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Recent Articles in Molecular Biology of the Cell

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Badowski C, Pawlak G, Grichine A, Chabadel A, Oddou C, Jurdic P, Pfaff M, Albigès-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]

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Recent Articles in Molecular and Cellular Biology

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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]

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Recent Articles in Journal of Cell Science

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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]

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Recent Articles in Journal of Cellular and Molecular Medicine

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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, Marchiò 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, Fariñas 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]

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Recent Articles in Molecules and Cells

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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 (http://human-genome.kribb.re.kr/stomach/). [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]

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Recent Articles in Cell Research

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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]

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Recent Articles in Cellular & Molecular Biology Letters

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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]

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Recent Articles in BMC Cell Biology

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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, Lelièvre 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]

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Recent Articles in Cell Communication and Signaling

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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, Delapeyrière 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]

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Recent Articles in Eukaryotic Cell

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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