy of the polysaccharide derived from black currant, named cassis polysaccharide (CAPS), for inhibiting Japanese cedar pollinosis symptoms and improving quality of life by a randomized double-blind, placebo-controlled trial in 2006. A total of 28 subjects were enrolled in the study, and 10 subjects in each group completed the trial. Although there was no significant difference between the CAPS and placebo group in the weekly mean value of any symptom in the daily symptom diary at any time, a smaller degree of final symptom aggravation was found in the CAPS group. Significant aggravation of the score was finally observed in the placebo group with inferior conch swelling and with sneezing, itchy nose, itchy eye and watery eye in the Japan rhino-conjunctivitis quality of life questionnaire assessment, while the changes observed in the CAPS group were not significant. In conclusion, our findings clearly indicate that CAPS would be useful as a food supplement in assisting the treatment of Japanese cedar pollinosis. [Abstract/Link to Full Text]

Tanaka R
New Polar Constituents of the Pupae of the Silkworm Bombyx mori L. I. Isolation and Identification of Methionine Sulfoxide, Methionine Sulfone, and gamma-Cyclic di-L-Glutamate.
Biosci Biotechnol Biochem. 2007 Dec 7;
In addition to serine (L:D = 68:32), methionine sulfoxide (MSO), L-methionine sulfone (L-MSO(2)), and disodium gamma-cyclic di-L-glutamate were identified in a methanol extract of Bombyx mori L. pupae. MSO was isolated in a diastereomeric mixture of L(+)- and D(+)-MSO in a ratio of 99:1. The presence of these compounds in other developmental stages, including eggs, larvae (1st, 4th, 5th, and mature 5th instar), adults, and excrement (feces and urine) was investigated. The L(+)-isomer of MSO was present in extracts of the 1st and 5th instar larvae, adults, and eggs, but was not detected in feces or urine. The D(+)-isomer was found only in pupal stage extracts, and was excreted into the meconium with L(+)-isomer. L-MSO(2) and gamma-cyclic di-L-glutamate were not detected at other insect life stages or in the insect excrement. gamma-Cyclic di-L-glutamate is thought be produced due to blockage of the glutamate synthetic pathway (glutamine synthetase) by L-MSO(2) and Mg(2+). The biochemical role of L-MSO(2) during the pupal life stage remains unknown, but importantly, the stage-specific expression suggests that it is a candidate molecule for the induction of diapause. [Abstract/Link to Full Text]

Son IS, Kim JH, Sohn HY, Son KH, Kim JS, Kwon CS
Antioxidative and Hypolipidemic Effects of Diosgenin, a Steroidal Saponin of Yam (Dioscorea spp.), on High-Cholesterol Fed Rats.
Biosci Biotechnol Biochem. 2007 Dec 7;
Diosgenin (a steroidal saponin of yam) has long been used as a raw material for the industrial production of steroid drugs, and reported to have a hypocholesterolemic effect by suppressing cholesterol absorption and increasing cholesterol secretion. Oxidative stress has been suggested as a main risk factor in the development of atherosclerosis. The aim of this study is to investigate the possible hypolipidemic and antioxidative effect of diosgenin on rats fed with a high-cholesterol diet supplemented with either 0.1% or 0.5% diosgenin for 6 weeks. We measured the lipid profile in the plasma and liver, lipid peroxidation and antioxidative enzyme activities in the plasma, erythrocyte and gene expression of antioxidative enzymes in the liver, and the oxidative DNA damage in lymphocytes. Diosgenin showed a decrease in the plasma and hepatic total cholesterol levels, but increased the plasma high-density lipoprotein (HDL) cholesterol level. Erythrocyte TBARS and lymphocyte DNA damage measured by the comet assay were decreased in the diosgenin supplemented group. Furthermore, diosgenin feeding enhanced the resistance to lymphocyte DNA damage caused by an oxidant challenge with H(2)O(2). The antioxidative enzyme activities were also affected by diosgenin supplementation. Total superoxide dismutase (SOD) in the plasma and liver, glutathione peroxidase (GSH-Px) in erythrocytes, and catalase (CAT) in erythrocytes and liver were significantly increased in the 0.5% diosgenin group. The expression of antioxidative enzymes was up-regulated by diosgenin, the expression of GSH-Px being the highest in the 0.5% diosgenin group. These results suggest that diosgenin could be a very useful compound to control hypercholesterolemia by both improving the lipid profile and modulating oxidative stress. [Abstract/Link to Full Text]

Fujita Y, Mita S, Ohtsuka H, Aiba H
Identification of a Fatty Acyl-CoA Synthetase Gene, lcf2(+), Which Affects Viability after Entry into the Stationary Phase in Schizosaccharomyces pombe.
Biosci Biotechnol Biochem. 2007 Dec 7;
The lcf1(+) gene, which encodes a long chain fatty acyl-CoA synthetase, is necessary for the maintenance of viability after entry into the stationary phase in Schizosaccharomyces pombe. In this study, we analyzed a paralogous gene, SPBP4H10.11c (named lcf2(+)), and we present evidence that the gene encodes a new fatty acyl-CoA synthetase. The enzyme preferentially recognized myristic acid as a substrate. A Deltalcf2 mutant showed increased viability after entry into the stationary phase in SD medium. A Deltalcf1Deltalcf2 double mutant showed a severe decrease in long-chain fatty acyl-CoA synthetase activity and a rapid loss of viability after entry into the stationary phase. These results suggest that fatty acid utilization and/or metabolism is important to determine viability in the stationary phase. [Abstract/Link to Full Text]

Shigematsu T, Ueno S, Tsuchida Y, Hayashi M, Okonogi H, Masaki H, Fujii T
Comparative Analyses of Viable Bacterial Counts in Foods and Seawater under Microplate Based Liquid- and Conventional Agar Plate Cultivation: Increased Culturability of Marine Bacteria under Liquid Cultivation.
Biosci Biotechnol Biochem. 2007 Dec 7;
Bacterial counts under liquid cultivation using 96-well microplates were performed. The counts under liquid and under solid cultivation were equivalent in foods, although the counts under liquid cultivation exceeded those under solid cultivation in seawater, suggesting that some bacteria in seawater were viable but did not form detectable colonies. Phylogenetic analysis of bacteria obtained under liquid cultivation was also performed. [Abstract/Link to Full Text]

Gullapalli P, Takata G, Poonperm W, Rao D, Morimoto K, Akimitsu K, Tajima S, Izumori K
Bioproduction of D-Psicose from Allitol with Enterobacter aerogenes IK7: A New Frontier in Rare Ketose Production.
Biosci Biotechnol Biochem. 2007 Dec 7;
D-Psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 degrees C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods. [Abstract/Link to Full Text]

Tsukagoshi T, Tokiwano T, Oikawa H
Studies on the Later Stage of the Biosynthesis of Pleuromutilin.
Biosci Biotechnol Biochem. 2007 Dec 7;
Mutilin (4) and deoxy analogues 2 and 3 are biosynthetic precursors of pleuromutilin (1) in the later stage of biosynthesis. Precursors 2 and 3 are required for studies on the oxygenation steps in biosynthesis, and were synthesized from readily available 1 via 4 by deoxygenation of the hydroxy groups. Feeding experiments with the (2)H-labeled precursors confirmed their microbial conversion into 1. [Abstract/Link to Full Text]

Matsui K, Kawaji I, Utsumi Y, Ukita Y, Asano T, Takeo M, Kato DI, Negoro S
Immunoassay Using Microfluid Filters Constructed by Deep X-Ray Lithography.
Biosci Biotechnol Biochem. 2007 Dec 7;
Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi40 mum) in 200 mum-thickness polymethylmethacrylate (PMMA) sheets (psi3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunogloblin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay. [Abstract/Link to Full Text]

Takata G, Poonperm W, Rao D, Souda A, Nishizaki T, Morimoto K, Izumori K
Cloning, Expression, and Transcription Analysis of L-Arabinose Isomerase Gene from Mycobacterium smegmatis SMDU.
Biosci Biotechnol Biochem. 2007 Dec 7;
The L-arabinose metabolic gene cluster, araA, araB, araD, araG, araH and araR, encoding L-arabinose isomerase (L-AI) and its accessory proteins was cloned from Mycobacterium smegmatis SMDU and sequenced. The deduced amino acid sequence of araA displayed highest identity with that of Bacillus subtilis (52%). These six genes comprised the L-arabinose operon, and its genetic arrangement was similar to that of B. subtilis. The L-AI gene (araA), encoding a 501 amino acid protein with a calculated molecular mass of 54,888 Da, was expressed in Escherichia coli. The productivity and overall enzymatic properties of the recombinant L-AI were almost same as the authentic L-AI from M. smegmatis. Although the recombinant L-AI showed high substrate specificity, as did L-AI from other organisms, this enzyme catalyzed not only isomerization of L-arabinose-L-ribulose and D-galactose-D-tagatose but also isomerization of L-altrose-L-psicose and L-erythrulose-L-threose. In combination with L-AI from M. smegmatis, L-threose and L-altrose can be produced from cheap and abundant erythritol and D-fructose respectively, indicating that this enzyme has great potential for biological application in rare sugar production. Transcription analysis using various sugars revealed that this enzyme was significantly induced not only by L-arabinose and D-galactose but also by L-ribose, galactitol, L-ribulose, and L-talitol. This different result of transcription mediated by sugars from that of E. coli suggests that the transcriptional regulation of araA from M. smegmatis against sugar is loose compared with that from E. coli, and that it depends on the hydroxyl configuration at C2, C3 and C4 positions of sugars. [Abstract/Link to Full Text]

Recent Articles in Experimental & Molecular Medicine

Park KH, Kim JS, Lee YR, Moon YJ, Hur H, Choi YH, Kim CH, Kim UH, Song EK, Yoo WH, Lee CS, Kim BS, Lee SH, Ryu PY, Han MK
Low-density lipoprotein protects Vibrio vulnificus-induced lethality through blocking lipopolysaccharide action.
Exp Mol Med. 2007 Oct 31;39(5):673-8.
Lipoprotein plays a role in the host defense against bacterial infection, and its serum level has been demonstrated to be an important prognosis factor of survival. We have previously demonstrated that LDL directly inactivates the hemolytic activity of Vibrio vulnificus cytolysin (VVC) in vitro. The object of this study was therefore to examine whether the LDL-mediated inactivation of VVC leads to protection against lethal infection of V. vulnificus in vivo, using wild and VVC-deficient V. vulnificus strains. Unexpectedly, we found that LDL protects mouse lethality induced by VVC-deficient as well as wild V. vulnificus strain. We also demonstrated that LDL blocks V. vulnificus LPS-induced lethality in mice. These results suggest that LDL preferentially act on endotoxin rather than exotoxin in the protection against V. vulnificus-induced mice lethality. [Abstract/Link to Full Text]

Jung MY, Thapa N, Kim JE, Yang JD, Cho BC, Kim IS
Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing.
Exp Mol Med. 2007 Oct 31;39(5):663-72.
An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9(th) and 10(th) type III domains of fibronectin, and 4(th) FAS1 domain of betaig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9(th) and 10(th) type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4(th) FAS1 domain of betaig-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4(th) FAS1 domain of betaig-h3 or 9(th) and 10(th) type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug. [Abstract/Link to Full Text]

Yoon J, Choi SC, Park CY, Shim WJ, Lim do S
Cardiac side population cells exhibit endothelial differentiation potential.
Exp Mol Med. 2007 Oct 31;39(5):653-62.
Recent studies have shown that side population (SP) cells, isolated from adult myocardium, represent a distinct cardiac progenitor cell population that exhibits functional cardiomyogenic differentiation. However, information on the intrinsic characteristics and endothelial potential, of cardiac SP cells, is limited. The present study was designed to investigate whether cardiac SP cells exhibit endothelial differentiation potential. The cardiac SP cells more highly expressed the early cardiac transcription factors as well as endothelial cell markers compared to the bone marrow-SP cells. After treatment with VEGF, for 28 days, cardiac SP cells were able to differentiate into endothelial cells expressing von Willebrand factor as determined by immunocytochemistry. Furthermore, expression of endothelial cell markers increased several-fold in VEGF-treated cardiac SP cells compared to the control group when assessed by real-time PCR. We also confirmed that cardiac SP cells provided a significantly augmented ratio of ischemic/normal blood flow, in the cardiac SP cell-transplanted group compared with saline-treated controls on postoperative days 7, 14, 21 and 28, in a murine model. These results show that cardiac SP cells may contribute to regeneration of injured heart tissues partly by transdifferentiation into angiogenic lineages. [Abstract/Link to Full Text]

Yu GR, Kim SH, Park SH, Cui XD, Xu DY, Yu HC, Cho BH, Yeom YI, Kim SS, Kim SB, Chu IS, Kim DG
Identification of molecular markers for the oncogenic differentiation of hepatocellular carcinoma.
Exp Mol Med. 2007 Oct 31;39(5):641-52.
The aim of this study was to identify molecular markers associated with oncogenic differentiation in hepatocellular carcinoma (HCC). Using an unsupervised clustering method with a cDNA microarray, HCC (T) gene expression profiles and corresponding non-tumor tissues (NT) from 40 patients were analyzed. Of total 217 genes, 72 were expressed preferentially in HCC tissues. Among 186 differentially regulated genes, there were molecular chaperone and tumor suppressor gene clusters in the Edmondson grades I and II (GI/II) subclass compared with the liver cirrhosis (LC) subclass. The Edmondson grades III and IV (GIII/IV) subclass with a poor survival (P=0.0133) contained 122 differentially regulated genes with a cluster containing various metastasis- and invasion-related genes compared with the GI/II subclass. Immunohistochemical analysis revealed that ANXA2, one of the 72 genes preferentially expressed in HCC, was over-expressed in the sinusoidal endothelium and in malignant hepatocytes in HCC. The genes identified in the HCC subclasses will be useful molecular markers for the genesis and progression of HCC. In addition, ANXA2 might be a novel marker for tumor angiogenesis in HCC. [Abstract/Link to Full Text]

Park SJ, Lee KS, Kim SR, Min KH, Lee KY, Choe YH, Park SY, Hong SH, Lee YC
Change of connexin 37 in allergen-induced airway inflammation.
Exp Mol Med. 2007 Oct 31;39(5):629-40.
Gap junction channels formed with connexins directly link to the cytoplasm of adjacent cells and have been implicated in intercellular signaling. Connexin 37 (Cx37) is expressed in the gas-exchange region of the lung. Recently, Cx37 has been reported to be involved in the pathogenesis of inflammatory disease. However, no data are available on the role of Cx37 in allergic airway inflammatory disease. In the present study, we used a murine model of ovalbumin (OVA)- induced allergic airway disease and primary murine epithelial cells to examine the change of Cx37 in allergic airway disease. These mice develop the following typical pathophysiological features of asthma: airway hyperresponsiveness, airway inflammation, and increased IL-4, IL-5, IL-13, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, eotaxin, and RANTES levels in lungs. Cx37 protein and mRNA expression were decreased in OVA-induced allergic airway disease. Immunoreactive Cx37 localized in epithelial layers around the bronchioles in control mice, which dramatically disappeared in allergen-induced asthmatic lungs. Moreover, the levels of Cx37 protein in lung tissues showed significantly negative correlations with airway inflammation, airway responsiveness, and levels of Th2 cytokines in lungs. These findings indicate that change of Cx37 may be associated with the asthma phenotype. [Abstract/Link to Full Text]

Jeon JH, Shin DM, Cho SY, Song KY, Park NH, Kang HS, Kim YD, Kim IG
Immunocytochemical detection of HPV16 E7 in cervical smear.
Exp Mol Med. 2007 Oct 31;39(5):621-8.
Cervical cancer is characterized by a long period of preclinical dysplasia or carcinoma in situ progressing into invasive cancer. Although Papanicolaou (Pap) smear test has contributed significantly to the early detection of precursor lesions, the cytological screening has inherent problems that produce considerable false negative/positive results. Since the infection of high-risk type of human papillomavirus (HPV) is strongly associated with cervical cancer, we investigated the feasibility of an immunostaining test to detect cells infected by HPV in cervical smear. We produced monoclonal antibodies against HPV16 E7 in mice by repeated injections with the recombinant HPV16 E7. Western blot analysis and immunocytochemical assay demonstrated that the selected monoclonal antibody, mAb (130-9-7), reacts specifically with cultured cervical cancer cell lines infected by HPV16. Specific staining was observable with the HPV16-positive smear specimens obtained from the cervical cancer patients, whereas no staining was detected with the HPV-negative smear specimens. To achieve the desired sensitivity, specificity and reproducibility, we modified and optimized the conventional immunocytochemical procedure for cervical smear specimens. Our results suggest that this immunostaining method for detecting high-risk HPV in cervical smear may be used as a strategy to distinguish a high-risk group, especially those patients with low grade cytological abnormality. [Abstract/Link to Full Text]

Gamze K, Mehmet HM, Deveci F, Turgut T, Ilhan F, Ozercan I
Effect of bosentan on the production of proinflammatory cytokines in a rat model of emphysema.
Exp Mol Med. 2007 Oct 31;39(5):614-20.
Endothelin (ET) receptor antagonists have been developed to produce a reduction of ET related effects in various diseases, as well as in animal models of airway inflammation. We aimed to investigate the anti-inflammatory potential of bosentan on a rat model of emphysema. Thirty Wistar male rats were classified as control group (group 1), intratracheally (i.t.) instilled with saline, treated with vehicle solution; elastase group (group 2), i.t. instilled with porcine pancreatic elastase (PPE), treated with vehicle solution; and PPE+bosentan group (group 3), i.t. instilled with PPE, treated with bosentan. The levels of TNF-alpha, IL-1beta, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) and lung tissue, cell counts in BALF, and histologic analysis of all groups were evaluated. Neutrophile granulocytes (NG) and alveolar macrophages (AM) were increased more in group 2 than in group 1 (P<0.001, P=0.04, respectively). Compared with group 2, neutrophil granulocyte (NG) and alveolar macrophages (AM) counts were decreased in group 3 (P<0.001). Histological examination confirmed a diffuse neutrophilic inflammation and irregular alveolar air space enlargement in group 2. Treatment with bosentan partially reduced the enlarged lung volumes. Compared with group 1, the BALF levels of TNF-alpha and IL-6, and the lung tissue levels of IL-1beta, IL-6, and IL-8 were increased in group 2 (P=0.028, P=0.005, P=0.001, P=0.019, P<0.001, respectively). The TNF-alpha and IL-8 levels of BALF (P=0.007, P=0.001, respectively), and the TNF-alpha, IL-1beta, IL-6, and the IL-8 levels of lung tissue (P=0.031, P=0.017, P=0.007, P<0.001) were decreased in group 3 compared to group 2. In conclusion, bosentan decreased the inflammatory response by reducing numbers of inflammatory cells and proinflammatory cytokines. [Abstract/Link to Full Text]

Jeon S, Kim NH, Koo BS, Lee HJ, Lee AY
Bee venom stimulates human melanocyte proliferation, melanogenesis, dendricity and migration.
Exp Mol Med. 2007 Oct 31;39(5):603-13.
Pigmentation may result from melanocyte proliferation, melanogenesis, migration or increases in dendricity. Recently, it has been reported that secreted phospholipase A(2)(sPLA(2)) known as a component of bee venom (BV), stimulates melanocyte dendricity and pigmentation. BV has been used clinically to control rheumatoid arthritis and to ameliorate pain via its anti-inflammatory and antinociceptive properties. Moreover, after treatment with BV, pigmentation around the injection sites was occasionally observed and the pigmentation lasted a few months. However, no study has been done about the effect of BV on melanocytes. Thus, in the present study, we examined the effect of BV on the proliferation, melanogenesis, dendricity and migration in normal human melanocytes and its signal transduction. BV increased the number of melanocytes dose and time dependently through PKA, ERK, and PI3K/Akt activation. The level of cAMP was also increased by BV treatment. Moreover, BV induced melanogenesis through increased tyrosinase expression. Furthermore, BV induced melanocyte dendricity and migration through PLA(2) activation. Overall, in this study, we demonstrated that BV may have an effect on the melanocyte proliferation, melanogenesis, dendricity and migration through complex signaling pathways in vitro, responsible for the pigmentation. Thus, our study suggests a possibility that BV may be developed as a therapeutic drug for inducing repigmentation in vitiligo skin. [Abstract/Link to Full Text]

Lee EH, Allen PD
Homo-dimerization of RyR1 C-terminus via charged residues in random coils or in an alpha-helix.
Exp Mol Med. 2007 Oct 31;39(5):594-602.
To investigate the mechanism by which the C-terminus (4,938-5,037) of the ryanodine receptor 1 (RyR1) homo-tetramerizes, forming a functional Ca(2+)-release channel, the structural requirements for the tetramerization were studied using site-directed mutagenesis. Alanine-substitutions at five charged residues, E4976, H5003, D5026, E5033 and D5034, significantly decreased the formation of homo-dimers (reduced by >50%). Interaction between the C-terminus and cytoplasmic loop I (4,821-4,835) required two positively charged residues, H4832 and K4835. Based on the predicted protein secondary structures, all seven charged residues are located in random coils. Paired alanine-substitutions at six negatively charged residues (E4942A/D4953A, D4945A/E4952A and E4948A/ E4955A) of the alpha-helix (4,940-4,956) in the C-terminus increased homo-dimerization. Therefore, the homo-tetramerization of RyR1 may be mediated by intra- and/or inter-monomer electrostatic interactions among the C-terminal charged residues in random coils or in an alpha-helix. [Abstract/Link to Full Text]

Kim SY, Seo M, Oh JM, Cho EA, Juhnn YS
Inhibition of gamma ray-induced apoptosis by stimulatory heterotrimeric GTP binding protein involves Bcl-xL down-regulation in SH-SY5Y human neuroblastoma cells.
Exp Mol Med. 2007 Oct 31;39(5):583-93.
Heterotrimeric GTP-binding proteins (G proteins) transduce extracellular signals into intracellular signals by activating effector molecules including adenylate cyclases that catalyze cAMP formation, and thus regulate various cellular responses such as metabolism, proliferation, and apoptosis. cAMP signaling pathways have been reported to protect cells from ionizing radiation-induced apoptosis, but however, the protective mechanism is not clear. Therefore, this study aimed to investigate the signaling molecules and the mechanism mediating the anti-apoptotic action of cAMP signaling system in radiation-induced apoptosis. Stable expression of a constitutively active mutant of Gas (GalphasQL) protected gamma ray-induced apoptosis which was assessed by analysis of the cleavages of PARP, caspase-9, and caspase-3 and cytochrome C release in SH-SY5Y human neuroblastoma cells. GasQL repressed the gamma ray-induced down-regulation of Bcl-xL protein, but transfection of Bcl-xL siRNA increased the gamma ray-induced apoptosis and abolished the anti-apoptotic effect of GasQL. GasQL decreased the degradation rate of Bcl-xL protein, and it also restrained the decrease in Bcl-xL mRNA by increasing the stability following ionizing irradiation. Furthermore, prostaglandin E2 that activates Gas was found to protect gamma ray-induced apoptosis, and the protective effect was abolished by treatment with prostanoid receptor antagonist specific to EP2/4R subtype. Moreover, specific agonists for adenosine A1 receptor that inhibits cAMP signaling pathway augmented gamma ray-induced apoptosis. From this study, it is concluded that Galphas-cAMP signaling system can protect SH-SY5Y cells from gamma ray-induced apoptosis partly by restraining down-regulation of Bcl-xL expression, suggesting that radiation-induced apoptosis can be modulated by GPCR ligands to improve the efficiency of radiation therapy. [Abstract/Link to Full Text]

Kim HJ, Im W, Kim S, Kim SH, Sung JJ, Kim M, Lee KW
Calcium-influx increases SOD1 aggregates via nitric oxide in cultured motor neurons.
Exp Mol Med. 2007 Oct 31;39(5):574-82.
Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells. [Abstract/Link to Full Text]

Huang CL, Cha SK, Wang HR, Xie J, Cobb MH
WNKs: protein kinases with a unique kinase domain.
Exp Mol Med. 2007 Oct 31;39(5):565-73.
Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells. [Abstract/Link to Full Text]

Choi YS, Cho KO, Kim EJ, Sung KW, Kim SY
Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia.
Exp Mol Med. 2007 Aug 31;39(4):556-63.
Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion. [Abstract/Link to Full Text]

Choi HK, Choi KC, Oh SY, Kang HB, Lee YH, Haam S, Ahn YH, Kim KS, Kim K, Yoon HG
The functional role of the CARM1-SNF5 complex and its associated HMT activity in transcriptional activation by thyroid hormone receptor.
Exp Mol Med. 2007 Aug 31;39(4):544-55.
We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking-down either CARM1 or SNF5 also inhibited the down-regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation. [Abstract/Link to Full Text]

Qiang W, Weiqiang K, Qing Z, Pengju Z, Yi L
Aging impairs insulin-stimulated glucose uptake in rat skeletal muscle via suppressing AMPKalpha.
Exp Mol Med. 2007 Aug 31;39(4):535-43.
Insufficient intracellular fat oxidation is an important contributor to aging-related insulin resistance, while the precise mechanism underlying is unclear. AMP-activated protein kinase (AMPK) is an important regulator of intracellular fat oxidation and was evidenced to play a key role in high-glucose and high-fat induced glucose intolerance. In the present study, we investigated whether altered AMPK expression or activity was also involved in aging-related insulin resistance. Insulin sensitivity of rats' skeletal muscles was evaluated using in-vitro glucose uptake assay. Activity of alpha subunit of AMPK (AMPKalpha) was evaluated by measuring the phosphorylation of both AMPKalpha (P-AMPKalpha) and acetyl-CoA carboxylase (P-ACC), while expression of AMPKalpha was assessed by determining the mRNA levels of AMPKalpha1 and AMPKalpha2, and protein contents of AMPKalpha. Compared with 4-month old rats, 24-month old rats exhibited obviously impaired insulin sensitivity. At the same time, AMPKalpha activity significantly decreased, while AMPKalpha expression did not alter during aging. Glucose transporter 4 expression also decreased in old rats. Compared with 24-month old rats, administration of the specific activator of AMPK, 5-aminoimidazole-4-carboxamide riboside (AICAR), significantly elevated AMPKalpha activity and GluT4 expression. Also, aging-related insulin resistance was significantly ameliorated by AICAR treatment. In conclusion, aging-related insulin resistance is associated with impaired AMPKalpha activity and could be ameliorated by AICAR, thus indicating a possible role of AMPK in aging-induced insulin resistance. [Abstract/Link to Full Text]

Cheon H, Woo YS, Lee JY, Kim HS, Kim HJ, Cho S, Won NH, Sohn J
Signaling pathway for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages.
Exp Mol Med. 2007 Aug 31;39(4):524-34.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha. [Abstract/Link to Full Text]

Kim SM, Kim N, Lee S, Kim do K, Lee YM, Ahn SH, Song JH, Choi BK, Wu C, Jung KY
TGF-beta1-induced PINCH-1-ILK-alpha-parvin complex formation regulates mesangial cell proliferation and hypertrophy.
Exp Mol Med. 2007 Aug 31;39(4):514-23.
TGF-beta1-induced glomerular mesangial cell (GMC) injury is a prominent characteristic of renal pathology in several kidney diseases, and a ternary protein complex consisting of PINCH-1, integrin-linked kinase (ILK) and alpha-parvin plays a pivotal role in the regulation of cell behavior such as cell proliferation and hypertrophy. We report here that PINCH-1-ILK-alpha-parvin (PIP) complex regulates the TGF-beta1-induced cell proliferation and hypertrophy in cultured rat GMCs. When GMCs were treated with TGF-beta1 for 1, 2 and 3 days, the PIP complex formation was up-regulated after 1 day, but it was down-regulated on day 2. Cell numbers were significantly elevated on day 2, but dramatically decreased on day 3. In contrast, a significant increase in cellular protein contents was observed 3 days after TGF-beta1-treatment. TGF-beta1 induced early increase of caspase-3 activity. In GMCs incubated with TGF-beta1 for 2 days, cytosolic expression of p27(Kip1) was dramatically reduced, but its nuclear expression was remarkably elevated. A significantly decreased expression of phospho-Akt (Ser 473) was observed in the cells treated with TGF-beta1 for 1 day. TGF-beta1 induced early increase of phospho-p27(Kip1) (Thr 157) expression with subsequent decrease, and similar responses to TGF-beta1 were observed in the p38 phosphorylation (Thr 180/Thr 182). Taken together, TGF-beta1 differently regulates the PIP complex formation of GMCs in an incubation period-dependant fashion. The TGF-beta1-induced up- and down-regulation of the PIP complex formation likely contributes to the pleiotropic effects of TGF-beta1 on mesangial cell proliferation and hypertrophy through cellular localization of p27(Kip1) and alteration of Akt and p38 phosphorylation. TGF-beta1-induced alteration of the PIP complex formation may be importantly implicated in the development and progression of glomerular failure shown in several kidney diseases. [Abstract/Link to Full Text]

Yun MY, Kim SB, Park S, Han CJ, Han YH, Yoon SH, Kim SH, Kim CM, Choi DW, Cho MH, Park GH, Lee KH
Mutation analysis of p31comet gene, a negative regulator of Mad2, in human hepatocellular carcinoma.
Exp Mol Med. 2007 Aug 31;39(4):508-13.
Failure of mitotic checkpoint machinery leads to the chromosomal missegregation and nuclear endoreduplication, thereby driving the emergence of aneuploidy and tetraploidy population. Although abnormal nuclear ploidy and the resulting impairment of mitotic checkpoint function are typical physiological event leading to human hepatocellular carcinoma, any mutational change of mitotic checkpoint regulators has not yet been discovered. Therefore, we investigated the mutation of p31(comet), a recently identified mitotic checkpoint regulator, in human hepatocellular carcinoma. Of 51 human hepatocellular carcinoma tissue and 6 cell lines tested, five samples exhibited nucleotide sequence variations dispersed on four sites within the entire coding sequence. Among these sites with sequence substitutions, three were found to be missense mutation accompanied with amino acid change but one was a silent mutation. Of these sequence substitutions, two were present in both tumor and non-tumor liver tissues, suggesting the possibility of polymorphism. The present findings indicate that p31(comet) does not have an impact on the formation of aneuploidy and tetraploidy found in human hepatocellular carcinoma. [Abstract/Link to Full Text]

Cho ML, Yoon BY, Ju JH, Jung YO, Jhun JY, Park MK, Park SH, Cho CS, Kim HY
Expression of CCR2A, an isoform of MCP-1 receptor, is increased by MCP-1, CD40 ligand and TGF-beta in fibroblast like synoviocytes of patients with RA.
Exp Mol Med. 2007 Aug 31;39(4):499-507.
Cytokine and chemokine receptors play a key role in inflammation caused by rheumatoid arthritis (RA). Two isoforms of human CC chemokine receptor R2 (CCR2), the receptor of monocyte chemoattractant protein 1 (MCP-1), have been identified but their relative expression in fibroblast-like synoviocytes (FLS) and their contribution to inflammatory responses mediated by MCP-1 or inflammatory cytokines in patients with RA remain uncertain. We examined the pattern of expression of two CCR2 isoforms upon stimulation by proinflammatory cytokines and CD40 ligation. FLS were prepared from the synovial tissues of RA patients and cultured in the presence of MCP-1, soluble CD40 ligand (sCD40L), TGF-beta, IL-1beta, IL-18, IL-15, and LPS. CCR2A and CCR2B expression was examined by immunohistochemistry, RT-PCR and western blot analysis. IL-15, TNF-alpha and MCP-1 production was determined by ELISA. Immunohistochemistry showed that CCR2A is highly expressed in RA synovium compared with OA synovium. Transcripts of both CCR2A and CCR2B were detected in FLS. Exogenous MCP-1, CD40L, TGF-beta, and IL-15 significantly increased the expression of CCR2A but not CCR2B. Exposure of FLS to sCD40L caused strong upregulation of CCR2A but not of CCR2B protein expression. MCP-1 increased the proliferation of FLS and the production of IL-15, TNF-alpha, and IL-18. Because CCR2A is the main target of regulation by cytokines and CD40 ligation, the relatively higher expression of CCR2A on the cell surface suggests that this isoform of MCP-1 receptor functions as the principal mediator of inflammatory signals in RA FLS. [Abstract/Link to Full Text]

Jeon SH, Jeong WJ, Cho JY, Lee KH, Choi KY
Akt is involved in the inhibition of cell proliferation by EGF.
Exp Mol Med. 2007 Aug 31;39(4):491-8.
Axin is a negative regulator of the Wnt/beta-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation. [Abstract/Link to Full Text]

Park JS, Kim S, Han DK, Lee JY, Ghil SH
Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells.
Exp Mol Med. 2007 Aug 31;39(4):483-90.
Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease. [Abstract/Link to Full Text]

Sung YK, Park MK, Hong SH, Hwang SY, Kwack MH, Kim JC, Kim MK
Regulation of cell growth by fatty acid-CoA ligase 4 in human hepatocellular carcinoma cells.
Exp Mol Med. 2007 Aug 31;39(4):477-82.
Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified free arachidonic acid (AA) level in cells and the free AA is known to induce apoptosis. We have recently reported that expression of FACL4 is upregulated in about 40% of human hepatocellular carcinoma (HCC) and 50% of HCC cell lines, suggesting that FACL4 may be involved in liver carcinogenesis. In this study, we investigated whether HCC cell growth is regulated by FACL4. Immunoblot analysis showed that SNU 398 cells express very low or no detectable level of FACL4. We, therefore, transfected the SNU 398 cells with FACL4 expression vector, and clones expressing FACL4 were pooled and analyzed. We found that forced expression of FACL4 in SNU 398 promotes the growth of cells. In addition, we observed that triacsin C, a FACL4 inhibitor, inhibits the growth of Hep 3B cell line which expresses high level of endogenous FACL4. We also found that the triacsin C-mediated growth inhibition in Hep 3B cells results from the induction of apoptosis with evidence of Bcl-2 reduction. Altogether, our data show that FACL4 affects HCC cell growth and suggest that modulation of FACL4 expression/activity is an approach for treatment of HCC. [Abstract/Link to Full Text]

Lee EJ, Choi EM, Kim SR, Park JH, Kim H, Ha KS, Kim YM, Kim SS, Choe M, Kim JI, Han JA
Cyclooxygenase-2 promotes cell proliferation, migration and invasion in U2OS human osteosarcoma cells.
Exp Mol Med. 2007 Aug 31;39(4):469-76.
Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E2 treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously. [Abstract/Link to Full Text]

Jung SY, Park YJ, Park YJ, Cha SH, Lee MZ, Suh CK
Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts.
Exp Mol Med. 2007 Aug 31;39(4):458-68.
Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content. [Abstract/Link to Full Text]

Lee SJ, Lee JR, Hahn HS, Kim YH, Ahn JH, Bae CD, Yang JM, Hahn MJ
PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity.
Exp Mol Med. 2007 Aug 31;39(4):450-7.
Zinc finger protein 133 (ZNF133) is composed of a Krüppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1. [Abstract/Link to Full Text]

Park HY, Jin JO, Song MG, Park JI, Kwak JY
Expression of dendritic cell markers on cultured neutrophils and its modulation by anti-apoptotic and pro-apoptotic compounds.
Exp Mol Med. 2007 Aug 31;39(4):439-49.
Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction. [Abstract/Link to Full Text]

Krishnan J, Selvarajo K, Tsuchiya M, Lee G, Choi S
Toll-like receptor signal transduction.
Exp Mol Med. 2007 Aug 31;39(4):421-38.
Toll-like receptors (TLRs) are the archetypal pattern recognition receptors in sensing exogenous pathogens. Activation of TLRs is a first line of defense of the immune system, leading to the activation and recruitment of neutrophils and macrophages to sites of infection and enhances antimicrobial activity. The TLR signaling through different intracellular molecules, such as MAP kinases and IkappaB kinases which are conserved signaling elements for many receptors, leads to a distinct set of proinflammatory gene expressions. However, how these pathways differentially and precisely control the transcription of identical genes remains largely unknown. Our review focuses on the details of up-to-date signaling molecules including negative regulators and their role in controlling innate immune response. We also stress the importance of developing systemic approaches for the global understanding of TLR signaling so that appropriate drug therapeutic targets can be identified for regulating inflammatory diseases. [Abstract/Link to Full Text]

Cho YH, Park H, Cho ES, Kim WJ, Kang BS, Park BY, Kim YJ, Lee YI, Chang SI, Park K
A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer.
Exp Mol Med. 2007 Jun 30;39(3):412-8.
To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound- healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis. [Abstract/Link to Full Text]

Kang JH, Kim SA, Chang SY, Hong S, Hong KJ
Inhibition of trichostatin A-induced antiangiogenesis by small-interfering RNA for thrombospondin-1.
Exp Mol Med. 2007 Jun 30;39(3):402-11.
Expression of thrombospondin-1 (TSP-1), which is a known inhibitor of tumor growth and angiogenesis, is reciprocally regulated by positive regulators, such as VEGF. Additionally, trichostatin A (TSA) suppresses tumor progression by altering VEGF levels and VEGF-mediated signaling. Thus, understanding TSA-regulated TSP-1 expression and the effects of altered TSP-1 levels might provide insights into the mechanism of action of TSA in anti-tumorigenesis, and provide an approach to cancer therapy. Here, we examined the effect of TSA on TSP-1 expression, and the effects of TSA-induced TSP-1 on cell motility and angiogenesis, in HeLa and bovine aortic endothelial cells. TSA remarkably increased TSP-1 expression at the mRNA and protein levels, by controlling the TSP-1 promoter activity. Both TSA and exogenous TSP-1 reduced cell migration and capillary-like tube formation and these activities were confirmed by blocking TSP-1 with its neutralizing antibody and small-interfering RNA. Our results suggest that TSP-1 is a potent mediator of TSA-induced anti- angiogenesis. [Abstract/Link to Full Text]

Oh YS, Kim HJ, Ryu SJ, Cho KA, Park YS, Park H, Kim M, Kim CK, Park SC
Exercise type and muscle fiber specific induction of caveolin-1 expression for insulin sensitivity of skeletal muscle.
Exp Mol Med. 2007 Jun 30;39(3):395-401.
It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifty week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor beta (IR beta) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity. [Abstract/Link to Full Text]

Recent Articles in BMC Biochemistry

Johansson M, Lundberg M
Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary.
BMC Biochem. 2007 Dec 10;8(1):26.
ABSTRACT: BACKGROUND: Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo has been unclear. RESULTS: We have studied glutathionylation of alpha- and beta-actin in vitro using a enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. Alpha- and beta-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, beta-actin was also glutathionylated by both oxidized glutathione (GSSG) and GSH in the absence of diamide whereas alpha-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. Beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin. CONCLUSIONS: We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms. [Abstract/Link to Full Text]

Cardozo T, Pagano M
Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes.
BMC Biochem. 2007;8 Suppl 1S9.
Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Shackelford J, Pagano JS
Role of the ubiquitin system and tumor viruses in AIDS-related cancer.
BMC Biochem. 2007;8 Suppl 1S8.
Tumor viruses are linked to approximately 20% of human malignancies worldwide. This review focuses on examples of human oncogenic viruses that manipulate the ubiquitin system in a subset of viral malignancies; those associated with AIDS. The viruses include Kaposi's sarcoma herpesvirus, Epstein-Barr virus and human papilloma virus, which are causally linked to Kaposi's sarcoma, certain B-cell lymphomas and cervical cancer, respectively. We discuss the molecular mechanisms by which these viruses subvert the ubiquitin system and potential viral targets for anti-cancer therapy from the perspective of this system. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Nury D, Doucet C, Coux O
Roles and potential therapeutic targets of the ubiquitin proteasome system in muscle wasting.
BMC Biochem. 2007;8 Suppl 1S7.
Muscle wasting, characterized by the loss of protein mass in myofibers, is in most cases largely due to the activation of intracellular protein degradation by the ubiquitin proteasome system (UPS). During the last decade, mechanisms contributing to this activation have been unraveled and key mediators of this process identified. Even though much remains to be understood, the available information already suggests screens for new compounds inhibiting these mechanisms and highlights the potential for pharmaceutical drugs able to treat muscle wasting when it becomes deleterious. This review presents an overview of the main pathways contributing to UPS activation in muscle and describes the present state of efforts made to develop new strategies aimed at blocking or slowing muscle wasting. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Scheffner M, Staub O
HECT E3s and human disease.
BMC Biochem. 2007;8 Suppl 1S6.
In a simplified view, members of the HECT E3 family have a modular structure consisting of the C-terminal HECT domain, which is catalytically involved in the attachment of ubiquitin to substrate proteins, and N-terminal extensions of variable length and sequence that mediate the substrate specificity of the respective HECT E3. Although the physiologically relevant substrates of most HECT E3s have remained elusive, it is becoming increasingly clear that HECT E3s play an important role in sporadic and hereditary human diseases including cancer, cardiovascular (Liddle's syndrome) and neurological (Angelman syndrome) disorders, and/or in disease-relevant processes including bone homeostasis, immune response and retroviral budding. Thus, molecular approaches to target the activity of distinct HECT E3s, regulators thereof, and/or of HECT E3 substrates could prove valuable in the treatment of the respective diseases. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Layfield R, Shaw B
Ubiquitin-mediated signalling and Paget's disease of bone.
BMC Biochem. 2007;8 Suppl 1S5.
Multiple steps in the RANK-NF-kappaB signalling pathway are regulated by ubiquitylation. Mutations affecting different components of this pathway, including the ubiquitin binding p62 signalling adapter protein, are found in patients with Paget's disease of bone or related syndromes. Here, we review the molecular defects and potential disease mechanisms in these conditions and conclude that the mutations may confer a common increased sensitivity of osteoclasts to cytokines, resulting in disordered NF-kappaB-dependent osteoclast function. Modulation of the osteoclast RANK-NF-kappaB signalling axis may represent a viable therapeutic strategy for Paget's disease and other conditions where excessive bone resorption or remodelling is a feature. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Corn PG
Role of the ubiquitin proteasome system in renal cell carcinoma.
BMC Biochem. 2007;8 Suppl 1S4.
Renal cell carcinoma (RCC) accounts for approximately 2.6% of all cancers in the United States. While early stage disease is curable by surgery, the median survival of metastatic disease is only 13 months. In the last decade, there has been considerable progress in understanding the genetics of RCC. The VHL tumor suppressor gene is inactivated in the majority of RCC cases. The VHL protein (pVHL) acts as an E3 ligase that targets HIF-1, the hypoxia inducible transcription factor, for degradation by the ubiquitin proteasome system (UPS). In RCC cases with mutant pVHL, HIF-1 is stabilized and aberrantly expressed in normoxia, leading to the activation of pro-survival genes such as vascular endothelial growth factor (VEGF). This review will focus on the defect in the UPS that underlies RCC and describe the development of novel therapies that target the UPS. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Dahlmann B
Role of proteasomes in disease.
BMC Biochem. 2007;8 Suppl 1S3.
A functional ubiquitin proteasome system is essential for all eukaryotic cells and therefore any alteration to its components has potential pathological consequences. Though the exact underlying mechanism is unclear, an age-related decrease in proteasome activity weakens cellular capacity to remove oxidatively modified proteins and favours the development of neurodegenerative and cardiac diseases. Up-regulation of proteasome activity is characteristic of muscle wasting conditions including sepsis, cachexia and uraemia, but may not be rate limiting. Meanwhile, enhanced presence of immunoproteasomes in aging brain and muscle tissue could reflect a persistent inflammatory defence and anti-stress mechanism, whereas in cancer cells, their down-regulation reflects a means by which to escape immune surveillance. Hence, induction of apoptosis by synthetic proteasome inhibitors is a potential treatment strategy for cancer, whereas for other diseases such as neurodegeneration, the use of proteasome-activating or -modulating compounds could be more effective. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Davies JE, Sarkar S, Rubinsztein DC
The ubiquitin proteasome system in Huntington's disease and the spinocerebellar ataxias.
BMC Biochem. 2007;8 Suppl 1S2.
Huntington's disease and several of the spinocerebellar ataxias are caused by the abnormal expansion of a CAG repeat within the coding region of the disease gene. This results in the production of a mutant protein with an abnormally expanded polyglutamine tract. Although these disorders have a clear monogenic cause, each polyglutamine expansion mutation is likely to cause the dysfunction of many pathways and processes within the cell. It has been proposed that the ubiquitin proteasome system is impaired in polyglutamine expansion disorders and that this contributes to pathology. However, this is controversial with some groups demonstrating decreased proteasome activity in polyglutamine expansion disorders, some showing no change in activity and others demonstrating an increase in proteasome activity. It remains unknown whether the ubiquitin proteasome system is a feasible therapeutic target in these disorders. Here we review the conflicting results obtained from different assays performed in a variety of different systems. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Guédat P, Colland F
Patented small molecule inhibitors in the ubiquitin proteasome system.
BMC Biochem. 2007;8 Suppl 1S14.
Deregulation of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the proteasome inhibitor Velcade(R) (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented small molecule inhibitors targeting more specific UPS components, such as E3 ubiquitin ligases and deubiquitylating enzymes. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Lim KL, Tan JM
Role of the ubiquitin proteasome system in Parkinson's disease.
BMC Biochem. 2007;8 Suppl 1S13.
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Recently, several lines of evidence have implicated an intimate link between aberrations in the ubiquitin proteasome system (UPS) and PD pathogenesis. Derangements of the UPS, which normally functions as a type of protein degradation machinery, lead to alterations in protein homeostasis that could conceivably promote the toxic accumulation of proteins detrimental to neuronal survival. Not surprisingly, various cellular and animal models of PD that are based on direct disruption of UPS function reproduce the most prominent features of PD. Although persuasive, new developments in the past few years have in fact raised serious questions about the link between the UPS and PD. Here I review current thoughts and controversies about their relationship and discuss whether strategies aimed at mitigating UPS dysfunction could represent rational ways to intervene in the disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Upadhya SC, Hegde AN
Role of the ubiquitin proteasome system in Alzheimer's disease.
BMC Biochem. 2007;8 Suppl 1S12.
Though Alzheimer's disease (AD) is a syndrome with well-defined clinical and neuropathological manifestations, an array of molecular defects underlies its pathology. A role for the ubiquitin proteasome system (UPS) was suspected in the pathogenesis of AD since the presence of ubiquitin immunoreactivity in AD-related neuronal inclusions, such as neurofibrillary tangles, is seen in all AD cases. Recent studies have indicated that components of the UPS could be linked to the early phase of AD, which is marked by synaptic dysfunction, as well as to the late stages of the disease, characterized by neurodegeneration. Insoluble protein aggregates in the brain of AD patients could result from malfunction or overload of the UPS, or from structural changes in the protein substrates, which prevent their recognition and degradation by the UPS. Defective proteolysis could cause the synaptic dysfunction observed early in AD since the UPS is known to play a role in the normal functioning of synapses. In this review, we discuss recent observations on possible links between the UPS and AD, and the potential for utilizing UPS components as targets for treatment of this disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Turnbull EL, Rosser MF, Cyr DM
The role of the UPS in cystic fibrosis.
BMC Biochem. 2007;8 Suppl 1S11.
CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the DeltaF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of 'corrector' drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Jacquemont C, Taniguchi T
The Fanconi anemia pathway and ubiquitin.
BMC Biochem. 2007;8 Suppl 1S10.
Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Madsen L, Schulze A, Seeger M, Hartmann-Petersen R
Ubiquitin domain proteins in disease.
BMC Biochem. 2007;8 Suppl 1S1.
The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable diversity with respect to UDP function. Here, we give a short summary of the biochemical and physiological roles of the UDPs, which have been linked to human diseases including neurodegeneration and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; [Abstract/Link to Full Text]

Bullinger D, Neubauer H, Fehm T, Laufer S, Gleiter CH, Kammerer B
Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling.
BMC Biochem. 2007 Nov 29;8(1):25.
ABSTRACT: BACKGROUND: Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer. RESULTS: Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines. 13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N2,N2,7-trimethylguanosine, N6-methyl-N6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells. CONCLUSION: The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo. [Abstract/Link to Full Text]

Senin II, Churumova VA, Philippov PP, Koch KW
Membrane binding of the neuronal calcium sensor recoverin - modulatory role of the charged carboxy-terminus.
BMC Biochem. 2007 Nov 22;8(1):24.
ABSTRACT: BACKGROUND: The Ca2+-binding protein recoverin operates as a Ca2+-sensor in vertebrate photoreceptor cells. It undergoes a so-called Ca2+-myristoyl switch when cytoplasmic Ca2+-concentrations fluctuate in the cell. Its covalently attached myristoyl-group is exposed at high Ca2+-concentrations and enables recoverin to associate with lipid bilayers and to inhibit its target rhodopsin kinase. At low Ca2+-concentrations the myristoyl group is inserted into a hydrophobic pocket of recoverin thereby relieving inhibitory constraint on rhodopsin kinase. Hydrophobic and electrostatic interactions of recoverin with membranes have not been clearly determined, in particular the function of the positively charged carboxy-terminus in recoverin 191QKVKEKLKEKKL202 in this context is poorly understood. RESULTS: Binding of myristoylated recoverin to lipid bilayer depends on the charge distribution in phospholipids. Binding was tested by equilibrium centrifugation and surface plasmon resonance (SPR) assays. It is enhanced to a certain degree by the inclusion of phosphatidylserine (up to 60 %) in the lipid mixture. However, a recoverin mutant that lacked the charged carboxy-terminus displayed the same relative binding amplitudes as wildtype (WT) recoverin when bound to neutral or acidic lipids. Instead, the charged carboxy-terminus of recoverin has a significant impact on the biphasic dissociation of recoverin from membranes. On the other hand, the nonmyristoylated WT and truncated mutant form of recoverin did not bind to lipid bilayers to a substantial amount as binding amplitudes observed in SPR measurements are similar to bulk refractive index changes. CONCLUSIONS: Our data indicate a small, but evident electrostatic contribution to the overall binding energy of recoverin association with lipid bilayer. Properties of the charged carboxy-terminus are consistent with a role of this region as an internal effector region that prolongs the time recoverin stays on the membrane by influencing its Ca2+-sensitivity. [Abstract/Link to Full Text]

Sanchez-Pulido L, Andrade-Navarro MA
The FTO (fat mass and obesity associated) gene codes for a novel member of the non-heme dioxygenase superfamily.
BMC Biochem. 2007 Nov 8;8(1):23.
ABSTRACT: BACKGROUND: Genetic variants in the FTO (fat mass and obesity associated) gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO. RESULTS: We performed a sequence similarity search using the human FTO protein as query and then generated a profile using the multiple sequence alignment of the homologous sequences. Profile-to-sequence and profile-to-profile based comparisons identified remote homologs of the non-heme dioxygenase family. CONCLUSIONS: Our analysis suggests that human FTO is a member of the non-heme dioxygenase (Fe(II)- and 2-oxoglutarate-dependent dioxygenases) superfamily. Amino acid conservation patterns support this hypothesis and indicate that both 2-oxoglutarate and iron should be important for FTO function. This computational prediction of the function of FTO should suggest further steps for its experimental characterization and help to formulate hypothesis about the mechanisms by which it relates to obesity in humans. [Abstract/Link to Full Text]

Benitex Y, Baranger AM
Recognition of essential purines by the U1A protein.
BMC Biochem. 2007 Nov 2;8(1):22.
ABSTRACT: BACKGROUND: The RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can specifically recognize RNA targets with diverse sequences and structures. Understanding the principles governing this specificity will be important for the rational modification and design of RRM-RNA complexes. RESULTS: In this paper we have investigated the origins of specificity of the N terminal RRM of the U1A protein for stem loop 2 (SL2) of U1 snRNA by substituting modified bases for essential purines in SL2 RNA. In one series of modified bases, hydrogen bond donors and acceptors were replaced by aliphatic groups to probe the importance of these functional groups to binding. In a second series of modified bases, hydrogen bond donors and acceptors were incorrectly placed on the purine bases to analyze the origins of discrimination between cognate and non-cognate RNA. The results of these experiments show that three different approaches are used by the U1A protein to gain specificity for purines. Specificity for A1 is based primarily on discrimination against RNA containing the incorrect base, specificity for G4 is based largely on recognition of the donors and acceptors of G4, while specificity for A6 results from a combination of direct recognition of the base and discrimination against incorrectly placed functional groups. CONCLUSIONS: These investigations identify different roles that hydrogen bond donors and acceptors on bases in both cognate and non-cognate RNA play in the specific recognition of RNA by the U1A protein. Taken together with investigations of other RNA-RRM complexes, the results contribute to a general understanding of the origins of RNA-RRM specificity and highlight, in particular, the contribution of steric and electrostatic repulsion to binding specificity. [Abstract/Link to Full Text]

Pham VL, Cadel MS, Gouzy-Darmon C, Hanquez C, Beinfeld MC, Nicolas P, Etchebest C, Foulon T
Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling.
BMC Biochem. 2007 Oct 31;8(1):21.
ABSTRACT: BACKGROUND: Aminopeptidase B (Ap-B; EC catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. RESULTS: The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. CONCLUSIONS: Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing. [Abstract/Link to Full Text]

Golestani A, Ramshini H, Nemat-Gorgani M
A study on the two binding sites of hexokinase on brain mitochondria.
BMC Biochem. 2007 Oct 20;8(1):20.
ABSTRACT: BACKGROUND: Type I hexokinase (HK-I) constitutes the predominant form of the enzyme in the brain, a major portion of which is associated with the outer mitochondrial membrane involving two sets of binding sites. In addition to the glucose-6-phosphate (G6P)-sensitive site (Type A), the enzyme is bound on a second set of sites (Type B) which are, while insensitive to G6P, totally releasable by use of high concentrations of chaotropic salts such as KSCN. Results obtained on release of HK-I from these sites suggested the possibility for the existence of distinct populations of the bound enzyme, differing in susceptibility to release by G6P. RESULTS: In the present study, the sensitivity of HK-I toward release by G6P (2 mM) and a low concentration of KSCN (45 mM) was investigated using rat brain, bovine brain and human brain mitochondria. Partial release from the G6P-insensitive site occurred without disruption of the mitochondrial membrane as a whole and as related to HK-I binding to the G6P-sensitive site. While, as expected, the sequential regime release-rebinding-release was observed on site A, no rebinding was detectable on site B, pre-treated with 45 mM KSCN. Also, no binding was detectable on mitochondria upon blocking site A for HK-I binding utilizing dicyclohexylcarbodiimide (DCCD), followed by subsequent treatment with KSCN. These observations while confirmed the previously-published results on the overall properties of the two sites, demonstrated for the first time that the reversible association of the enzyme on mitochondria is uniquely related to the Type A site. CONCLUSION: Use of very low concentrations of KSCN at about 10% of the level previously reported to cause total release of HK-I from the G6P- insensitive site, caused partial release from this site in a reproducible manner. In contrast to site A, no rebinding of the enzyme takes place on site B, suggesting that site A is ; the only physiologically-important site in relation to the release-rebinding of the enzyme which occur in response to the energy requirements of the brain. Based on the results presented, a possible physiological role for distribution of the enzyme between the two sites on the mitochondrion is proposed. [Abstract/Link to Full Text]

Petersen SV, Valnickova Z, Oury TD, Crapo JD, Chr Nielsen N, Enghild JJ
The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity.
BMC Biochem. 2007;819.
BACKGROUND: Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thřgersen IB, Hřjrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875-80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist. RESULTS: The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits. CONCLUSION: This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity. [Abstract/Link to Full Text]

Nini L, Waheed AA, Panicker LM, Czapiga M, Zhang JH, Simonds WF
R7-binding protein targets the G protein beta 5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain.
BMC Biochem. 2007;818.
BACKGROUND: Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of G alpha, G beta, and G gamma subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. G beta 5 is the most structurally divergent G beta isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of G beta 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of G beta 5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. RESULTS: We show that endogenous G beta 5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated G beta 5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous G beta 5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed G beta 5/R7-RGS/R7BP proteins. A fraction of endogenous G beta 5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. CONCLUSION: A fraction of G beta 5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed G beta 5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the G beta 5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the G beta 5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex. [Abstract/Link to Full Text]

Makarchikov AF, Brans A, Bettendorff L
Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate.
BMC Biochem. 2007;817.
BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells. [Abstract/Link to Full Text]

Hoke SM, Liang G, Mutiu AI, Genereaux J, Brandl CJ
C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex.
BMC Biochem. 2007;816.
BACKGROUND: Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of approximately 10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex. RESULTS: We have found that C-terminal processing of Spt7 to its SLIK form (Spt7SLIK) and to a distinct third form (Spt7Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7SLIK or Spt7Form3. Interestingly, reduced levels of Spt7SLIK and Spt7Form3 were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7. CONCLUSION: We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation. [Abstract/Link to Full Text]

Gonzalez-Fernandez F, Baer CA, Ghosh D
Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.
BMC Biochem. 2007;815.
BACKGROUND: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. RESULTS: The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules. CONCLUSION: Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle. [Abstract/Link to Full Text]

McDonald AG, Boyce S, Moss GP, Dixon HB, Tipton KF
ExplorEnz: a MySQL database of the IUBMB enzyme nomenclature.
BMC Biochem. 2007;814.
BACKGROUND: We describe the database ExplorEnz, which is the primary repository for EC numbers and enzyme data that are being curated on behalf of the IUBMB. The enzyme nomenclature is incorporated into many other resources, including the ExPASy-ENZYME, BRENDA and KEGG bioinformatics databases. DESCRIPTION: The data, which are stored in a MySQL database, preserve the formatting of chemical and enzyme names. A simple, easy to use, web-based query interface is provided, along with an advanced search engine for more complex queries. The database is publicly available at The data are available for download as SQL and XML files via FTP. CONCLUSION: ExplorEnz has powerful and flexible search capabilities and provides the scientific community with the most up-to-date version of the IUBMB Enzyme List. [Abstract/Link to Full Text]

Al-Ali H, Khachfe HM
The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling.
BMC Biochem. 2007;812.
BACKGROUND: Apolipoprotein B-100 (apo B-100) stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B. RESULTS: The structure of apo B-17 (the N-terminal 17% of apolipoprotein B-100) was predicted by homology modeling based on the structure of the N-terminal domain of lipovitellin (LV), a protein that shares not only sequence similarity with B17, but also a functional aspect of lipid binding and transport. The model structure was first induced to accommodate the six disulfide bonds found in that region, and then optimized using simulated annealing. CONCLUSION: The content of secondary structural elements in this model structure correlates well with the reported data from other biophysical probes. The overall topology of the model conforms with the structural outline corresponding to the apo B-17 domain as seen in the EM representation of the complete LDL structure. [Abstract/Link to Full Text]

Pohl T, Walter J, Stolpe S, Soufo JH, Grauman PL, Friedrich T
Effects of the deletion of the Escherichia coli frataxin homologue CyaY on the respiratory NADH:ubiquinone oxidoreductase.
BMC Biochem. 2007;813.
BACKGROUND: Frataxin is discussed as involved in the biogenesis of iron-sulfur clusters. Recently it was discovered that a frataxin homologue is a structural component of the respiratory NADH:ubiquinone oxidoreductase (complex I) in Thermus thermophilus. It was not clear whether frataxin is in general a component of complex I from bacteria. The Escherichia coli homologue of frataxin is coined CyaY. RESULTS: We report that complex I is completely assembled to a stable and active enzyme complex equipped with all known iron-sulfur clusters in a cyaY mutant of E. coli. However, the amount of complex I is reduced by one third compared to the parental strain. Western blot analysis and live cell imaging of CyaY engineered with a GFP demonstrated that CyaY is located in the cytoplasm and not attached to the membrane as to be expected if it were a component of complex I. CONCLUSION: CyaY plays a non-essential role in the assembly of complex I in E. coli. It is not a structural component but may transiently interact with the complex. [Abstract/Link to Full Text]

Langham AA, Waring AJ, Kaznessis YN
Comparison of interactions between beta-hairpin decapeptides and SDS/DPC micelles from experimental and simulation data.
BMC Biochem. 2007;811.
BACKGROUND: We applied a combined experimental and computational approach to ascertain how peptides interact with host and microbial membrane surrogates, in order to validate simulation methodology we hope will enable the development of insights applicable to the design of novel antimicrobial peptides. We studied the interactions of two truncated versions of the potent, but cytotoxic, antimicrobial octadecapeptide protegrin-1, PC-72 [LCYCRRRFCVC] and PC-73 [CYCRRRFCVC]. RESULTS: We used a combination of FTIR, fluorescence spectroscopy and molecular dynamics simulations to examine the peptides' interactions with sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles. The relative amounts of secondary structure determined by FTIR agreed with those from the simulations. Fluorescence spectroscopy, deuterium exchange experiments and the simulations all indicate that neither peptide embeds itself deeply into the micelle core. Although molecular simulations placed both peptides at the micelle-water interface, further examination revealed differences in how certain residues interacted with the micelle core. CONCLUSION: We demonstrate here the accuracy of molecular dynamics simulations methods through comparison with experiments, and have used the simulation results to enhance the understanding of how these two peptides interact with the two types of micelles. We find agreement between simulation and experimental results in the final structure of the peptides and in the peptides final conformation with respect to the micelle. Looking in depth at the peptide interactions, we find differences in the interactions between the two peptides from the simulation data; Leu-1 on PC-72 interacts strongly with the SDS micelle, though the interaction is not persistent--the residue withdraws and inserts into the micelle throughout the simulation. [Abstract/Link to Full Text]

Recent Articles in BMC Chemical Biology

Tanaka Y, Wood LA, Cooney RV
Enhancement of intracellular gamma-tocopherol levels in cytokine-stimulated C3H 10T1/2 fibroblasts: relation to NO synthesis, isoprostane formation, and tocopherol oxidation.
BMC Chem Biol. 2007;72.
BACKGROUND: Stimulation of C3H 10T1/2 murine fibroblasts with interferon-gamma(IFN) and bacterial lipopolysaccharide (LPS) generates reactive oxygen and nitrogen species leading to DNA damage, lipid oxidation, and tocopherol oxidation. The tocopherols possess unique chemical and biological properties that suggest they have important roles related to intracellular defense against radical-mediated damage. RESULTS: Despite increased levels of reactive oxidants and decreased media tocopherol, cellular levels of gamma-tocopherol, but not alpha-tocopherol, were observed to increase significantly when cells were treated with IFN/LPS. Inhibition of nitric oxide (NO) synthesis by a specific inhibitor of inducible NO synthase (iNOS) increased both intracellular alpha-tocopherol and gamma-tocopherol concentrations, but did not significantly alter the reduction in media tocopherol levels caused by IFN/LPS treatment. Both exposure to exogenous NO and cellular synthesis of NO in cell culture increased media levels of 8-epi-prostaglandin F2alpha, a marker of oxidative lipid damage, whereas inhibition of endogenous NO synthesis reduced media 8-epi-prostaglandin F2alpha formation to control levels. CONCLUSION: Elevated intracellular levels of gamma-tocopherol in response to the cellular inflammatory state may indicate that it serves a unique role in minimizing cellular damage resulting from endogenous NO synthesis. Results of the current study suggest that NO is an important mediator of damage within the cell, as well as in the oxidation of both alpha- and gamma-tocopherols. The paradoxical increase in cellular tocopherol associated with the induction of NO synthesis may indicate either enhanced cellular transport/decreased export for tocopherols or recruitment of free tocopherol from tocopherol storage molecules. [Abstract/Link to Full Text]

Flores-Ramírez G, Rivera M, Morales-Pablos A, Osuna J, Soberón X, Gaytán P
The effect of amino acid deletions and substitutions in the longest loop of GFP.
BMC Chem Biol. 2007;71.
BACKGROUND: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. RESULTS: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. CONCLUSION: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure. [Abstract/Link to Full Text]

Abdullah A, Huq F, Chowdhury A, Tayyem H, Beale P, Fisher K
Studies on the synthesis, characterization, binding with DNA and activities of two cis-planaramineplatinum(II) complexes of the form: cis-PtL(NH3)Cl2 where L = 3-hydroxypyridine and 2,3-diaminopyridine.
BMC Chem Biol. 2006;63.
BACKGROUND: Cis-planaramineplatinum(II) complexes like their trans isomers are often found to be active against cancer cell lines. The present study deals with the synthesis, characterization and determination of activity of new cis-planaramineplatinum(II) complexes. RESULTS: Two cis-planaramineplatinum(II) complexes: cis-(3-hydroxypyridine)(ammine)dichloroplatinum(II) (code named AH3) and cis-(2,3-diaminopyridine)(ammine)dichloroplatinum(II) (code named AH7) have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1H NMR spectral measurements. The interactions of the compounds with pBR322 plasmid DNA have been investigated and their activity against ovarian cancer cell lines: A2780, A2780cisR and A2780ZD047Rhave been determined. Like cisplatin, AH3 and AH7 are believed to form mainly monofunctional N7(G) and bifunctional intrastrand N7(G)N7(G) adducts with DNA, causing a local distortion of a DNA strand. As a result, gel mobility of the DNA changes. Both AH3 and AH7 are found to be less active than cisplatin against the three cell lines with AH3 being the more active compound of the two. The higher activity of AH3 is in line with its lower molar conductivity value corresponding to a lower degree of dissociation. CONCLUSION: The differences in activity of AH3, AH7 and cisplatin against the cell lines illustrate structure-activity relationship. [Abstract/Link to Full Text]

Maliga Z, Mitchison TJ
Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol.
BMC Chem Biol. 2006;62.
BACKGROUND: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain. RESULTS: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure. CONCLUSION: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5. [Abstract/Link to Full Text]

Heynekamp JJ, Hunsaker LA, Vander Jagt TA, Deck LM, Vander Jagt DL
Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator.
BMC Chem Biol. 2006;61.
BACKGROUND: Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. RESULTS: 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 microM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. CONCLUSION: This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors. [Abstract/Link to Full Text]

Lu Y, Irani NG, Grotewold E
Covalent attachment of the plant natural product naringenin to small glass and ceramic beads.
BMC Chem Biol. 2005 Oct 10;53.
BACKGROUND: Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products. RESULTS: The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads. CONCLUSION: Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins. [Abstract/Link to Full Text]

Dwyer DS
Electronic properties of amino acid side chains: quantum mechanics calculation of substituent effects.
BMC Chem Biol. 2005 Aug 3;52.
BACKGROUND: Electronic properties of amino acid side chains such as inductive and field effects have not been characterized in any detail. Quantum mechanics (QM) calculations and fundamental equations that account for substituent effects may provide insight into these important properties. PM3 analysis of electron distribution and polarizability was used to derive quantitative scales that describe steric factors, inductive effects, resonance effects, and field effects of amino acid side chains. RESULTS: These studies revealed that: (1) different semiempirical QM methods yield similar results for the electronic effects of side chain groups, (2) polarizability, which reflects molecular deformability, represents steric factors in electronic terms, and (3) inductive effects contribute to the propensity of an amino acid for alpha-helices. CONCLUSION: The data provide initial characterization of the substituent effects of amino acid side chains and suggest that these properties affect electron density along the peptide backbone. [Abstract/Link to Full Text]

Green HM, Alberola-Ila J
Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity.
BMC Chem Biol. 2005 Jul 5;51.
BACKGROUND: Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. RESULTS: ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. CONCLUSION: EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies. [Abstract/Link to Full Text]

Dinakarpandian D, Morrissette V, Chaudhary S, Amini K, Bennett B, Van Horn JD
An informatics search for the low-molecular weight chromium-binding peptide.
BMC Chem Biol. 2004 Dec 16;4(1):2.
BACKGROUND: The amino acid composition of a low molecular weight chromium binding peptide (LMWCr), isolated from bovine liver, is reportedly E:G:C:D::4:2:2:2, though its sequence has not been discovered. There is some controversy surrounding the exact biochemical forms and the action of Cr(III) in biological systems; the topic has been the subject of many experimental reports and continues to be investigated. Clarification of Cr-protein interactions will further understanding Cr(III) biochemistry and provide a basis for novel therapies based on metallocomplexes or small molecules. RESULTS: A genomic search of the non-redundant database for all possible decapeptides of the reported composition yields three exact matches, EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. The first two sequences are found in ADAM 19 (A Disintegrin and Metalloproteinase domain 19) proteins in man and mouse; the last is found in a protein kinase in rice (Oryza sativa). A broader search for pentameric sequences (and assuming a disulfide dimer) corresponding to the stoichiometric ratio E:D:G:C::2:1:1:1, within the set of human proteins and the set of proteins in, or related to, the insulin signaling pathway, yields a match at an acidic region in the alpha-subunit of the insulin receptor (-EECGD-, residues 175-184). A synthetic peptide derived from this sequence binds chromium(III) and forms a metal-peptide complex that has properties matching those reported for isolated LMWCr and Cr(III)-containing peptide fractions. CONCLUSION: The search for an acidic decameric sequence indicates that LMWCr may not be a contiguous sequence. The identification of a distinct pentameric sequence in a significant insulin-signaling pathway protein suggests a possible identity for the LMWCr peptide. This identification clarifies directions for further investigation of LMWCr peptide fractions, chromium bio-coordination chemistry and a possible role in the insulin signaling pathway. Implications for models of chromium action in the insulin-signaling pathway are discussed. [Abstract/Link to Full Text]

Hendry P, McCall MJ, Stewart TS, Lockett TJ
Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability.
BMC Chem Biol. 2004 Dec 9;4(1):1.
BACKGROUND: Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. RESULTS: A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent. CONCLUSION: Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum. [Abstract/Link to Full Text]

Bui CT, Lambrinakos A, Babon JJ, Cotton RG
Chemical cleavage reactions of DNA on solid support: application in mutation detection.
BMC Chem Biol. 2003 May 13;3(1):1.
BACKGROUND: The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. RESULTS: DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. CONCLUSIONS: The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations. [Abstract/Link to Full Text]

Wang Y, Yuan H, Wright SC, Wang H, Larrick JW
Synthesis and cytotoxicity of a biotinylated CC-1065 analogue.
BMC Chem Biol. 2002;2(1):1.
BACKGROUND: The use of pretargeting technology for cancer imaging and treatment has made significant progress in the last few years. This approach takes advantage of the fact that biotin binds strongly to proteins avidin and streptavidin. Thus, a non-toxic tumor cell specific antibody is conjugated with avidin/streptavidin, and is administered to patients. After the antibody binds to tumor cells (usually 24--48 h); a clearing agent is given to remove the residual circulating antibodies in blood. Lastly, a toxic biotin-radioisotope conjugate is administered. Due to the small size of the biotin-radioisotope molecule and tight binding between biotin and avidin/streptavidin, the biotin-radioisotope rapidly binds to tumor cells with high specificity. CC-1065 (1) is one of a few classes of extremely potent antitumor agents, and a biotinalyted CBI-bearing CC-1065 analogue is a promising candidate to be used in the pretargeting technology to treat cancer. RESULTS: A biotinalyted CBI-bearing CC-1065 analogue, 6, was synthesized. The IC50 of 6 was 0.7 nM against U937 cells. Compound 6 caused apototsis of U937 cells. CONCLUSIONS: For the first time, a biotinalyted CBI-bearing CC-1065 analogue, 6, was synthesized. The biotinylated 6 can serve as a model compound to explore the usefulness of non-radioactive small molecule anticancer drugs in the pretargeting strategy for cancer imaging and therapy. [Abstract/Link to Full Text]

Wang Y, Yuan H, Wright SC, Wang H, Larrick JW
Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug.
BMC Chem Biol. 2001;1(1):4.
BACKGROUND: Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects. RESULTS: A CC-1065 analogue was conjugated to a cephalosporin affording prodrug 2. The prodrug and its corresponding free drug, 1, have IC50 values of 0.9 and 0.09 nM, respectively, against U937 leukemia cells in vitro. CONCLUSIONS: For the first time, a prodrug comprised of a cephalosporin and a CC-1065 analogue has been synthesized. The preliminary in vitro studies show that the prodrug was 10-fold less toxic than the free drug. Prodrug 2 has the potential to be useful in cancer treatment using the ADEPT approach. [Abstract/Link to Full Text]

Lakdawala A, Wang M, Nevins N, Liotta DC, Rusinska-Roszak D, Lozynski M, Snyder JP
Calculated conformer energies for organic molecules with multiple polar functionalities are method dependent: Taxol (case study).
BMC Chem Biol. 2001;1(1):2.
BACKGROUND: Molecular mechanics (MM) and quantum chemical (QM) calculations are widely applied and powerful tools for the stereochemical and conformational investigations of molecules. The same methods have been extensively used to probe the conformational profile of Taxol (Figure 1) both in solution and at the beta-tubulin protein binding site. RESULTS: In the present work, the relative energies of seven conformations of Taxol derived from NMR and X-ray analyses were compared with a set of widely used force fields and semiempirical MO methods coupled to a continuum solvent treatment. The procedures not only diverge significantly in their assessment of relative conformational energies, but none of them provide satisfactory agreement with experiment. CONCLUSIONS: For Taxol, molecular mechanics and semiempirical QM methods are unable to provide a consistent energetic ranking of side-chain conformations. For similar highly polar organic structures, "energy-free" conformational search methods are advised. [Abstract/Link to Full Text]

Varnum JM, Baraniak J, Kaczmarek R, Stec WJ, Brenner C
Di-, tri- and tetra-5'-O-phosphorothioadenosyl substituted polyols as inhibitors of Fhit: Importance of the alpha-beta bridging oxygen and beta phosphorus replacement.
BMC Chem Biol. 2001;1(1):3.
BACKGROUND: The human FHIT gene is inactivated early in the development of many human cancers and loss of Fhit in mouse predisposes to cancer while reintroduction of FHIT suppresses tumor formation via induction of apoptosis. Fhit protein, a diadenosine polyphosphate hydrolase, does not require hydrolase activity to function in tumor suppression and may signal for apoptosis as an enzyme-substrate complex. Thus, high affinity nonhydrolyzable substrate analogs may either promote or antagonize Fhit function, depending on their features, in Fhit + cells. Previously synthesized analogs with phosphorothioadenosyl substitutions and "supercharged" branches do not bind better than natural substrates and thus have limited potential as cellular probes. RESULTS: Here we link adenosine 5'-O-phosphates and phosphorothioates to short-chain polyols to generate a series of substrate analogs. We obtain structure-activity data in the form of in vitro Fhit inhibition for four types of analog substitutions and describe two compounds, inhibitory constants for which are 65 and 75-fold lower than natural substrates. CONCLUSIONS: The best Fhit inhibitors obtained to date separate two or more 5'-O-phosphoromonothioadenosyl moieties with as many bond lengths as in AppppA, maintain oxygen at the location of the alpha-beta bridging oxygen, and replace carbon for the beta phosphorus. [Abstract/Link to Full Text]

Bennett VJ, Simmons MA
Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation.
BMC Chem Biol. 2001;1(1):1.
BACKGROUND: Substance P (SP) is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. RESULTS: Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. CONCLUSIONS: The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling. [Abstract/Link to Full Text]

Recent Articles in BMC Molecular Biology

Moncini S, Bevilacqua A, Venturin M, Fallini C, Ratti A, Nicolin A, Riva P
The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability.
BMC Mol Biol. 2007 Dec 3;8(1):111.
ABSTRACT: BACKGROUND: CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. RESULTS: The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. CONCLUSIONS: Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders. [Abstract/Link to Full Text]

Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, Tang Q, Kong D, Ma D
Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells.
BMC Mol Biol. 2007 Dec 3;8(1):110.
ABSTRACT: BACKGROUND: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines. RESULTS: We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter. CONCLUSIONS: In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence. [Abstract/Link to Full Text]

Amit M, Sela N, Keren H, Melamed Z, Muler I, Shomron N, Izraeli S, Ast G
Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene.
BMC Mol Biol. 2007 Nov 29;8(1):109.
ABSTRACT: BACKGROUND: Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. RESULTS: Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. CONCLUSIONS: The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains. [Abstract/Link to Full Text]

Bahar B, Monahan FJ, Moloney AP, Schmidt O, Machugh DE, Sweeney T
Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues.
BMC Mol Biol. 2007 Nov 29;8(1):108.
ABSTRACT: BACKGROUND: Recovering high quality intact RNA from post-mortem tissue is of major concern for gene expression studies in animals and humans. Since the availability of post-mortem tissue is often associated with substantial delay, it is important that we understand the temporal variation in the stability of total RNA and of individual gene transcripts so as to be able to appropriately interpret the data generated from such studies. Hence, the objective of this experiment was to qualitatively and quantitatively assess the integrity of total and messenger RNA extracted from bovine skeletal muscle, subcutaneous adipose tissue and liver stored at 4 degree C at a range of time points up to 22 days post-mortem. These conditions were designed to mimic the environment prevailing during the transport of beef from the abattoir to retail outlets. RESULTS: The 28S and 18S rRNA molecules of total RNA were intact for up to 24 h post-mortem in liver and adipose tissues and up to 8 days post-mortem in skeletal muscle. The mRNA of housekeeping genes (GAPDH and ACTB) and two diet-related genes (RBP5 and SCD) were detectable up to 22 days post-mortem in skeletal muscle. While the mRNA stability of the two housekeeping genes was different in skeletal muscle and liver, they were similar to each other in adipose tissue. After 22 days post-mortem, the relative abundance of RBP5 gene was increased in skeletal muscle and in adipose tissue and decreased in liver. During this period, the relative abundance of SCD gene also increased in skeletal muscle whereas it decreased in both adipose tissue and liver. CONCLUSION: Stability of RNA in three tissues (skeletal muscle, subcutaneous adipose tissue and liver) subjected to long-term post-mortem storage at refrigeration temperature indicated that skeletal muscle can be a suitable tissue for recovering biologically useful RNA for gene expression studies even if the tissue is subjected to post-mortem storage for weeks, whereas adipose tissue and liver should be processed within 24 h post-mortem. [Abstract/Link to Full Text]

McNeill RE, Miller N, Kerin MJ
Evaluation and validation of candidate endogenous control genes in real-time quantitative PCR studies of breast cancer.
BMC Mol Biol. 2007 Nov 27;8(1):107.
ABSTRACT: BACKGROUND: Real-time quantitative PCR (RQ-PCR) forms the basis of many breast cancer biomarker studies and novel prognostic assays paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC) genes used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1) was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. RESULTS: The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4) was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. CONCLUSIONS: We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the quantitation of gene expression data, facilitating the detection of smaller changes in gene expression than otherwise possible. The combination identified here is a good candidate for use as a two-gene endogenous control in a broad spectrum of future research and diagnostic applications in breast cancer. [Abstract/Link to Full Text]

Steffensen KR, Bouzga M, Skjeldal F, Kasi C, Karahasan A, Matre V, Bakke O, Guerin S, Eskild W
Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator.
BMC Mol Biol. 2007 Nov 16;8(1):106.
ABSTRACT: BACKGROUND: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. RESULTS: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It consists of 6 exons localized on chromosome 1. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required the presence of the footprint 1 element. In transiently transfected S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under the control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter. CONCLUSIONS: We propose that NCU-G1 is a dual-function protein functioning as a transcription factor as well as a nuclear receptor co-activator. [Abstract/Link to Full Text]

Winter J, Kunath M, Roepcke S, Krause S, Schneider R, Schweiger S
Alternative polyadenylation signals and promoters act in concert to control tissue-specific expression of the Opitz Syndrome gene MID1.
BMC Mol Biol. 2007 Nov 15;8(1):105.
ABSTRACT: BACKGROUND: Mutations in the X-linked MID1 gene are responsible for Opitz G/BBB syndrome, a malformation disorder of developing midline structures. Previous Northern blot analyses revealed the existence of at least three MID1 transcripts of differing lengths. RESULTS: Here we show that alternative polyadenylation generates the size differences observed in the Northern blot analyses. Analysis of EST data together with additional Northern blot analyses proved tissue-specific usage of the alternative polyadenylation sites. Bioinformatic characterization of the different 3'UTRs of MID1 revealed numerous RNA-protein interaction motifs, several of which turned out to be conserved between different species. Furthermore, our data suggest that mRNA termination at different polyadenylation sites is predetermined by the choice of alternative 5'UTRs and promoters of the MID1 gene, a mechanism that efficiently allows synergistic function of 5' and 3'UTRs. CONCLUSIONS: MID1 expression is tightly regulated through concerted action of alternative promoters and alternative polyadenylation signals both during embryonic development and in the adult. [Abstract/Link to Full Text]

Gaigalat L, Schlueter JP, Hartmann M, Mormann S, Tauch A, Puehler A, Kalinowski J
The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum.
BMC Mol Biol. 2007 Nov 15;8(1):104.
ABSTRACT: BACKGROUND: The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. RESULTS: Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6-P) and glucose-6-phosphate (G-6-P) also negatively affect SugR-binding, but in millimolar concentrations. CONCLUSION: In C. glutamicum ATCC 13032 the DeoR-type regulator SugR acts as a pleiotropic transcriptional repressor of all described PTS genes. Thus, in contrast to most DeoR-type repressors described, SugR is able to act also on the transcription of the distantly located genes ptsG and ptsS of C. glutamicum. Transcriptional repression of the fructose-PTS gene cluster is observed during growth on acetate and transcription is derepressed in the presence of the PTS sugars glucose and fructose. This derepression of the fructose-PTS gene cluster is mainly modulated by the negative effector F-1-P, but reduced sensitivity to the other effectors, F-1,6-P or G-6-P might cause differential transcriptional regulation of genes of the general part of the PTS (ptsI, ptsH) and associated genes encoding sugar-specific functions (ptsF, ptsG, ptsS). [Abstract/Link to Full Text]

Lohmann JS, Stougaard M, Koch J
Detection of short DNA sequence elements on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis.
BMC Mol Biol. 2007 Nov 13;8(1):103.
ABSTRACT: BACKGROUND: In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. METHODS: Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. RESULTS: An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1) on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. CONCLUSIONS: Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as represented by metaphase spreads. An optimized protocol for chromosome spreading in diagnostic well-slides was used for the detection of circularized padlock probes amplified by target primed rolling circle DNA synthesis from condensed metaphase chromosomes. We were able to detect a 40 nucleotide sequence in the male specific repetitive satellite I sequence and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene. Our overall conclusion is that whilst this type of reaction indeed can be brought to work on nuclear genomes, including metaphase chromosomes, the total efficiency of this multistep reaction is at present relatively low (1-10% of target sites picked up), meaning that it is best suited for the detection of targets that exist in multiple copies per cell. Changing this will require substantial efforts to systematically increase the efficiency in each step. [Abstract/Link to Full Text]

Yochum GS, Rajaraman V, Cleland R, McWeeney S
Localization of TFIIB binding regions using serial analysis of chromatin occupancy.
BMC Mol Biol. 2007 Nov 12;8(1):102.
ABSTRACT: BACKGROUND: RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome. RESULTS: A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20-22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of protein-coding genes in the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID (an addition component of PICs) bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse. CONCLUSION: Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated. [Abstract/Link to Full Text]

Yang J, Everett AD
Hepatoma derived growth factor binds DNA through the N-terminal PWWP domain.
BMC Mol Biol. 2007 Oct 31;8(1):101.
ABSTRACT: BACKGROUND: Hepatoma Derived Growth Factor (HDGF) is a nuclear protein with nuclear targeting required for mitogenic activity. Recently we demonstrated that HDGF is a transcriptional repressor, but whether HDGF binds DNA, the specificity of DNA binding and what protein domain is required are still unknown. In this study, we aimed to identify if HDGF is a DNA binding protein, map the functional DNA binding domain and DNA binding element for HDGF. RESULTS: Using chromatin immunoprecipitation (ChIP) of human DNA, we isolated 10 DNA sequences sharing a conserved ~200 bp element. Homology analysis identified the binding sequences as a motif within the promoter of the SMYD1 gene, a HDGF target gene. Electrophoretic Mobility Shift Assays (EMSA) confirmed the binding of HDGF to this conserved sequence. As a result, an 80 bp conserved sequence located in the SMYD1 promoter bound GST-HDGF tightly. The binding core sequence for HDGF was narrowed down to 37 bp using a deletion mapping strategy from both the 5' and 3' ends. Moreover, ChIP and DNase I footprinting analysis revealed that HDGF binds this 80bp DNA fragment specifically. Functionally overexpression of HDGF represses a reporter gene which is controlled by an SV-40 promoter containing the 80 bp DNA element. Using serial truncations of GST-HDGF, we mapped the DNA binding domain of HDGF to the N-terminal PWWP domain. CONCLUSIONS: HDGF is a DNA binding protein, binds DNA specifically, and prefers a minimum of 37 bp long DNA fragment. The N-terminal PWWP domain of HDGF is required for DNA binding. HDGF exerts its transcription repressive effect through binding to a conserved DNA element in the promoter of target genes. [Abstract/Link to Full Text]

Vimberg V, Tats A, Remm M, Tenson T
Translation initiation region sequence preferences in Escherichia coli.
BMC Mol Biol. 2007 Oct 31;8(1):100.
ABSTRACT: BACKGROUND: The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation. RESULTS: We found that during bacterial growth at 37C, a six-nucleotide SD (AGGAGG) is more efficient than shorter or longer sequences. The A/U-rich enhancer contributes strongly to the efficiency of initiation, having the greatest stimulatory effect in the exponential growth phase of the bacteria. The SD sequences and the A/U-rich enhancer stimulate translation co-operatively: strong SDs are stimulated by the enhancer much more than weak SDs. The bacterial growth rate does not have a major influence on the TIR selection pattern. On the other hand, temperature affects the TIR preference pattern: shorter SD sequences are preferred at lower growth temperatures. We also performed an in silico analysis of the TIRs in all E. coli mRNAs. The base pairing potential of the SD sequences does not correlate with the codon adaptation index, which is used as an estimate of gene expression level. CONCLUSIONS: In E.coli the SD selection preferences are influenced by the growth temperature and not influenced by the growth rate. The A/U rich enhancers stimulate translation considerably by acting co-operatively with the SD sequences. [Abstract/Link to Full Text]

Habran L, El Mjiyad N, Di Valentin E, Sadzot-Delvaux C, Bontems S, Piette J
Varicella Zoster Virus Immediate-Early 63 protein acts as an epigenetic factor regulating gene transcription in a cell-type dependent manner.
BMC Mol Biol. 2007 Oct 30;8(1):99.
ABSTRACT: BACKGROUND: Varicella Zoster Virus Immediate Early 63 protein (IE63) has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. RESULTS: In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-kB dependent genes such as IL-8, ICAM-1, and IkBa, it modulates transcription of these genes upon TNFa induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. CONCLUSIONS: While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-kB dependent genes by the accelerated resynthesis of the inhibitor IkBa. [Abstract/Link to Full Text]

Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID
A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.
BMC Mol Biol. 2007 Oct 30;8(1):98.
ABSTRACT: BACKGROUND: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. RESULTS: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. CONCLUSIONS: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells. [Abstract/Link to Full Text]

Karlsson KH, Stenerlow B
Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase.
BMC Mol Biol. 2007 Oct 26;8(1):97.
ABSTRACT: BACKGROUND: Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. RESULTS: In this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at DNA double-strand breaks in NHEJ deficient cells. At repair times > 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50oC) are used. CONCLUSIONS: We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required. [Abstract/Link to Full Text]

Zaniolo K, Desnoyers S, Leclerc S, Guerin SL
Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1.
BMC Mol Biol. 2007 Oct 25;8(1):96.
ABSTRACT: BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that play critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity. RESULTS: Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but not with Sp3 nor NFI, in PARP-1+/+ cells. In addition, in vitro PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1+/+ and PARP-1-/- cells revealed that the lack of PARP-1 expression in PARP-1-/- cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1-/- cells. Subjecting PARP-1+/+ cells to an oxydative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function. CONCLUSIONS: Our results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription. [Abstract/Link to Full Text]

Ushizawa K, Takahashi T, Hosoe M, Ohkoshi K, Hashizume K
Expression and characterization of novel ovine orthologs of bovine placental prolactin-related proteins.
BMC Mol Biol. 2007 Oct 25;8(1):95.
ABSTRACT: BACKGROUND: The prolactin-related proteins (PRPs) are non-classical placental-specific members of the growth hormone/prolactin family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes. RESULTS: We cloned two novel ovine PRPs, named oPRP1 and oPRP2. oPRP2 had a typical PRP sequence similar to bovine PRP1 (bPRP1). oPRP1 had a short sequence identical with bovine or caprine type PRP but the reading frame was shifted. Both oPRPs were expressed in trophoblast giant binucleate cells (BNC) as in cattle and goat. oPRP1 expression declined from the early to the middle stage of gestation. In contrast, oPRP2 expression remained constant throughout the gestation period. oPRP2 was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35kDa for the FLAG-tag fusion oPRP2 protein. This recombinant protein and bPRP1 were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant PRP had lactogenic activity because the Nb2 lymphoma cells did not proliferate. CONCLUSION: We have identified two novel PRPs, oPRP1 and oPRP2, in ovine placenta. Both these ovine PRPs were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the oPRP2 molecule. It is anticipated that novel and known ruminant PRPs have common functions, except for lactogenic activity. [Abstract/Link to Full Text]

Forbes EM, Nieduszynska SR, Brunton FK, Gibson J, Glover LA, Stansfield I
Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.
BMC Mol Biol. 2007 Oct 25;8(1):94.
ABSTRACT: BACKGROUND: In the C.albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. RESULTS: The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C.albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C.albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. CONCLUSIONS: This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S.cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2. [Abstract/Link to Full Text]

Levesque-Sergerie JP, Duquette M, Thibault C, Delbecchi L, Bissonnette N
Detection limits of several commercial reverse transcriptase enzymes: impact on the low and high abundant transcript levels assessed by quantitative RT-PCR.
BMC Mol Biol. 2007 Oct 22;8(1):93.
ABSTRACT: BACKGROUND: In functional genomics, transcript measurement is of fundamental importance. Quantitative RT-PCR assays are the most popular technology and depends on the initial molecular step, reverse transcription (RT). This study depicts a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used reverse transcription (RT) systems and measured the production of PCR amplifiable products and the influence of PCR inhibitor contents. RESULTS: The qRT-PCR assays were conducted using the TaqMan system but we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundant transcripts, the SuperScript II system generated more detectable molecules than the 4 other systems tested: SensiScript, OmniScript, SuperScript III and PowerScript (P < 0.05). However, the SensiScript and PowerScript systems were more efficient for detecting high-abundant transcripts in presence of 1-2 ug of background RNA (P < 0.05). The most striking aspect was the influence of the dilution of the RT reaction on the subsequent PCR. Indeed, some inhibition was released when diluted RT reactions were used for the qPCR measurements. Furthermore, the amount of background RNA in the RT reaction was also a major component influencing a downstream step in qRT-PCR, the PCR reaction. Whereas the SensiScript was less biased, other systems contained an important source of PCR inhibitors interfering up to 70% in the qRT-PCR. CONCLUSION: This study depicts a complex overview of the influence of elements such as RT systems, qRT-PCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors contained in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray), the RT step is critical and should be revisited with care. [Abstract/Link to Full Text]

Scheibe M, Bonin S, Hajnsdorf E, Betat H, Morl M
Hfq stimulates the activity of the CCA-Adding Enzyme.
BMC Mol Biol. 2007 Oct 18;8(1):92.
ABSTRACT: BACKGROUND: The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A) polymerase I (PAP). As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme). Therefore, it was assumed that Hfq might not only influence the poly(A) polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. RESULTS: Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA addition. CONCLUSIONS: The increase of the CCA addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq). So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme. [Abstract/Link to Full Text]

Hughes S, Jones LJ
The use of whole genome amplified DNA as a control for methylation specific PCR, pyrosequencing, bisulfite sequencing and methylation-sensitive restriction enzyme PCR.
BMC Mol Biol. 2007 Oct 16;8(1):91.
ABSTRACT: BACKGROUND: Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples. RESULTS: We have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA. CONCLUSION: Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult. [Abstract/Link to Full Text]

Kleino I, Ortiz RM, Huovila AP
ADAM15 gene structure and differential alternative exon use in human tissues.
BMC Mol Biol. 2007 Oct 15;8(1):90.
ABSTRACT: BACKGROUND: ADAM15 is a metalloprotease-disintegrin implicated in ectodomain shedding and cell adhesion. Aberrant ADAM15 expression has been associated with human cancer and other disorders. We have previously shown that the alternative splicing of ADAM15 transcripts is mis-regulated in cancer cells. To gain a better understanding of ADAM15 regulation, its genomic organization and regulatory elements as well as the alternative exon use in human tissues were characterized. RESULTS: Human ADAM15, flanked by the FLJ32785/DCST1 and ephrin-A4 genes, spans 11.4 kb from the translation initiation codon to the polyadenylation signal, being the shortest multiple-exon ADAM gene. The gene contains 23 exons varying from 63 to 316 bp and 22 introns from 79 to 1283 bp. The gene appeared to have several transcription start sites and their location suggested the promoter location within a CpG island proximal to the translation start. Reporter expression experiments confirmed the location of functional GC-rich, TATAless and CAATless promoter, with the most critical transcription-supporting elements located -266 to -23 bp relative to the translation start. Normal human tissues showed different complex patterns of at least 13 different ADAM15 splice variants arising from the alternative use of the cytosolic-encoding exons 19, 20a/b, and 21a/b. The deduced ADAM15 protein isoforms have different combinations of cytosolic regulatory protein interaction motifs. CONCLUSION: Characterization of human ADAM15 gene and identification of elements involved in the regulation of transcription and alternative splicing provide important clues for elucidation of physiological and pathological roles of ADAM15. The present results also show that the alternative exon use is a physiological post-transcriptional mechanism regulating ADAM15 expression in human tissues. [Abstract/Link to Full Text]

Wang HY, Chang HT, Pai TW, Wu CI, Lee YH, Chang YH, Tai HL, Tang CY, Chou WY, Chang MD
Transcriptional Regulation of Human Eosinophil RNases by an Evolutionary- Conserved Sequence Motif in Primate Genome.
BMC Mol Biol. 2007 Oct 11;8(1):89.
ABSTRACT: BACKGROUND: Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported. RESULTS: In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors. CONCLUSION: Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters. [Abstract/Link to Full Text]

Chen H, Mo W, Su H, Zhang Y, Song H
Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities.
BMC Mol Biol. 2007 Oct 7;8(1):88.
ABSTRACT: BACKGROUND: Although staphylokianse (SAK) is among the most promising blood dissolving agents, it is far from ideal. It is interesting to hypothesize that the clot lysis efficacy of SAK can be enhanced with direct active platelet binding ability, and at the same time the rethrombosis complication after successful recanalization can be minimized with an antiplatelet aggregation activity. The present study was performed to characterize the functional properties of RGD-SAK, a novel mutant of staphylokinase (SAK). RESULTS: By using site-directed mutagenesis, an Arg-Gly-Asp (RGD) motif was engineered in the staphylokinase (SAK). This mutant of SAK designated RGD-SAK was expressed, purified and characterized. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic function of SAK. Measurement of platelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK. In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis and the concentration required to obtain 50% clot lysis (C50) were reduced significantly across different concentrations of RGD-SAK comparing with SAK. Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation. CONCLUSIONS: RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted thrombolytic and antiplatelet aggregation activities in compared with SAK. [Abstract/Link to Full Text]

Ho P, Kong KF, Chan YH, Tsang JS, Wong JT
An unusual S-adenosylmethionine synthetase gene from dinoflagellates is methylated.
BMC Mol Biol. 2007 Oct 4;8(1):87.
ABSTRACT: BACKGROUND: S-Adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-Adenosylmethionine (AdoMet), the major methyl group donor in cells. AdoMet-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure. Transcription of environmental-responsive genes was demonstrated to be mediated via DNA methylation in dinoflagellates. RESULTS: A full-length cDNA encoding AdoMetS was cloned from the dinoflagellate Crypthecodinium cohnii. Phylogenetic analysis suggests that the CcAdoMetS gene, is associated with the clade of higher plant orthrologues, and not to the clade of the animal orthrologues. Surprisingly, three extra stretches of residues (8 to 19 amino acids) were found on CcAdoMetS, when compared to other members of this usually conserved protein family. Modeled on the bacterial AdeMetS, two of the extra loops are located close to the methionine binding site. Despite this, the CcAdoMetS was able to rescue the corresponding mutant of budding yeast. Southern analysis, coupled with methylation-sensitive and insensitive enzyme digestion of C.cohnii genomic DNA, demonstrated that the AdoMetS gene is itself methylated. The increase in digestibility of methylation-sensitive enzymes on AdoMet synthetase gene observed following the addition of DNA methylation inhibitors L-ethionine and 5-azacytidine suggests the presence of cytosine methylation sites within CcAdoMetS gene. During the cell cycle, both the transcript and protein levels of CcAdoMetS peaked at the G1 phase. L-ethionine was able to delay the cell cycle at the entry of S phase. A cell cycle delay at the exit of G2/M phase was induced by 5-azacytidine. CONCLUSION: The present study demonstrates a major role of AdoMet-mediated DNA methylation in the regulation of cell proliferation and that the CcAdoMetS gene is itself methylated. [Abstract/Link to Full Text]

Jia D, Cai L, He H, Skogerbo G, Li T, Aftab MN, Chen R
Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans.
BMC Mol Biol. 2007 Sep 29;8(1):86.
ABSTRACT: BACKGROUND: The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans. RESULTS: The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU45GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins. CONCLUSIONS: Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs. [Abstract/Link to Full Text]

Borrelli S, Testoni B, Callari M, Alotto D, Castagnoli C, Romano RA, Sinha S, Vigano AM, Mantovani R
Reciprocal regulation of p63 by C/EBPdelta in human keratinocytes.
BMC Mol Biol. 2007 Sep 28;8(1):85.
ABSTRACT: BACKGROUND: Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. RESULTS: C/EBPdelta, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPdelta as a primary target of DeltaNp63alpha by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPdelta is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the DeltaNp63 promoter. Overexpression of C/EBPdelta&#61472;leads to alteration in the normal profile of p63 isoforms, with the emergence of DeltaNp63alpha and beta, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPdelta leads to gene expression modifications, in part due to the concomitant repression of DeltaNp63alpha. Finally, C/EBPdelta is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. CONCLUSIONS: Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. [Abstract/Link to Full Text]

Sakazume S, Sorokina E, Iwamoto Y, Semina EV
Functional analysis of human mutations in homeodomain transcription factor PITX3.
BMC Mol Biol. 2007;884.
BACKGROUND: The homeodomain-containing transcription factor PITX3 was shown to be essential for normal eye development in vertebrates. Human patients with point mutations in PITX3 demonstrate congenital cataracts along with anterior segment defects in some cases when one allele is affected and microphthalmia with brain malformations when both copies are mutated. The functional consequences of these human mutations remain unknown. RESULTS: We studied the PITX3 mutant proteins S13N and G219fs to determine the type and severity of functional defects. Our results demonstrate alterations in DNA-binding profiles and/or transactivation activities and suggest a partial loss-of-function in both mutants with the G219fs form being more severely affected. No anomalies in cellular distribution and no dominant-negative effects were discovered for these mutants. Interestingly, the impairment of the G219fs activity varied between different ocular cell lines. CONCLUSION: The G219fs mutation was found in multiple families affected with congenital cataracts along with anterior segment malformations in many members. Our data suggest that the presence/severity of anterior segment defects in families affected with G219fs may be determined by secondary factors that are expressed in the developing anterior segment structures and may modify the effect(s) of this mutation. The S13N mutant showed only minor alteration of transactivation ability and DNA binding pattern and may represent a rare polymorphism in the PITX3 gene. A possible contribution of this mutation to human disease needs to be further investigated. [Abstract/Link to Full Text]

Jurado J, Fuentes-Almagro CA, Prieto-Alamo MJ, Pueyo C
Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species.
BMC Mol Biol. 2007;883.
BACKGROUND: Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. RESULTS: A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. CONCLUSION: We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant. [Abstract/Link to Full Text]

Romero-Díaz M, Gómez C, López-Reyes I, Martínez MB, Orozco E, Rodríguez MA
Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter.
BMC Mol Biol. 2007;882.
BACKGROUND: The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. RESULTS: To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. CONCLUSION: The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress. [Abstract/Link to Full Text]