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Recent Articles in Molecular Biology of the Cell

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James BP, Bunch TA, Krishnamoorthy S, Perkins LA, Brower DL
Nuclear localization of the ERK MAP kinase mediated by Drosophila alphaPS2betaPS integrin and importin-7.
Mol Biol Cell. 2007 Oct;18(10):4190-9.
The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein. [Abstract/Link to Full Text]

Yu HY, Bement WM
Multiple myosins are required to coordinate actin assembly with coat compression during compensatory endocytosis.
Mol Biol Cell. 2007 Oct;18(10):4096-105.
Actin is involved in endocytosis in organisms ranging from yeast to mammals. In activated Xenopus eggs, exocytosing cortical granules (CGs) are surrounded by actin "coats," which compress the exocytosing compartments, resulting in compensatory endocytosis. Here, we examined the roles of two myosins in actin coat compression. Myosin-2 is recruited to exocytosing CGs late in coat compression. Inhibition of myosin-2 slows coat compression without affecting actin assembly. This differs from phenotype induced by inhibition of actin assembly, where exocytosing CGs are trapped at the plasma membrane (PM) completely. Thus, coat compression is likely driven in part by actin assembly itself, but it requires myosin-2 for efficient completion. In contrast to myosin-2, the long-tailed myosin-1e is recruited to exocytosing CGs immediately after egg activation. Perturbation of myosin-1e results in partial actin coat assembly and induces CG collapse into the PM. Intriguingly, simultaneous inhibition of actin assembly and myosin-1e prevents CG collapse. Together, the results show that myosin-1e and myosin-2 are part of an intricate machinery that coordinates coat compression at exocytosing CGs. [Abstract/Link to Full Text]

Chen Y, McQuade KJ, Guan XJ, Thomason PA, Wert MS, Stock JB, Cox EC
Isoprenylcysteine carboxy methylation is essential for development in Dictyostelium discoideum.
Mol Biol Cell. 2007 Oct;18(10):4106-18.
Members of the Ras superfamily of small GTPases and the heterotrimeric G protein gamma subunit are methylated on their carboxy-terminal cysteine residues by isoprenylcysteine methyltransferase. In Dictyostelium discoideum, small GTPase methylation occurs seconds after stimulation of starving cells by cAMP and returns quickly to basal levels, suggesting an important role in cAMP-dependent signaling. Deleting the isoprenylcysteine methyltransferase-encoding gene causes dramatic defects. Starving mutant cells do not propagate cAMP waves in a sustained manner, and they do not aggregate. Motility is rescued when cells are pulsed with exogenous cAMP, or coplated with wild-type cells, but the rescued cells exhibit altered polarity. cAMP-pulsed methyltransferase-deficient cells that have aggregated fail to differentiate, but mutant cells plated in a wild-type background are able to do so. Localization of and signaling by RasG is altered in the mutant. Localization of the heterotrimeric Ggamma protein subunit was normal, but signaling was altered in mutant cells. These data indicate that isoprenylcysteine methylation is required for intercellular signaling and development in Dictyostelium. [Abstract/Link to Full Text]

George VT, Brooks G, Humphrey TC
Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast.
Mol Biol Cell. 2007 Oct;18(10):4168-79.
We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress. [Abstract/Link to Full Text]

Chattopadhyay S, Bielinsky AK
Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication.
Mol Biol Cell. 2007 Oct;18(10):4085-95.
In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-alpha and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-alpha is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-alpha, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-alpha subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to "recycle" it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity. [Abstract/Link to Full Text]

Sun Y, Shestakova A, Hunt L, Sehgal S, Lupashin V, Storrie B
Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis.
Mol Biol Cell. 2007 Oct;18(10):4129-42.
We used multiple approaches to investigate the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. We conclude that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG. [Abstract/Link to Full Text]

Martin SG, Rincón SA, Basu R, Pérez P, Chang F
Regulation of the formin for3p by cdc42p and bud6p.
Mol Biol Cell. 2007 Oct;18(10):4155-67.
Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Delta cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition. [Abstract/Link to Full Text]

Zhu H, Zhu G, Liu J, Liang Z, Zhang XC, Li G
Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell.
Mol Biol Cell. 2007 Oct;18(10):4119-28.
Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5. [Abstract/Link to Full Text]

Yorimitsu T, Zaman S, Broach JR, Klionsky DJ
Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae.
Mol Biol Cell. 2007 Oct;18(10):4180-9.
Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1'-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA-Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1. [Abstract/Link to Full Text]

Jin H, Wang JY
Abl tyrosine kinase promotes dorsal ruffles but restrains lamellipodia extension during cell spreading on fibronectin.
Mol Biol Cell. 2007 Oct;18(10):4143-54.
The nonreceptor Abl tyrosine kinase stimulates F-actin microspikes and membrane ruffles in response to adhesion and growth factor signals. We show here that induced dimerization of Abl-FKBP, but not the kinase-defective AblKD-FKBP, inhibits cell spreading on fibronectin. Conversely, knockdown of cellular Abl by shRNA stimulates cell spreading. The Abl kinase inhibitor, imatinib, also stimulates cell spreading and its effect is overridden by the imatinib-resistant AblT315I. Expression of Abl but not AbkKD in Abl/Arg-deficient cells again inhibits spreading. Furthermore, Abl inhibits spreading of cells that express the activated Rac, RacV12, correlating with RacV12 localization to dorsal membrane protrusions. Ectopic expression of CrkII, a Rac activator that is inactivated by Abl-mediated tyrosine phosphorylation, antagonizes Abl-mediated dorsal membrane localization of RacV12. Ectopic expression of a dynamin-2 mutant, previously shown to induce Rac-GTP localization to the dorsal membrane, abolishes the stimulatory effect of imatinib on cell spreading. These results suggest that Abl tyrosine kinase, through CrkII phosphorylation and in collaboration with dynamin-2 can regulate the partitioning of Rac-GTP to favor dorsal ruffles during cell spreading. The Abl-dependent dorsal membrane localization of activated Rac explains its positive role in ruffling and negative role in cell spreading and migration. [Abstract/Link to Full Text]

Oztan A, Silvis M, Weisz OA, Bradbury NA, Hsu SC, Goldenring JR, Yeaman C, Apodaca G
Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.
Mol Biol Cell. 2007 Oct;18(10):3978-92.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A. [Abstract/Link to Full Text]

Barco R, Hunt LB, Frump AL, Garcia CB, Benesh A, Caldwell RL, Eid JE
The synovial sarcoma SYT-SSX2 oncogene remodels the cytoskeleton through activation of the ephrin pathway.
Mol Biol Cell. 2007 Oct;18(10):4003-12.
Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway. [Abstract/Link to Full Text]

Barriere H, Nemes C, Du K, Lukacs GL
Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery.
Mol Biol Cell. 2007 Oct;18(10):3952-65.
Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting. [Abstract/Link to Full Text]

Galdeen SA, Stephens S, Thomas DD, Titus MA
Talin influences the dynamics of the myosin VII-membrane interaction.
Mol Biol Cell. 2007 Oct;18(10):4074-84.
Myosin VII (M7) and talin are ancient and ubiquitous actin-binding proteins with conserved roles in adhesion. Talin serves to link membrane receptors to the underlying actin cytoskeleton and forms a complex with M7 in Dictyostelium. The levels of talinA are tightly linked to M7 levels in Dictyostelium. Cells lacking M7 exhibit an 80% decrease in steady-state levels of talinA, whereas increased levels of M7 result in concomitant increases in total talinA. In contrast, changes in talinA levels do not affect M7 levels. Immunoprecipitation reveals that talinA and M7 are associated with each other in membrane fractions. Fluorescence recovery after photobleaching experiments on green fluorescent protein (GFP)-M7 cells expressing different levels of the M7 and talinA show that changes in the overall amounts of these two proteins influences the dynamics of membrane-associated M7. The recovery of GFP-M7 on the membrane is faster in cells lacking talinA and limited in the presence of excess amounts of talinA and M7. These results establish that M7 stabilizes talinA in the cytosol and, in return, talinA regulates the residence time of M7 at the plasma membrane, suggesting that these two proteins are both part of the same dynamic adhesion complex on the plasma membrane. [Abstract/Link to Full Text]

Juo P, Harbaugh T, Garriga G, Kaplan JM
CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans.
Mol Biol Cell. 2007 Oct;18(10):3883-93.
The proline-directed kinase Cdk5 plays a role in several aspects of neuronal development. Here, we show that CDK-5 activity regulates the abundance of the glutamate receptor GLR-1 in the ventral cord of Caenorhabditis elegans and that it produces corresponding changes in GLR-1-dependent behaviors. Loss of CDK-5 activity results in decreased abundance of GLR-1 in the ventral cord, accompanied by accumulation of GLR-1 in neuronal cell bodies. Genetic analysis of cdk-5 and the clathrin adaptin unc-11 AP180 suggests that CDK-5 functions prior to endocytosis at the synapse. The scaffolding protein LIN-10/Mint-1 also regulates GLR-1 abundance in the nerve cord. CDK-5 phosphorylates LIN-10/Mint-1 in vitro and bidirectionally regulates the abundance of LIN-10/Mint-1 in the ventral cord. We propose that CDK-5 promotes the anterograde trafficking of GLR-1 and that phosphorylation of LIN-10 may play a role in this process. [Abstract/Link to Full Text]

Boca M, D'Amato L, Distefano G, Polishchuk RS, Germino GG, Boletta A
Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion.
Mol Biol Cell. 2007 Oct;18(10):4050-61.
Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1-/- mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and beta-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated beta-catenin through activation of GSK3beta. Expression of a nondegradable form of beta-catenin in PC-1 MDCK cells restores strong cell-cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. [Abstract/Link to Full Text]

Qiu J, Huang Y, Chen G, Chen Z, Tweardy DJ, Dong S
Aberrant chromatin remodeling by retinoic acid receptor alpha fusion proteins assessed at the single-cell level.
Mol Biol Cell. 2007 Oct;18(10):3941-51.
Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)alpha and promyelocytic leukemia zinc finger (PLZF)-RARalpha (X-RARalpha). In this study, we have applied lac operator array systems to study the effects of X-RARalpha versus wild-type RARalpha on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RARalpha-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RARalpha; however, cells expressing X-RARalpha, especially PML-RARalpha, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RARalpha versus X-RARalpha, the hormone-independent association between SRC-1 and X-RARalpha on the array has been identified. Rather, compared with cells expressing RARalpha, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RARalpha-bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators. [Abstract/Link to Full Text]

Lopes VS, Wasmeier C, Seabra MC, Futter CE
Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis.
Mol Biol Cell. 2007 Oct;18(10):3914-27.
Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases. [Abstract/Link to Full Text]

Aratyn YS, Schaus TE, Taylor EW, Borisy GG
Intrinsic dynamic behavior of fascin in filopodia.
Mol Biol Cell. 2007 Oct;18(10):3928-40.
Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers. [Abstract/Link to Full Text]

Ferenz NP, Wadsworth P
Prophase microtubule arrays undergo flux-like behavior in mammalian cells.
Mol Biol Cell. 2007 Oct;18(10):3993-4002.
In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5-4.5 microm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation. [Abstract/Link to Full Text]

Zhang J, Megraw TL
Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1.
Mol Biol Cell. 2007 Oct;18(10):4037-49.
Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. We have identified a conserved motif (Motif 1) near the amino terminus of Cnn that is essential for its function in vivo. Cnn Motif 1 is necessary for proper recruitment of gamma-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn. [Abstract/Link to Full Text]

Mankouri HW, Ngo HP, Hickson ID
Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3.
Mol Biol Cell. 2007 Oct;18(10):4062-73.
CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination repair (HRR), their precise role(s) within this pathway remains poorly understood. Here, we have identified a specific role for the Shu proteins in a Rad51/Rad54-dependent HRR pathway(s) to repair MMS-induced lesions during S-phase. We show that, although mutation of RAD51 or RAD54 prevented the formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex. [Abstract/Link to Full Text]

Santamaria A, Neef R, Eberspächer U, Eis K, Husemann M, Mumberg D, Prechtl S, Schulze V, Siemeister G, Wortmann L, Barr FA, Nigg EA
Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.
Mol Biol Cell. 2007 Oct;18(10):4024-36.
Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis. [Abstract/Link to Full Text]

Weigert A, Tzieply N, von Knethen A, Johann AM, Schmidt H, Geisslinger G, Brüne B
Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate.
Mol Biol Cell. 2007 Oct;18(10):3810-9.
Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization. [Abstract/Link to Full Text]

Peacock JG, Miller AL, Bradley WD, Rodriguez OC, Webb DJ, Koleske AJ
The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin.
Mol Biol Cell. 2007 Oct;18(10):3860-72.
In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions. [Abstract/Link to Full Text]

Thomas S, Kaplan KB
A Bir1p Sli15p kinetochore passenger complex regulates septin organization during anaphase.
Mol Biol Cell. 2007 Oct;18(10):3820-34.
Kinetochore-passenger complexes in metazoans have been proposed to coordinate the segregation of chromosomes in anaphase with the induction of cytokinesis. Passenger protein homologues in the budding yeast Saccharomyces cerevisiae play a critical role early in mitosis, ensuring proper biorientation of kinetochore-microtubule attachments. Our recent work has implicated the passenger protein Bir1p (Survivin) and the inner kinetochore complex centromere binding factor 3 (CBF3) in the regulation of septin dynamics during anaphase. Here, we present data that is consistent with there being multiple passenger protein complexes. Our data show that Bir1p links together a large passenger complex containing Ndc10p, Sli15p (INCENP), and Ipl1p (Aurora B) and that the interaction between Bir1p and Sli15p is specifically involved in regulating septin dynamics during anaphase. Neither conditional alleles nor mutants of BIR1 that disrupt the interaction between Bir1p and Sli15p resulted in mono-attached kinetochores, suggesting that the Bir1p-Sli15p complex functions in anaphase and independently from Sli15p-Ipl1p complexes. We present a model for how discrete passenger complexes coordinate distinct aspects of mitosis. [Abstract/Link to Full Text]

Baars TL, Petri S, Peters C, Mayer A
Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium.
Mol Biol Cell. 2007 Oct;18(10):3873-82.
Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics. [Abstract/Link to Full Text]

Mekhail K, Rivero-Lopez L, Al-Masri A, Brandon C, Khacho M, Lee S
Identification of a common subnuclear localization signal.
Mol Biol Cell. 2007 Oct;18(10):3966-77.
Proteins share peptidic sequences, such as a nuclear localization signal (NLS), which guide them to particular membrane-bound compartments. Similarities have also been observed within different classes of signals that target proteins to membrane-less subnuclear compartments. Common localization signals affect spatial and temporal subcellular organization and are thought to allow the coordinated response of different molecular networks to a given signaling cue. Here we identify a higher-order and predictive code, {[RR(I/L)X(3)r]((n, n > or = 1))+[L(phi/N)(V/L)]((n,n>1))}, that establishes high-affinity interactions between a group of proteins and the nucleolus in response to a specific signal. This position-independent code is referred to as a nucleolar detention signal regulated by H(+) (NoDS(H+)) and the class of proteins includes the cIAP2 apoptotic regulator, VHL ubiquitylation factor, HSC70 heat shock protein and RNF8 transcription regulator. By identifying a common subnuclear targeting consensus sequence, our work reveals rules governing the dynamics of subnuclear organization and ascribes new modes of regulation to several proteins with diverse steady-state distributions and dynamic properties. [Abstract/Link to Full Text]

Lee K, Kang MJ, Kwon SJ, Kwon YK, Kim KW, Lim JH, Kwon H
Expansion of chromosome territories with chromatin decompaction in BAF53-depleted interphase cells.
Mol Biol Cell. 2007 Oct;18(10):4013-23.
Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism. [Abstract/Link to Full Text]

Nadeau PJ, Charette SJ, Toledano MB, Landry J
Disulfide Bond-mediated multimerization of Ask1 and its reduction by thioredoxin-1 regulate H(2)O(2)-induced c-Jun NH(2)-terminal kinase activation and apoptosis.
Mol Biol Cell. 2007 Oct;18(10):3903-13.
Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH(2)-terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H(2)O(2) caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H(2)O(2). During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-DeltaCys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H(2)O(2) treatment. [Abstract/Link to Full Text]

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Recent Articles in Molecular and Cellular Biology

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Zeng H, Di L, Fu G, Chen Y, Gao X, Xu L, Lin X, Wen R
Phosphorylation of Bcl10 negatively regulates T-cell receptor-mediated NF-kappaB activation.
Mol Cell Biol. 2007 Jul;27(14):5235-45.
Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation. [Abstract/Link to Full Text]

Papadaki C, Alexiou M, Cecena G, Verykokakis M, Bilitou A, Cross JC, Oshima RG, Mavrothalassitis G
Transcriptional repressor erf determines extraembryonic ectoderm differentiation.
Mol Cell Biol. 2007 Jul;27(14):5201-13.
Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment. [Abstract/Link to Full Text]

Yeh N, Miller JP, Gaur T, Capellini TD, Nikolich-Zugich J, de la Hoz C, Selleri L, Bromage TG, van Wijnen AJ, Stein GS, Lian JB, Vidal A, Koff A
Cooperation between p27 and p107 during endochondral ossification suggests a genetic pathway controlled by p27 and p130.
Mol Cell Biol. 2007 Jul;27(14):5161-71.
Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107(-/-) p27(D51/D51) and p107(-/-) p130(-/-) mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation. [Abstract/Link to Full Text]

Shi B, Liang J, Yang X, Wang Y, Zhao Y, Wu H, Sun L, Zhang Y, Chen Y, Li R, Zhang Y, Hong M, Shang Y
Integration of estrogen and Wnt signaling circuits by the polycomb group protein EZH2 in breast cancer cells.
Mol Cell Biol. 2007 Jul;27(14):5105-19.
Essential for embryonic development, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in breast and prostate cancers and is implicated in the growth and aggression of the tumors. The tumorigenic mechanism underlying EZH2 overexpression is largely unknown. It is believed that EZH2 exerts its biological activity as a transcription repressor. However, we report here that EZH2 functions in gene transcriptional activation in breast cancer cells. We show that EZH2 transactivates genes that are commonly targeted by estrogen and Wnt signaling pathways. We demonstrated that EZH2 physically interacts directly with estrogen receptor alpha and beta-catenin, thus connecting the estrogen and Wnt signaling circuitries, functionally enhances gene transactivation by estrogen and Wnt pathways, and phenotypically promotes cell cycle progression. In addition, we identified the transactivation activity of EZH2 in its two N-terminal domains and demonstrated that these structures serve as platforms to connect transcription factors and the Mediator complex. Our experiments indicated that EZH2 is a dual function transcription regulator with a dynamic activity, and we provide a mechanism for EZH2 in tumorigenesis. [Abstract/Link to Full Text]

Amir-Zilberstein L, Ainbinder E, Toube L, Yamaguchi Y, Handa H, Dikstein R
Differential regulation of NF-kappaB by elongation factors is determined by core promoter type.
Mol Cell Biol. 2007 Jul;27(14):5246-59.
NF-kappaB transcription factors activate genes important for immune response, inflammation, and cell survival. P-TEFb and DSIF, which are positive and negative transcription elongation factors, respectively, both regulate NF-kappaB-induced transcription, but the mechanism underlying their recruitment to NF-kappaB target genes is unknown. We show here that upon induction of NF-kappaB, a subset of target genes is regulated differentially by either P-TEFb or DSIF. The regulation of these genes and their occupancy by these elongation factors are dependent on the NF-kappaB enhancer and the core promoter type. Converting a TATA-less promoter to a TATA promoter switches the regulation of NF-kappaB from DSIF to P-TEFb. Accumulation or displacement of DSIF and P-TEFb is dictated by the formation of distinct initiation complexes (TFIID dependent or independent) on the two types of core promoter. The underlying mechanism for the dissociation of DSIF from TATA promoters upon NF-kappaB activation involves the phosphorylation of RNA polymerase II by P-TEFb. The results highlight a regulatory link between the initiation and the elongation phases of the transcription reaction and broaden our comprehension of the NF-kappaB pathway. [Abstract/Link to Full Text]

Nakamura K, Hirai H, Torashima T, Miyazaki T, Tsurui H, Xiu Y, Ohtsuji M, Lin QS, Tsukamoto K, Nishimura H, Ono M, Watanabe M, Hirose S
CD3 and immunoglobulin G Fc receptor regulate cerebellar functions.
Mol Cell Biol. 2007 Jul;27(14):5128-34.
The immune and nervous systems display considerable overlap in their molecular repertoire. Molecules originally shown to be critical for immune responses also serve neuronal functions that include normal brain development, neuronal differentiation, synaptic plasticity, and behavior. We show here that FcgammaRIIB, a low-affinity immunoglobulin G Fc receptor, and CD3 are involved in cerebellar functions. Although membranous CD3 and FcgammaRIIB are crucial regulators on different cells in the immune system, both CD3epsilon and FcgammaRIIB are expressed on Purkinje cells in the cerebellum. Both CD3epsilon-deficient mice and FcgammaRIIB-deficient mice showed an impaired development of Purkinje neurons. In the adult, rotarod performance of these mutant mice was impaired at high speed. In the two knockout mice, enhanced paired-pulse facilitation of parallel fiber-Purkinje cell synapses was shared. These results indicate that diverse immune molecules play critical roles in the functional establishment in the cerebellum. [Abstract/Link to Full Text]

Weaver BK, Bohn E, Judd BA, Gil MP, Schreiber RD
ABIN-3: a molecular basis for species divergence in interleukin-10-induced anti-inflammatory actions.
Mol Cell Biol. 2007 Jul;27(13):4603-16.
Whereas interleukin-10 (IL-10) is an anti-inflammatory cytokine known to regulate macrophage activation, its full mechanism of action remains incompletely defined. In a screen to identify novel IL-10-induced genes, we cloned the mouse ortholog of human ABIN-3 (also termed LIND). ABIN-3 expression was induced selectively by IL-10 in both mouse and human mononuclear phagocytes coordinately undergoing proinflammatory responses. In contrast to the previously characterized ABINs, mouse ABIN-3 was incapable of inhibiting NF-kappaB activation by proinflammatory stimuli. Generation and analysis of ABIN-3-null mice demonstrated that ABIN-3 is unnecessary for the anti-inflammatory effects of IL-10 as well as for proper negative regulation of NF-kappaB. Conversely, human ABIN-3 was capable of inhibiting NF-kappaB activation in response to signaling from Toll-like receptor, IL-1, and tumor necrosis factor. Enforced expression of human ABIN-3 in human monocytic cells suppressed the cytoplasmic degradation of IkappaBalpha, the activation of NF-kappaB, and the induction of proinflammatory genes. Comparative sequence analyses revealed that mouse ABIN-3 lacks a complete ABIN homology domain, which was required for the functional activity of human ABIN-3. ABIN-3 is, thus, an IL-10-induced gene product capable of attenuating NF-kappaB in human macrophages yet is inoperative in mice and represents a basis for species-specific differences in IL-10 actions. [Abstract/Link to Full Text]

Jaworski A, Smith CL, Burden SJ
GA-binding protein is dispensable for neuromuscular synapse formation and synapse-specific gene expression.
Mol Cell Biol. 2007 Jul;27(13):5040-6.
The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses. [Abstract/Link to Full Text]

Pirinen E, Kuulasmaa T, Pietilä M, Heikkinen S, Tusa M, Itkonen P, Boman S, Skommer J, Virkamäki A, Hohtola E, Kettunen M, Fatrai S, Kansanen E, Koota S, Niiranen K, Parkkinen J, Levonen AL, Ylä-Herttuala S, Hiltunen JK, Alhonen L, Smith U, Jänne J, Laakso M
Enhanced polyamine catabolism alters homeostatic control of white adipose tissue mass, energy expenditure, and glucose metabolism.
Mol Cell Biol. 2007 Jul;27(13):4953-67.
Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes. [Abstract/Link to Full Text]

Zhu J, Fu X, Koo YD, Zhu JK, Jenney FE, Adams MW, Zhu Y, Shi H, Yun DJ, Hasegawa PM, Bressan RA
An enhancer mutant of Arabidopsis salt overly sensitive 3 mediates both ion homeostasis and the oxidative stress response.
Mol Cell Biol. 2007 Jul;27(14):5214-24.
The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3. [Abstract/Link to Full Text]

Pathak RU, Rangaraj N, Kallappagoudar S, Mishra K, Mishra RK
Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.
Mol Cell Biol. 2007 Jul;27(13):4796-806.
Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains. [Abstract/Link to Full Text]

Berthet C, Rodriguez-Galan MC, Hodge DL, Gooya J, Pascal V, Young HA, Keller J, Bosselut R, Kaldis P
Hematopoiesis and thymic apoptosis are not affected by the loss of Cdk2.
Mol Cell Biol. 2007 Jul;27(14):5079-89.
Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2(-)(/)(-) and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2(-)(/)(-) quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy. [Abstract/Link to Full Text]

Liang F, Wang Y
DNA damage checkpoints inhibit mitotic exit by two different mechanisms.
Mol Cell Biol. 2007 Jul;27(14):5067-78.
Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1. [Abstract/Link to Full Text]

Cowling VH, D'Cruz CM, Chodosh LA, Cole MD
c-Myc transforms human mammary epithelial cells through repression of the Wnt inhibitors DKK1 and SFRP1.
Mol Cell Biol. 2007 Jul;27(14):5135-46.
c-myc is frequently amplified in breast cancer; however, the mechanism of myc-induced mammary epithelial cell transformation has not been defined. We show that c-Myc induces a profound morphological transformation in human mammary epithelial cells and anchorage-independent growth. c-Myc suppresses the Wnt inhibitors DKK1 and SFRP1, and derepression of DKK1 or SFRP1 reduces Myc-dependent transforming activity. Myc-dependent repression of DKK1 and SFRP1 is accompanied by Wnt target gene activation and endogenous T-cell factor activity. Myc-induced mouse mammary tumors have repressed SFRP1 and increased expression of Wnt target genes. DKK1 and SFRP1 inhibit the transformed phenotype of breast cancer cell lines, and DKK1 inhibits tumor formation. We propose a positive feedback loop for activation of the c-myc and Wnt pathways in breast cancer. [Abstract/Link to Full Text]

McArthur T, Ohtoshi A
A brain-specific homeobox gene, Bsx, is essential for proper postnatal growth and nursing.
Mol Cell Biol. 2007 Jul;27(14):5120-7.
To investigate in vivo roles of a murine hypothalamic homeobox gene, Bsx, we generated and analyzed two mutant alleles, Bsx(DeltaHD) and Bsx(lacZ). Bsx(DeltaHD) lacks the homeodomain, and Bsx(lacZ) is an insertion of a lacZ reporter gene. Bsx-lacZ expression was detected in the hypothalamus and pineal gland and reiterates Bsx expression. Bsx homozygous mutant mice were born at the expected Mendelian ratio, but their growth was impaired. Offspring from Bsx homozygous mutant females exhibited a low survival rate due to a nursing defect. Mammary glands of the mutant females developed normally during pregnancy; however, they involuted quickly after parturition. These results demonstrate that Bsx is required for postnatal growth and maintenance of lactating mammary glands. Thus, mouse Bsx is likely involved in systemic control of suppression of apoptosis of postpartum mammary epithelial cells. [Abstract/Link to Full Text]

Senese S, Zaragoza K, Minardi S, Muradore I, Ronzoni S, Passafaro A, Bernard L, Draetta GF, Alcalay M, Seiser C, Chiocca S
Role for histone deacetylase 1 in human tumor cell proliferation.
Mol Cell Biol. 2007 Jul;27(13):4784-95.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer. [Abstract/Link to Full Text]

Ciubotaru M, Kriatchko AN, Swanson PC, Bright FV, Schatz DG
Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.
Mol Cell Biol. 2007 Jul;27(13):4745-58.
A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage. [Abstract/Link to Full Text]

Fang J, Hogan GJ, Liang G, Lieb JD, Zhang Y
The Saccharomyces cerevisiae histone demethylase Jhd1 fine-tunes the distribution of H3K36me2.
Mol Cell Biol. 2007 Jul;27(13):5055-65.
Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units. [Abstract/Link to Full Text]

Moubarak RS, Yuste VJ, Artus C, Bouharrour A, Greer PA, Menissier-de Murcia J, Susin SA
Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.
Mol Cell Biol. 2007 Jul;27(13):4844-62.
Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program. [Abstract/Link to Full Text]

Dmitriev SE, Andreev DE, Terenin IM, Olovnikov IA, Prassolov VS, Merrick WC, Shatsky IN
Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.
Mol Cell Biol. 2007 Jul;27(13):4685-97.
Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event. [Abstract/Link to Full Text]

Luo L, Uerlings Y, Happel N, Asli NS, Knoetgen H, Kessel M
Regulation of geminin functions by cell cycle-dependent nuclear-cytoplasmic shuttling.
Mol Cell Biol. 2007 Jul;27(13):4737-44.
The geminin protein functions both as a DNA rereplication inhibitor through association with Cdt1 and as a repressor of Hox gene transcription through the polycomb pathway. Here, we report that the functions of avian geminin are coordinated with and regulated by cell cycle-dependent nuclear-cytoplasmic shuttling. In S phase, geminin enters nuclei and inhibits both loading of the minichromosome maintenance (MCM) complex onto chromatin and Hox gene transcription. At the end of mitosis, geminin is exported from nuclei by the exportin protein Crm1 and is unavailable in the nucleus during the next G(1) phase, thus ensuring proper chromatin loading of the MCM complex and Hox gene transcription. This mechanism for regulating the functions of geminin adds to distinct mechanisms, such as protein degradation and ubiquitination, applied in other vertebrates. [Abstract/Link to Full Text]

Miyamoto J, Matsumoto T, Shiina H, Inoue K, Takada I, Ito S, Itoh J, Minematsu T, Sato T, Yanase T, Nawata H, Osamura YR, Kato S
The pituitary function of androgen receptor constitutes a glucocorticoid production circuit.
Mol Cell Biol. 2007 Jul;27(13):4807-14.
Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland. [Abstract/Link to Full Text]

Frydrychova RC, Biessmann H, Konev AY, Golubovsky MD, Johnson J, Archer TK, Mason JM
Transcriptional activity of the telomeric retrotransposon HeT-A in Drosophila melanogaster is stimulated as a consequence of subterminal deficiencies at homologous and nonhomologous telomeres.
Mol Cell Biol. 2007 Jul;27(13):4991-5001.
Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions. [Abstract/Link to Full Text]

Lin X, Liu CC, Gao Q, Zhang X, Wu G, Lee WH
RINT-1 serves as a tumor suppressor and maintains Golgi dynamics and centrosome integrity for cell survival.
Mol Cell Biol. 2007 Jul;27(13):4905-16.
Faithful mitotic partitioning of the Golgi apparatus and the centrosome is critical for proper cell division. Although these two cytoplasmic organelles are probably coordinated during cell division, supporting evidence of this coordination is still largely lacking. Here, we show that the RAD50-interacting protein, RINT-1, is localized at the Golgi apparatus and the centrosome in addition to the endoplasmic reticulum. To examine the biological roles of RINT-1, we found that the homozygous deletion of Rint-1 caused early embryonic lethality at embryonic day 5 (E5) to E6 and the failure of blastocyst outgrowth ex vivo. About 81% of the Rint-1 heterozygotes succumbed to multiple tumor formation with haploinsufficiency during their average life span of 24 months. To pinpoint the cellular function of RINT-1, we found that RINT-1 depletion by RNA interference led to the loss of the pericentriolar positioning and dispersal of the Golgi apparatus and concurrent centrosome amplification during the interphase. Upon mitotic entry, RINT-1-deficient cells exhibited multiple abnormalities, including aberrant Golgi dynamics during early mitosis and defective reassembly at telophase, increased formation of multiple spindle poles, and frequent chromosome missegregation. Mitotic cells often underwent cell death in part due to the overwhelming cellular defects. Taken together, these findings suggest that RINT-1 serves as a novel tumor suppressor essential for maintaining the dynamic integrity of the Golgi apparatus and the centrosome, a prerequisite to their proper coordination during cell division. [Abstract/Link to Full Text]

Kasler HG, Verdin E
Histone deacetylase 7 functions as a key regulator of genes involved in both positive and negative selection of thymocytes.
Mol Cell Biol. 2007 Jul;27(14):5184-200.
Histone deacetylase 7 (HDAC7) is highly expressed in CD4(+)/CD8(+) thymocytes and functions as a signal-dependent repressor of gene transcription during T-cell development. In this study, we expressed HDAC7 mutant proteins in a T-cell line and use DNA microarrays to identify transcriptional targets of HDAC7 in T cells. The changes in gene expression levels were compared to differential gene expression profiles associated with positive and negative thymic selection. This analysis reveals that HDAC7 regulates an extensive set of genes that are differentially expressed during both positive and negative thymic selection. Many of these genes play important functional roles in thymic selection, primarily via modulating the coupling between antigen receptor engagement and downstream signaling events. Consistent with the model that HDAC7 may play an important role in both positive and negative thymic selection, the expression of distinct HDAC7 mutants or the abrogation of HDAC7 expression can either enhance or inhibit the signal-dependent differentiation of a CD4(+)/CD8(+) cell line. [Abstract/Link to Full Text]

Hwang CK, Song KY, Kim CS, Choi HS, Guo XH, Law PY, Wei LN, Loh HH
Evidence of endogenous mu opioid receptor regulation by epigenetic control of the promoters.
Mol Cell Biol. 2007 Jul;27(13):4720-36.
The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis- and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5'-aza-2'-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression. [Abstract/Link to Full Text]

Lim C, Lee J, Choi C, Kim J, Doh E, Choe J
Functional role of CREB-binding protein in the circadian clock system of Drosophila melanogaster.
Mol Cell Biol. 2007 Jul;27(13):4876-90.
Rhythmic histone acetylation underlies the oscillating expression of clock genes in the mammalian circadian clock system. Cellular factors that contain histone acetyltransferase and histone deacetylase activity have been implicated in these processes by direct interactions with clock genes, but their functional relevance remains to be assessed by use of appropriate animal models. Here, using transgenic fly models, we show that CREB-binding protein (CBP) participates in the transcriptional regulation of the Drosophila CLOCK/CYCLE (dCLK/CYC) heterodimer. CBP knockdown in pigment dispersing factor-expressing cells lengthens the period of adult locomotor rhythm with the prolonged expression of period and timeless genes, while CBP overexpression in timeless-expressing cells causes arrhythmic circadian behaviors with the impaired expression of these dCLK/CYC-induced clock genes. In contrast to the mammalian circadian clock system, CBP overexpression attenuates the transcriptional activity of the dCLK/CYC heterodimer in cultured cells, possibly by targeting the PER-ARNT-SIM domain of dCLK. Our data suggest that the Drosophila circadian clock system has evolved a distinct mechanism to tightly regulate the robust transcriptional potency of the dCLK/CYC heterodimer. [Abstract/Link to Full Text]

Fu J, Yoon HG, Qin J, Wong J
Regulation of P-TEFb elongation complex activity by CDK9 acetylation.
Mol Cell Biol. 2007 Jul;27(13):4641-51.
P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function. [Abstract/Link to Full Text]

Nakamura K, Johnson GL
Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling.
Mol Cell Biol. 2007 Jun;27(12):4566-77.
MEKK2 and MEK5 encode Phox/Bem1p (PB1) domains that heterodimerize with one another. MEKK2, MEK5, and extracellular signal-related kinase 5 (ERK5) form a ternary complex through interactions involving the MEKK2 and MEK5 PB1 domains and a 34-amino-acid C-terminal extension of the MEK5 PB1 domain. This C-terminal extension encodes an ERK5 docking site required for MEK5 activation of ERK5. The PB1 domains bind in a front-to-back arrangement, with a cluster of basic amino acids in the front of the MEKK2 PB1 domain binding to the back-end acidic clusters of the MEK5 PB1 domain. The C-terminal moiety, including the acidic cluster of the MEKK2 PB1 domain, is not required for MEK5 binding and binds MKK7. Quiescent MEKK2 preferentially binds MEK5, and MEKK2 activation results in ERK5 activation. Activated MEKK2 binds and activates MKK7, leading to JNK activation. The findings define how the MEKK2 and MEK5 PB1 domains are uniquely used for differential binding of two mitogen-activated protein kinase kinases, MEK5 and MKK7, for the coordinated control of ERK5 and c-Jun N-terminal kinase activation. [Abstract/Link to Full Text]

Fukuda S, Abematsu M, Mori H, Yanagisawa M, Kagawa T, Nakashima K, Yoshimura A, Taga T
Potentiation of astrogliogenesis by STAT3-mediated activation of bone morphogenetic protein-Smad signaling in neural stem cells.
Mol Cell Biol. 2007 Jul;27(13):4931-7.
Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis. [Abstract/Link to Full Text]

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Recent Articles in Journal of Cell Science

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Fessart D, Simaan M, Zimmerman B, Comeau J, Hamdan FF, Wiseman PW, Bouvier M, Laporte SA
Src-dependent phosphorylation of beta2-adaptin dissociates the beta-arrestin-AP-2 complex.
J Cell Sci. 2007 May 15;120(Pt 10):1723-32.
Beta-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between beta-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors. [Abstract/Link to Full Text]

Verbrugghe KJ, White JG
Cortical centralspindlin and G alpha have parallel roles in furrow initiation in early C. elegans embryos.
J Cell Sci. 2007 May 15;120(Pt 10):1772-8.
Evidence from various systems suggests that either asters or the midzone of the mitotic spindle are the predominant determinants of cleavage plane position. Disrupting spindle midzone formation in the one-cell Caenorhabditis elegans embryo, such as by using mutants of the centralspindlin component ZEN-4, prevents completion of cytokinesis but does not inhibit furrowing. However, furrowing is inhibited by the simultaneous depletion of ZEN-4 with either PAR-2 or G alpha, which are required for asymmetric divisions. Through studies of other genes required for the presence of an intact spindle midzone containing microtubule bundles, we found that furrowing failed in the absence of PAR-2 or G alpha only when centralspindlin was absent from the furrow. We also found spindle length or microtubule distribution did not correlate with furrow initiation. We propose that centralspindlin acts redundantly with G alpha to regulate furrow initiation. [Abstract/Link to Full Text]

Bone HK, Welham MJ
Phosphoinositide 3-kinase signalling regulates early development and developmental haemopoiesis.
J Cell Sci. 2007 May 15;120(Pt 10):1752-62.
Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class I(A) PI3K isoforms has implicated PI3K-mediated signalling in development of the early embryo and lymphohaemopoietic system. We have used embryonic stem (ES) cells as an in vitro model to study the involvement of PI3K-dependent signalling during early development and haemopoiesis. Both pharmacological inhibition and genetic manipulation of PI3K-dependent signalling demonstrate that PI3K-mediated signals, most likely via 3-phosphoinositide-dependent protein kinase 1 (PDK1), are required for proliferation of cells within developing embryoid bodies (EBs). Surprisingly, the haemopoietic potential of EB-derived cells was not blocked upon PI3K inhibition but rather enhanced, correlating with modest increases in expression of haemopoietic marker genes. By contrast, PDK1-deficient EB-derived progeny failed to generate terminally differentiated haemopoietic lineages. This deficiency appeared to be due to a requirement for PI3K signalling during the proliferative phase of blast-colony-forming cell (BL-CFC) expansion, rather than as a result of effects on differentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation of erythroid and myeloid progenitors and their differentiation into mature haemopoietic colony types. These data demonstrate that PI3K-dependent signals play important roles at different stages of haemopoietic development. [Abstract/Link to Full Text]

Chicheportiche A, Bernardino-Sgherri J, de Massy B, Dutrillaux B
Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse.
J Cell Sci. 2007 May 15;120(Pt 10):1733-42.
Meiotic DNA double strand breaks (DSBs) are indicated at leptotene by the phosphorylated form of histone H2AX (gamma-H2AX). In contrast to previous studies, we identified on both zygotene and pachytene chromosomes two distinct types of gamma-H2AX foci: multiple small (S) foci located along autosomal synaptonemal complexes (SCs) and larger signals on chromatin loops (L-foci). The S-foci number gradually declined throughout pachytene, in parallel with the repair of DSBs monitored by repair proteins suggesting that S-foci mark DSB repair events. We validated this interpretation by showing the absence of S-foci in Spo11(-/-) spermatocytes. By contrast, the L-foci number was very low through pachytene. Based on the analysis of gamma-H2AX labeling after irradiation of spermatocytes, the formation of DSBs clearly induced L-foci formation. Upon DSB repair, these foci appear to be processed and lead to the above mentioned S-foci. The presence of L-foci in wild-type pachytene and diplotene could therefore reflect delayed or unregulated DSB repair events. Interestingly, their distribution was different in Spo11(+/-) spermatocytes compared with Spo11(+/+) spermatocytes, where DSB repair might be differently regulated as a response to homeostatic control of crossing-over. The presence of these L-foci in Spo11(-/-) spermatocytes raises the interesting possibility of yet uncharacterized alterations in DNA or chromosome structure in Spo11(-/-) cells. [Abstract/Link to Full Text]

Cai L, Makhov AM, Bear JE
F-actin binding is essential for coronin 1B function in vivo.
J Cell Sci. 2007 May 15;120(Pt 10):1779-90.
Coronins are conserved F-actin binding proteins that have been implicated in a variety of processes including fibroblast migration, phagocytosis, and chemotaxis. Recent data from our lab indicate that coronin 1B coordinates Arp2/3-dependent actin filament nucleation and cofilin-mediated filament turnover at the leading edge of migrating fibroblasts. Analysis of coronin function has been hampered by the lack of a clear understanding of how coronin interacts with F-actin. Here, we identify a surface-exposed conserved arginine residue at position 30 (R30), which is crucial for coronin 1B binding to F-actin both in vitro and in vivo. Using actin co-sedimentation, we demonstrate that coronin 1B binds with high affinity to ATP/ADP-P(i)-F-actin (170 nM) and with 47-fold lower affinity to ADP-F-actin (8 microM). In contrast to a previous study, we find no evidence for enhanced cofilin binding to F-actin in the presence of either coronin 1B or coronin 1A. Instead, we find that coronin 1B protects actin filaments from cofilin-induced depolymerization. Consistent with an important role for interactions between coronin 1B and F-actin in vivo, an R30D coronin mutant that does not bind F-actin localizes inefficiently to the leading edge. Furthermore, our analysis indicates that F-actin binding is absolutely required for coronin 1B to exert its effects on whole-cell motility and lamellipodial dynamics. [Abstract/Link to Full Text]

Rathke C, Baarends WM, Jayaramaiah-Raja S, Bartkuhn M, Renkawitz R, Renkawitz-Pohl R
Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila.
J Cell Sci. 2007 May 1;120(Pt 9):1689-700.
In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. Here, we focus on post-meiotic stages and show that also after meiosis, histone H3 shows a high overall methylation of K9 and K27 and we hypothesise that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, we show that histones are lost during the early canoe stage and that just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. We believe that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, we report the first transition protein-like chromosomal protein, Tpl(94D), to be found in Drosophila. We propose that Tpl(94D)--an HMG box protein--and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, we show that histone modifications and removal are independent of protamine synthesis. [Abstract/Link to Full Text]

Oikawa D, Kimata Y, Kohno K
Self-association and BiP dissociation are not sufficient for activation of the ER stress sensor Ire1.
J Cell Sci. 2007 May 1;120(Pt 9):1681-8.
Ire1 is a type I transmembrane protein located on the endoplasmic reticulum (ER). Upon ER stress, Ire1 releases the ER chaperone BiP and self-associates. This activates Ire1 and triggers the unfolded protein response in the yeast Saccharomyces cerevisiae. We isolated and characterized an Ire1 luminal domain mutant lacking both the N-terminal and the juxtamembrane loosely folded subregions. Although this 'core' mutant was able to self-associate and failed to bind BiP even under nonstressed conditions, its activation was still dependent on ER stress. Furthermore, although substitution of Pro for Ser103 (S103P) in the luminal domain of full-length Ire1 caused neither BiP dissociation nor a change in self-association, the substitution in combination with the core mutation resulted in constitutive activation. This phenotype of the S103P mutation required a cluster of positively charged amino acid residues (Arg or Lys) located close to the mutation site in the Ire1 sequence. These observations indicate that in addition to BiP dissociation and self-association of Ire1, another unknown change on the luminal side is crucial for Ire1 activation. [Abstract/Link to Full Text]

Liao Z, Seye CI, Weisman GA, Erb L
The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12.
J Cell Sci. 2007 May 1;120(Pt 9):1654-62.
The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis. [Abstract/Link to Full Text]

Nawshad A, Medici D, Liu CC, Hay ED
TGFbeta3 inhibits E-cadherin gene expression in palate medial-edge epithelial cells through a Smad2-Smad4-LEF1 transcription complex.
J Cell Sci. 2007 May 1;120(Pt 9):1646-53.
Dissociation of medial-edge epithelium (MEE) during palate development is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFbeta3 upon adherence of opposing palatal shelves, because loss of E-cadherin causes the MEE seam to break into small epithelial islands. To investigate the molecular mechanisms that cause this E-cadherin loss, we isolated and cultured murine embryonic primary MEE cells from adhered or non-adhered palates. Here, we provide the first evidence that lymphoid enhancer factor 1 (LEF1), when functionally activated by phosphorylated Smad2 (Smad2-P) and Smad4 (rather than beta-catenin), binds with the promoter of the E-cadherin gene to repress its transcription in response to TGFbeta3 signaling. Furthermore, we found that TGFbeta3 signaling stimulates epithelial-mesenchymal transformation (EMT) and cell migration in these cells. LEF1 and Smad4 were found to be necessary for up-regulation of the mesenchymal markers vimentin and fibronectin, independently of beta-catenin. We proved that TGFbeta3 signaling induces EMT in MEE cells by forming activated transcription complexes of Smad2-P, Smad4 and LEF1 that directly inhibit E-cadherin gene expression. [Abstract/Link to Full Text]

Skoumpla K, Coulton AT, Lehman W, Geeves MA, Mulvihill DP
Acetylation regulates tropomyosin function in the fission yeast Schizosaccharomyces pombe.
J Cell Sci. 2007 May 1;120(Pt 9):1635-45.
Tropomyosin is an evolutionarily conserved alpha-helical coiled-coil protein that promotes and maintains actin filaments. In yeast, Tropomyosin-stabilised filaments are used by molecular motors to transport cargoes or to generate motile forces by altering the dynamics of filament growth and shrinkage. The Schizosaccharomyces pombe tropomyosin Cdc8 localises to the cytokinetic actomyosin ring during mitosis and is absolutely required for its formation and function. We show that Cdc8 associates with actin filaments throughout the cell cycle and is subjected to post-translational modification that does not vary with cell cycle progression. At any given point in the cell cycle 80% of Cdc8 molecules are acetylated, which significantly enhances their affinity for actin. Reconstructions of electron microscopic images of actin-Cdc8 filaments establish that the majority of Cdc8 strands sit in the 'closed' position on actin filaments, suggesting a role in the regulation of myosin binding. We show that Cdc8 regulates the equilibrium binding of myosin to actin without affecting the rate of myosin binding. Unacetylated Cdc8 isoforms bind actin, but have a reduced ability to regulate myosin binding to actin. We conclude that although acetylation of Cdc8 is not essential, it provides a regulatory mechanism for modulating actin filament integrity and myosin function. [Abstract/Link to Full Text]

Lombardi ML, Knecht DA, Dembo M, Lee J
Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement.
J Cell Sci. 2007 May 1;120(Pt 9):1624-34.
Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA-) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE-) and mhcA- cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it. [Abstract/Link to Full Text]

Yin X, Ouyang S, Xu W, Zhang X, Fok KL, Wong HY, Zhang J, Qiu X, Miao S, Chan HC, Wang L
YWK-II protein as a novel G(o)-coupled receptor for Müllerian inhibiting substance in cell survival.
J Cell Sci. 2007 May 1;120(Pt 9):1521-8.
Müllerian inhibiting substance (MIS) has recently been implicated in multiple cellular functions including promotion of cell survival, but the receptor(s) and signaling pathways involved remain elusive. We have investigated the possibility of YWK-II protein, previously shown to interact physically with MIS and G(o) protein, being a receptor mediating the cell survival effect of MIS. In YWK-II-overexpressing CHO cells, MIS activates the G(o)-coupled ERK1/2 signaling pathway and promotes cell survival with altered levels of p53 and caspase-3. YWK-II antibody is found to interfere with the ability of MIS to promote viability of mouse sperm and affect MIS-activated ERK1/2 phosphorylation. In vivo studies involving injection of YWK-II antibody into the seminiferous tubule of the mouse testis, where MIS is known to be produced, show significant reduction in the sperm count with accumulation of p53 and cleaved caspase-3 in testicular nuclei. Taken together, the present study has demonstrated a new G(o)-coupled receptor for MIS in mediating ERK1/2 activation leading to anti-apoptotic activity or cell survival. [Abstract/Link to Full Text]

Matter K, Balda MS
Epithelial tight junctions, gene expression and nucleo-junctional interplay.
J Cell Sci. 2007 May 1;120(Pt 9):1505-11.
Tight junctions are components of the junctional complex linking neighbouring epithelial cells and are important for barrier formation. Recent evidence suggests that tight junctions also participate in signal transduction mechanisms that regulate epithelial cell proliferation, gene expression, differentiation and morphogenesis. One important class of tight-junction-associated signal transduction mechanism is based on dual localisation of certain proteins both at junctions and in the nucleus. These proteins and their partners participate in various steps of gene expression, ranging from regulation of transcription and chromatin structure to mRNA processing and translation. In cancer tissues, their expression is often deregulated in a manner that suggests that tight junctions function as suppressors of proliferation and transformation. [Abstract/Link to Full Text]

Watt F
JCS Prize. 2006 winner: Satomi Matsuoka.
J Cell Sci. 2007 May 1;120(Pt 9):1503. [Abstract/Link to Full Text]

Richter K, Nessling M, Lichter P
Experimental evidence for the influence of molecular crowding on nuclear architecture.
J Cell Sci. 2007 May 1;120(Pt 9):1673-80.
Many compounds in the cell nucleus are structurally organized. To assess the influence of structural organization on nuclear function, we investigated the physical mechanisms of structure formation by using molecular crowding as a parameter for nuclear integrity. Molecular crowding promotes compaction of macromolecular compounds depending on their size and shape without the need for site-specific interactions. HeLa and MCF7 cells were incubated with hypertonic medium to increase crowding of their macromolecular content as a result of the osmotic loss of water. Supplementation of sucrose, sorbitol or NaCl to the growth medium shifted nuclear organization, observed by fluorescence and electron microscopy, towards compaction of chromatin and segregation of other nuclear compounds. With increasing hypertonic load and incubation time, this nuclear re-organization proceeded gradually, irrespective of the substances used, and reversibly relaxed to a regular phenotype upon re-incubation of cells in isotonic growth medium. Gradual and reversible re-organization are major features of controlled de-mixing by molecular crowding. Of fundamental importance for nuclear function, we discuss how macromolecular crowding could account for the stabilization of processes that involve large, macromolecular machines. [Abstract/Link to Full Text]

Millard TH, Dawson J, Machesky LM
Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties.
J Cell Sci. 2007 May 1;120(Pt 9):1663-72.
IRSp53 is a scaffold protein that contains an IRSp53/MIM homology domain (IMD) that bundles actin filaments and interacts with the small GTPase Rac. IRSp53 also binds to the small GTPase Cdc42 and to Scar/WAVE and Mena/VASP proteins to regulate the actin cytoskeleton. We have characterised a novel IMD-containing protein, insulin receptor tyrosine kinase substrate (IRTKS), which has widespread tissue distribution, is a substrate for the insulin receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters of short actin bundles rather than filopodia-like protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which resembles a WASP-homology 2 (WH2) motif. Addition of the Ct extension to IRSp53 causes an apparent shortening of bundles induced by the IMD in vitro, and in cultured cells, suggesting that the Ct extension of IRTKS modulates the organising activity of the IMD. Lastly, we could not detect actin monomer sequestration by the Ct extension of IRTKS as would be expected with a conventional WH2 motif, but it did interact with actin filaments. [Abstract/Link to Full Text]

Yana I, Sagara H, Takaki S, Takatsu K, Nakamura K, Nakao K, Katsuki M, Taniguchi S, Aoki T, Sato H, Weiss SJ, Seiki M
Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells.
J Cell Sci. 2007 May 1;120(Pt 9):1607-14.
The membrane-anchored matrix metalloproteinase MT1-MMP (also known as Mmp14) plays a key role in the angiogenic process, but the mechanisms underlying its spatiotemporal regulation in the in vivo setting have not been defined. Using whole-mount immunohistochemical analysis and the lacZ gene inserted into the Mmp14 gene, we demonstrate that MT1-MMP vascular expression in vivo is confined largely to the sprouting tip of neocapillary structures where endothelial cell proliferation and collagen degradation are coordinately localized. During angiogenesis in vitro, wherein endothelial cells are stimulated to undergo neovessel formation in the presence or absence of accessory mural cells, site-specific MT1-MMP expression is shown to be controlled by crosstalk between endothelial cells and vascular smooth muscle cells (VSMC). When vessel maturation induced by VSMCs is inhibited by introducing a soluble form of the receptor tyrosine kinase Tek, MT1-MMP distribution is no longer restricted to the endothelial tip cells, but instead distributes throughout the neovessel network in vitro as well as ex vivo. Taken together, these data demonstrate that vascular maturation coordinated by endothelial cell/mural cell interactions redirects MT1-MMP expression to the neovessel tip where the protease regulates matrix remodeling at the leading edge of the developing vasculature. [Abstract/Link to Full Text]

Cho JH, Ko KM, Singaruvelu G, Lee W, Kang GB, Rho SH, Park BJ, Yu JR, Kagawa H, Eom SH, Kim do H, Ahnn J
Functional importance of polymerization and localization of calsequestrin in C. elegans.
J Cell Sci. 2007 May 1;120(Pt 9):1551-8.
Dual roles of calsequestrin (CSQ-1) being the Ca2+ donor and Ca2+ acceptor make it an excellent Ca2+-buffering protein within the sarcoplasmic reticulum (SR). We have isolated and characterized a calsequestrin (csq-1)-null mutant in Caenorhabditis elegans. To our surprise, this mutant csq-1(jh109) showed no gross defects in muscle development or function but, however, is highly sensitive to perturbation of Ca2+ homeostasis. By taking advantage of the viable null mutant, we investigated the domains of CSQ-1 that are important for polymerization and cellular localization, and required for its correct buffering functions. In transgenic animals rescued with various CSQ-1 constructs, the in vivo patterns of polymerization and localization of several mutated calsequestrins were observed to correlate with the structure-function relationship. Our results suggest that polymerization of CSQ-1 is essential but not sufficient for correct cellular localization and function of CSQ-1. In addition, direct interaction between CSQ-1 and the ryanodine receptor (RyR) was found for the first time, suggesting that the cellular localization of CSQ-1 in C. elegans is indeed modulated by RyR through a physical interaction. [Abstract/Link to Full Text]

Sleeman J
A regulatory role for CRM1 in the multi-directional trafficking of splicing snRNPs in the mammalian nucleus.
J Cell Sci. 2007 May 1;120(Pt 9):1540-50.
Distinct pathways of ribonucleoprotein transport exist within the nucleus, connected to their biogenesis and maturation. These occur despite evidence that the major mechanism for their movement within the nucleus is passive diffusion. Using fusions of Sm proteins to YFP, CFP and photoactivatable GFP, I have demonstrated that pathways with uni-directional bulk flow of complexes can be maintained within the nucleus despite multi-directional exchange of individual complexes. Newly imported splicing small nuclear ribonucleoproteins (snRNPs) exchange between Cajal bodies (CBs) within a nucleus and access the cytoplasm, but are unable to accumulate in speckles. By contrast, snRNPs at steady-state exchange freely in any direction between CBs and speckles, but cannot leave the nucleus. In addition to these surprising qualitative observations in the behaviour of nuclear complexes, sensitive live-cell microscopy techniques can detect subtle quantitative disturbances in nuclear dynamics before they have had an effect on overall nuclear organization. Inhibition of the nuclear export factor, CRM1, using leptomycin B results in a change in the dynamics of interaction of newly imported snRNPs with CBs. Together with the detection of interactions of CRM1 with Sm proteins and the survival of motor neurons (SMN) protein, these studies suggest that the export receptor CRM1 is a key player in the molecular mechanism for maintaining these pathways. Its role in snRNP trafficking, however, appears to be distinct from its previously identified role in small nucleolar RNP (snoRNP) maturation. [Abstract/Link to Full Text]

Zhang X, Sejas DP, Qiu Y, Williams DA, Pang Q
Inflammatory ROS promote and cooperate with the Fanconi anemia mutation for hematopoietic senescence.
J Cell Sci. 2007 May 1;120(Pt 9):1572-83.
The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) inhibits hematopoietic stem cell (HSC) expansion, interferes with HSC self-renewal and compromises the ability of HSC to reconstitute hematopoiesis. We have investigated mechanisms by which TNFalpha suppresses hematopoiesis using the genomic instability syndrome Fanconi anemia mouse model deficient for the complementation-group-C gene (Fancc). Examination of senescence makers, such as senescence-associated beta-galactosidase, HP1-gamma, p53 and p16(INK4A) shows that TNFalpha induces premature senescence in bone marrow HSCs and progenitor cells as well as other tissues of Fancc-/- mice. TNFalpha-induced senescence correlates with the accumulation of reactive oxygen species (ROS) and oxidative DNA damage. Neutralization of TNFalpha or deletion of the TNF receptor in Fancc-/- mice (Fancc-/-;Tnfr1-/-) prevents excessive ROS production and hematopoietic senescence. Pretreatment of TNFalpha-injected Fancc-/- mice with a ROS scavenger significantly reduces oxidative base damage, DNA strand breaks and senescence. Furthermore, HSCs and progenitor cells from TNFalpha-treated Fancc-/- mice show increased chromosomal aberrations and have an impaired oxidative DNA-damage repair. These results indicate an intimate link between inflammatory reactive oxygen species and DNA-damage-induced premature senescence in HSCs and progenitor cells, which may play an important role in aging and anemia. [Abstract/Link to Full Text]

Kanda T, Kamiya M, Maruo S, Iwakiri D, Takada K
Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids.
J Cell Sci. 2007 May 1;120(Pt 9):1529-39.
In eukaryotes, many latent viruses replicate as extrachromosomal molecules, called episomes, and efficiently segregate to daughter cells by noncovalently attaching to mitotic chromosomes. To understand the mechanism governing the processes, we analyzed the detailed subcellular localization of Epstein-Barr virus (EBV) genomes and a viral protein EBNA1, a bridging molecule between viral genomes and cellular chromatin. In the cells that were infected with a recombinant EBV expressing epitope-tagged EBNA1, EBNA1 localized to intranuclear punctate dots, which coincided with the localization of EBV genomes as revealed by fluorescence in situ hybridization (FISH). A significant number of EBNA1 dots were found to localize symmetrically on sister chromatids of mitotic chromosomes. Such symmetrical localization of EBNA1 dots was observed in prematurely condensed G2 chromosomes as well, correlating with the presence of closely spaced double dots of EBNA1 in G2-phase-enriched cells. The EBNA1 double dots were occasionally interconnected by the FISH signals of EBV episomes, exhibiting a dumbbell-like appearance. Thus, we propose that the partitioning of EBNA1 molecules onto sister chromatids during cellular DNA replication underlies the non-stochastic segregation of extrachromosomally replicating viral genomes. [Abstract/Link to Full Text]

Neel NF, Lapierre LA, Goldenring JR, Richmond A
RhoB plays an essential role in CXCR2 sorting decisions.
J Cell Sci. 2007 May 1;120(Pt 9):1559-71.
The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling--events that are required for optimal chemotaxis. [Abstract/Link to Full Text]

van Tijn P, de Vrij FM, Schuurman KG, Dantuma NP, Fischer DF, van Leeuwen FW, Hol EM
Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease.
J Cell Sci. 2007 May 1;120(Pt 9):1615-23.
The ubiquitin-proteasome system is the main regulated intracellular proteolytic pathway. Increasing evidence implicates impairment of this system in the pathogenesis of diseases with ubiquitin-positive pathology. A mutant ubiquitin, UBB(+1), accumulates in the pathological hallmarks of tauopathies, including Alzheimer's disease, polyglutamine diseases, liver disease and muscle disease and serves as an endogenous reporter for proteasomal dysfunction in these diseases. UBB(+1) is a substrate for proteasomal degradation, however it can also inhibit the proteasome. Here, we show that UBB(+1) properties shift from substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB(+1) expression system. At low expression levels, UBB(+1) was efficiently degraded by the proteasome. At high levels, the proteasome failed to degrade UBB(+1), causing its accumulation, which subsequently induced a reversible functional impairment of the ubiquitin-proteasome system. Also in brain slice cultures, UBB(+1) accumulation and concomitant proteasome inhibition was only induced at high expression levels. Our findings show that by varying UBB(+1) expression levels, the dual proteasome substrate and inhibitory properties can be optimally used to serve as a research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease. [Abstract/Link to Full Text]

Chandramouly G, Abad PC, Knowles DW, Lelièvre SA
The control of tissue architecture over nuclear organization is crucial for epithelial cell fate.
J Cell Sci. 2007 May 1;120(Pt 9):1596-606.
The remodeling of nuclear organization during differentiation and the dramatic alteration of nuclear organization associated with cancer development are well documented. However, the importance of tissue architecture in the control of nuclear organization remains to be determined. Differentiation of mammary epithelial cells into functional tissue structures, in three-dimensional culture, is characterized by a specific tissue architecture (i.e. a basoapical polarity axis), cell cycle exit and maintenance of cell survival. Here we show that induction of partial differentiation (i.e. basal polarity only, cell cycle exit and cell survival) by epigenetic mechanisms in malignant breast cells is sufficient to restore features of differentiation-specific nuclear organization, including perinucleolar heterochromatin, large splicing factor speckles, and distinct nuclear mitotic apparatus protein (NuMA) foci. Upon alteration of nuclear organization using an antibody against NuMA, differentiated non-neoplastic cells undergo apoptosis, whereas partially differentiated malignant cells enter the cell cycle. Non-neoplastic cells cultured under conditions that prevent the establishment of apical polarity also enter the cell cycle upon NuMA antibody treatment. These findings demonstrate that the differentiation status rather than the non-neoplastic or neoplastic origin of cells controls nuclear organization and suggest a link between nuclear organization and epigenetic mechanisms dictated by tissue architecture for the control of cell behavior. [Abstract/Link to Full Text]

Baron DM, Kabututu ZP, Hill KL
Stuck in reverse: loss of LC1 in Trypanosoma brucei disrupts outer dynein arms and leads to reverse flagellar beat and backward movement.
J Cell Sci. 2007 May 1;120(Pt 9):1513-20.
Axonemal dyneins are multisubunit molecular motors that provide the driving force for flagellar motility. Dynein light chain 1 (LC1) has been well studied in Chlamydomonas reinhardtii and is unique among all dynein components as the only protein known to bind directly to the catalytic motor domain of the dynein heavy chain. However, the role of LC1 in dynein assembly and/or function is unknown because no mutants have previously been available. We identified an LC1 homologue (TbLC1) in Trypanosoma brucei and have investigated its role in trypanosome flagellar motility using epitope tagging and RNAi studies. TbLC1 is localized along the length of the flagellum and partitions between the axoneme and soluble fractions following detergent and salt extraction. RNAi silencing of TbLC1 gene expression results in the complete loss of the dominant tip-to-base beat that is a hallmark of trypanosome flagellar motility and the concomitant emergence of a sustained reverse beat that propagates base-to-tip and drives cell movement in reverse. Ultrastructure analysis revealed that outer arm dyneins are disrupted in TbLC1 mutants. Therefore LC1 is required for stable dynein assembly and forward motility in T. brucei. Our work provides the first functional analysis of LC1 in any organism. Together with the recent findings in T. brucei DNAI1 mutants [Branche et al. (2006). Conserved and specific functions of axoneme components in trypanosome motility. J. Cell Sci. 119, 3443-3455], our data indicate functionally specialized roles for outer arm dyneins in T. brucei and C. reinhardtii. Understanding these differences will provide a more robust description of the fundamental mechanisms underlying flagellar motility and will aid efforts to exploit the trypanosome flagellum as a drug target. [Abstract/Link to Full Text]

Castillo-Lluva S, Alvarez-Tabarés I, Weber I, Steinberg G, Pérez-Martín J
Sustained cell polarity and virulence in the phytopathogenic fungus Ustilago maydis depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family.
J Cell Sci. 2007 May 1;120(Pt 9):1584-95.
Cyclin-dependent kinases from the Cdk5/Pho85 family are thought to play important roles in morphogenesis in organisms as diverse as yeast and humans. Here we used the corn smut fungus Ustilago maydis to address the role of Cdk5/Pho85 kinases in the morphogenesis and virulence of dimorphic phytopathogens. We found that Cdk5 is essential for growth in U. maydis. A temperature-sensitive cdk5 mutant caused cell wall and morphology defects at the restrictive temperature. Actin patches labeled with a fimbrin-GFP fusion protein were delocalized and a GFP-Myo5 fusion was directed towards the growing cell pole and rapidly dissociated from the tip. These defects were found to be due to an impairment in the maintenance of cell polarity. Our results indicated that Cdk5 is required for the activity of Rac1, probably at the level of the localization of its GEF, Cdc24. Cdk5 was required for full virulence, probably because mutant cells are unable to sustain the dramatic polar growth required for the formation of the infective structures. These results support a major role for morphogenesis in the virulence program of dimorphic fungi. [Abstract/Link to Full Text]

Schaub S, Meister JJ, Verkhovsky AB
Analysis of actin filament network organization in lamellipodia by comparing experimental and simulated images.
J Cell Sci. 2007 Apr 15;120(Pt 8):1491-500.
Protrusion of lamellipodia during cell migration depends on the assembly of actin network. The assembly mechanism, based on dendritic filament branching, has been investigated in reconstituted in vitro systems, but little is known about the dynamical and structural properties of the actin network in the lamellipodia of migrating cells. The length and orientation of filaments are difficult to measure directly in either optical or electron microscopy images because of the high filament density and overlapping of individual filaments. Here, we use the non-uniformity of optical images of the lamellipodia to extract information about the structural and dynamical properties of the underlying actin network. To determine the relationship between the image features and the properties of the network, we performed simulations of actin network assembly, based on the hypothesis of stochastic branching and capping of filaments, and produced computed ;fluorescence' and ;electron microscopy' images of the simulated network. By varying simulation parameters, in particular the actin filament density, length and orientation, we closely reproduced the contrast and the characteristic diagonal criss-cross pattern observed in the experimental optical images. Thus, matching the images of the simulated network to the experimental images allowed us to estimate parameters of actin filament network in lamellipodia. [Abstract/Link to Full Text]

Jakymiw A, Pauley KM, Li S, Ikeda K, Lian S, Eystathioy T, Satoh M, Fritzler MJ, Chan EK
The role of GW/P-bodies in RNA processing and silencing.
J Cell Sci. 2007 Apr 15;120(Pt 8):1317-23.
GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. The formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures. [Abstract/Link to Full Text]

Tscharntke M, Pofahl R, Chrostek-Grashoff A, Smyth N, Niessen C, Niemann C, Hartwig B, Herzog V, Klein HW, Krieg T, Brakebusch C, Haase I
Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1.
J Cell Sci. 2007 Apr 15;120(Pt 8):1480-90.
To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of keratinocyte proliferation and migration. [Abstract/Link to Full Text]

Li Y, Clough N, Sun X, Yu W, Abbott BL, Hogan CJ, Dai Z
Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway.
J Cell Sci. 2007 Apr 15;120(Pt 8):1436-46.
Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function. [Abstract/Link to Full Text]

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Recent Articles in Journal of Cellular and Molecular Medicine

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Beretta L, Santaniello A, Cappiello F, Barili M, Scorza R
No evidence for a role of the proximal IL-6 G/C -174 single nucleotide polymorphism in Italian patients with systemic sclerosis.
J Cell Mol Med. 2007 Jul-Aug;11(4):896-8; author reply 898-9. [Abstract/Link to Full Text]

Sfrent-Cornateanu R, Mihai C, Balan S, Ionescu R, Moldoveanu E
The IL-6 promoter polymorphism is associated with disease activity and disability in systemic sclerosis.
J Cell Mol Med. 2006 Oct-Dec;10(4):955-9.
Systemic sclerosis (SSc) is a rare, autoimmune disease characterized by cutaneous and visceral fibrosis. Interleukin- 6 (IL-6) is involved in the pathogenesis of many immune-mediated diseases. IL-6 plays an important role in the initiation and promotion of fibrosis. The polymorphism in the position -174 (G/C) of the promoter region of the IL-6 gene (IL-6pr) may alter the expression of the gene. Complete linkage disequilibrium was observed between the -174 and -597 alleles. The aim of this study is to investigate the possible influence of -597 (-174) IL-6pr polymorphism on the susceptibility and/or the clinical course of SSc in Romanian population. Genotyping of -597 variant was performed by an RFLP method on 20 SSc patients and 26 healthy subjects. Patients having the homozygous GG (-597) genotype had higher disease activity and disability scores than heterozygous GA patients: the European Scleroderma Study Group (EScSG) disease activity score was 5.0 +/- 3.3 in homozygous GG subjects vs. 2.4 +/- 3.6 in heterozygous GA patients (p < 0.05), and the Disability Index of the Health Assessment Questionnaire (HAQ-DI) was 1.42 +/- 1.04 in homozygous GG subjects vs. 0.53 +/- 0.55 in heterozygous GA patients (p < 0.05). No difference was observed in the distribution of allele frequencies between SSc patients and healthy controls. Conclusions: The GG homozygosis was found to be associated with a higher degree of illness activity and disability in SSc patients. No statistically significant differences were found between SSc patients and healthy controls with respect to the -597 allele distribution. [Abstract/Link to Full Text]

Kostin S
Zonula occludens-1 and connexin 43 expression in the failing human heart.
J Cell Mol Med. 2007 Jul-Aug;11(4):892-5.
Focal disorganization of gap junctional distribution and down-regulation of the major gap junctional protein connexin 43 are typical features of myocardial remodelling in the failing human heart. Increasing evidence indicates that connexin 43 interacts with zonula-occludens-1 (ZO-1), and it has recently been shown that ZO-1 promotes the formation and growth of gap junctional plaques. In the present study, distribution patterns of ZO-1 and connexin 43 were studied in normal and in heart failure patients using double-label immunohistochemistry and confocal microscopy. ZO-1 was found to be co-localized with connexin 43 at intercalated disks. Importantly, in patients with heart failure due to dilated or ischaemic cardiomyopathy, areas of diminished connexin 43 expression were characterized by a markedly reduced ZO-1 staining. Based on these data it is concluded that in patients with heart failure, down-regulation of ZO-1 matches the diminished expression levels of connexin 43, suggesting that ZO-1 plays an important role in gap junction formation and gap junction plaque stability. [Abstract/Link to Full Text]

Depuydt CE, Boulet GA, Horvath CA, Benoy IH, Vereecken AJ, Bogers JJ
Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.
J Cell Mol Med. 2007 Jul-Aug;11(4):881-91.
The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool. [Abstract/Link to Full Text]

Kuttler B, Wanka H, Klöting N, Gerstmayer B, Volk HD, Sawitzki B, Ritter T
Ex vivo gene transfer of viral interleukin-10 to BB rat islets: no protection after transplantation to diabetic BB rats.
J Cell Mol Med. 2007 Jul-Aug;11(4):868-80.
Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation. [Abstract/Link to Full Text]

Mas ID, Biscardi A, Schnitzler CM, Ripamonti U
Bone loss in the ovariectomized baboon Papio ursinus: densitometry, histomorphometry and biochemistry.
J Cell Mol Med. 2007 Jul-Aug;11(4):852-67.
To develop a non-human primate model of systemic bone loss after ovariectomy, 24 ovariectomized (OVX) and eight control (non-OVX) female baboons Papio ursinus were investigated over a period of 48 months using bone mineral density (BMD), iliac crest bone histomorphometry, bone turnover markers, and variables of calcium metabolism. Lumbar spine (L1-L4) BMD measured by dual energy X-ray absorptiometry (DXA) decreased in OVX animals in the first 12 months (-7.6%) and showed a slow trend towards recovery after 24 months. Controls showed a slow increase in spinal BMD over 4 years (+9.7%). Total hip BMD decreased slowly up to 48 months in all animals (OVX -12.6%versus controls -10%); this indicated that OVX had a limited effect on total hip BMD. Forearm BMD did not change. The significant decrease in trabecular bone volume (TBV) of the iliac crest from baseline to 12 months was followed by some recovery. Microarchitectural deterioration of trabecular bone in OVX animals was demonstrated by a decline in trabecular number and an increase in trabecular spacing. These changes were also evident on sections of whole vertebrae, proximal femora and iliac crests. Changes in iliac TBV reflected spinal but not hip BMD changes in the OVX animals. Static and dynamic histomorphometric variables indicated that bone turnover was increased for 36 months following OVX. Controls showed no changes in histomorphometric variables. Bone specific alkaline phosphatase (ALPs) in OVX animals remained elevated throughout the study; osteocalcin (OC) was significantly elevated only at 6 and 12 months, and deoxypyridinoline (Pyr-D) was elevated at 12 months but declined after 24 months. ALPs was thus more sensitive to the long-term effects of OVX than were OC or Pyr-D. Controls showed no changes in bone turnover markers. This study showed consistent deleterious changes in lumbar BMD, bone histomorphometry with microarchitectural deterioration together with altered biochemical markers of bone turnover in the first 12 months after OVX. Since these changes resemble those in post-menopausal women, the non-human primate Papio ursinus is suitable for the study of bone loss in post-menopausal women. [Abstract/Link to Full Text]

Sultana R, Reed T, Perluigi M, Coccia R, Pierce WM, Butterfield DA
Proteomic identification of nitrated brain proteins in amnestic mild cognitive impairment: a regional study.
J Cell Mol Med. 2007 Jul-Aug;11(4):839-51.
Oxidative stress is an imbalance between the level of antioxidants and oxidants in a cell. Oxidative stress has been shown in brain of subjects with mild cognitive impairment (MCI) as well Alzheimer's disease (AD). MCI is considered as a transition phase between control and AD. The focus of the current study was to identify nitrated proteins in the hippocampus and inferior parietal lobule (IPL) brain regions of subjects with amnestic MCI using proteomics. The identified nitrated proteins in MCI brain were compared to those previously reported to be nitrated and oxidatively modified in AD brain, a comparison that might provide an invaluable insight into the progression of the disease. [Abstract/Link to Full Text]

Jones J, Berkhoff S, Weich E, Engl T, Wedel S, Relja B, Jonas D, Blaheta RA
Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes.
J Cell Mol Med. 2007 Jul-Aug;11(4):826-38.
Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive. [Abstract/Link to Full Text]

Oprica M, Hjorth E, Spulber S, Popescu BO, Ankarcrona M, Winblad B, Schultzberg M
Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic overexpression of IL-1 receptor antagonist.
J Cell Mol Med. 2007 Jul-Aug;11(4):810-25.
Inflammation is associated with both acute and chronic neurological disorders, including stroke and Alzheimer's disease (AD). Cytokines such as interleukin (IL)-1 have several activities in the brain both under physiological and pathophysiological conditions. The objective of this study was to evaluate consequences of the central blockade of IL-1 transmission in a previously developed transgenic mouse strain with brain-directed overexpression of human soluble IL-1 receptor antagonist (Tg hsIL-1ra). Effects on brain morphology and brain levels of the AD-related proteins beta-amyloid precursor protein (APP) and presenilin 1(PS1), as well as the levels of IL-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were analysed in homozygotic and heterozygotic mice and wild type (WT) controls, of both genders and of young (30-40 days) and adult (13-14 months) age. A marked reduction in brain volume was observed in transgenic mice as determined by volumetry. Western blot analysis showed higher levels of APP, but lower levels of PS1, in adult animals than in young ones. In the cerebellum, heterozygotic (Tg hsIL-1ra(+/-)) mice had lower levels of APP and PS1 than WT mice. With one exception, there were no genotypic differences in the levels of IL-1beta, IL-6 and TNF-alpha. The cytokine levels were generally higher in adult than in young mice. In conclusion, the chronic blockade of IL-1 signalling in the brain was associated with an atrophic phenotype of the brain, and with modified levels of APP and PS1. Brain-directed overexpression of hsIL-1ra was not followed by major compensatory changes in the levels of pro-inflammatory cytokines. [Abstract/Link to Full Text]

Kumar S, Kasseckert S, Kostin S, Abdallah Y, Piper HM, Steinhoff G, Reusch HP, Ladilov Y
Importance of bicarbonate transport for ischaemia-induced apoptosis of coronary endothelial cells.
J Cell Mol Med. 2007 Jul-Aug;11(4):798-809.
Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia-induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas-pase- 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 300 mumol/l) or exposed to ischaemia in bicarbonate free, 4-(2-hydroxyethyl)-I-piperazi-neethanesulphonic acid (HEPES)-buffered medium. Simulated ischaemia in bicarbonate-buffered medium (Bic) increased caspase-3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase-3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate-free, HEPES-buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca(2+) accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor-dependent pathway (caspase-8 cleavage) and endoplasmic reticulum- dependent pathway (caspase-12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia-induced apoptosis of coronary EC by suppression of mitochondria-dependent apoptotic pathway. [Abstract/Link to Full Text]

Das M, Das S, Das DK
Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium.
J Cell Mol Med. 2007 Jul-Aug;11(4):788-97.
Angiotensin II (Ang II) has been found to exert preconditioning (PC)-like effect in mammalian hearts. The present investigation reported for the first time a unique mitogen activated protein (MAP) kinase signalling in Ang II PC of the heart involving lipid rafts, which generated a survival signal by differentially associating MAP kinases with caveolin. A group of rat hearts was treated with Ang II in the absence or presence of NADPH oxidase inhibitor, apocynin or a cell permeable reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (NAC). Ang II pre-treatment improved post-ischaemic ventricular recovery, myocardial infraction and decreased the number of cardiomyocyte apoptosis indicating PC effect of Ang II. Both apocynin and NAC abolished the PC ability of Ang II. In Ang II treated heart, there was a decreased association of p38MAPKbeta & extracellular-signal regulated kinase (ERK) 1/ 2 (anti-death signalling component) with caveolin while there was an increased association of p38MAPKalpha & Jun N-terminal kinase (JNK) (death signalling component) indicating reduced amount of death signal components and increased amount of anti-death signalling components being available to the Ang II treated heart to generate a survival signal, which was reversed with NAC or apocynin. The survival signal was also demonstrated by increased phosphorylation of serine/threonine-protein kinase B (AKT) and enhanced induction of expression of Bcl-2 during Ang II PC and its reversal with NAC & apocynin treated heart. [Abstract/Link to Full Text]

Junquera C, Martínez-Ciriano C, Castiella T, Serrano P, Azanza MJ, Junquera SR
Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium.
J Cell Mol Med. 2007 Jul-Aug;11(4):776-87.
Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duodenum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce perinuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular contacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium showing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-Kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells. [Abstract/Link to Full Text]

Pucovský V, Harhun MI, Povstyan OV, Gordienko DV, Moss RF, Bolton TB
Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell.
J Cell Mol Med. 2007 Jul-Aug;11(4):764-75.
This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle alpha-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries. [Abstract/Link to Full Text]

Braet F, Ratinac K
Creating next-generation microscopists: structural and molecular biology at the crossroads.
J Cell Mol Med. 2007 Jul-Aug;11(4):759-63.
This paper highlights the importance of advanced microscopy and microanalysis in the pursuit of quality research in the biological and life sciences.With the growing complexity of modern microscopes, there is substantial risk of incorrect use or misinterpretation of data by the inexperienced researcher. This paper emphasizes the need for collaboration between biological microscopists and molecular biologists, within the context of centralized facilities and supported by first-class training, to fully realize the power of these unique instruments in modern biology and to create the next generation of molecular microscopists. [Abstract/Link to Full Text]

Lindstedt KA, Mäyränpää MI, Kovanen PT
Mast cells in vulnerable atherosclerotic plaques--a view to a kill.
J Cell Mol Med. 2007 Jul-Aug;11(4):739-58.
The aim of the present review is to discuss the participation of mast cells in the pathogenesis of erosion and rupture of atherosclerotic plaques, the major causes behind acute coronary syndromes and myocardial infarction. We present ex vivo observations describing mast cells and their activation in human atherosclerotic plaques and discuss in vitro and in vivo data showing that mast cells are potential regulators of inflammation, immunity and adverse remodeling, including matrix remodeling and cell death. Furthermore, we focus on studies that have been performed with human tissues and human mast cells, but when appropriate, we also discuss observations made in animal models. Finally, we present potential pharmacological means to modulate mast cell responses in the arterial vessel walls. [Abstract/Link to Full Text]

Presta M, Camozzi M, Salvatori G, Rusnati M
Role of the soluble pattern recognition receptor PTX3 in vascular biology.
J Cell Mol Med. 2007 Jul-Aug;11(4):723-38.
Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with short-pentraxins C-reactive protein and serum amyloid P component and possesses an unique N-terminal domain. These structural features suggest that PTX3 may have both overlapping and distinct biological/ligand recognition properties when compared to short-pentraxins. PTX3 serves as a mechanism of amplification of inflammation and innate immunity. Indeed, vessel wall elements produce high amounts of PTX3 during inflammation and the levels of circulating PTX3 increase in several pathological conditions affecting the cardiovascular system. PTX3 exists as a free or extracellular matrix-associated molecule and it binds the complement fraction C1q. PTX3 binds also apoptotic cells and selected pathogens, playing a role in innate immunity processes. In endothelial cells and macrophages, PTX3 upregulates tissue factor expression, suggesting its action as a regulator of endothelium during thrombogenesis and ischaemic vascular disease. Finally, PTX3 binds the angiogenic fibroblast growth factor-2, thus inhibiting its biological activity. Taken together, these properties point to a role for PTX3 during vascular damage, angiogenesis, atherosclerosis, and restenosis. [Abstract/Link to Full Text]

Herrmann J, Gressner AM, Weiskirchen R
Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?
J Cell Mol Med. 2007 Jul-Aug;11(4):704-22.
At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts. [Abstract/Link to Full Text]

Rhee HJ, Kim EJ, Lee JK
Physiological polyamines: simple primordial stress molecules.
J Cell Mol Med. 2007 Jul-Aug;11(4):685-703.
Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression, cell proliferation and modulation of cell signalling. Reports that the polyamines with cytoprotective activities were induced by diverse stresses raised the hypothesis that physiological polyamines may play a role in inducing stress response. In a wide range of organisms, physiological polyamines were not only induced by diverse stresses, such as reactive oxygen species (ROS), heat, ultraviolet (UV) and psychiatric stress but were able to confer beneficial effects for survival. Recent biochemical and genetic evidences show that polyamines can function as an ROS scavenger, acid tolerance factor and chemical chaperone, and positive regulators for expression of stress response genes which may explain their protective functions against diverse stresses. Taken together, these data suggest that physiological polyamines can function as primordial stress molecules in bacteria, plants and mammals, and may play an essential role in regulation of pathogen-host interactions. [Abstract/Link to Full Text]

Jones AT
Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides.
J Cell Mol Med. 2007 Jul-Aug;11(4):670-84.
Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides. [Abstract/Link to Full Text]

Hutmacher DW, Cool S
Concepts of scaffold-based tissue engineering--the rationale to use solid free-form fabrication techniques.
J Cell Mol Med. 2007 Jul-Aug;11(4):654-69.
A paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds combined with cells or biological molecules to repair and/or regenerate tissues. One of the potential benefits offered by solid free-form fabrication technology (SFF) is the ability to create scaffolds with highly reproducible architecture and compositional variation across the entire scaffold, due to its tightly controlled computer-driven fabrication. In this review, we define scaffold properties and attempt to provide some broad criteria and constraints for scaffold design in bone engineering.We also discuss the application-specific modifications driven by surgeon's requirements in vitro and/or in vivo. Next, we review the current use of SFF techniques in scaffold fabrication in the context of their clinical use in bone regeneration. Lastly, we comment on future developments in our groups, such as the functionalization of novel composite scaffolds with combinations of growth factors; and more specifically the promising area of heparan sulphate polysaccharide immobilization within the bone tissue engineering arena. [Abstract/Link to Full Text]

Lajoie P, Nabi IR
Regulation of raft-dependent endocytosis.
J Cell Mol Med. 2007 Jul-Aug;11(4):644-53.
Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveolae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1. [Abstract/Link to Full Text]

Stan RV
Endothelial stomatal and fenestral diaphragms in normal vessels and angiogenesis.
J Cell Mol Med. 2007 Jul-Aug;11(4):621-43.
Vascular endothelium lines the entire cardiovascular system where performs a series of vital functions including the control of microvascular permeability, coagulation inflammation, vascular tone as well as the formation of new vessels via vasculogenesis and angiogenesis in normal and disease states. Normal endothelium consists of heterogeneous populations of cells differentiated according to the vascular bed and segment of the vascular tree where they occur. One of the cardinal features is the expression of specific subcellular structures such as plas-malemmal vesicles or caveolae, transendothelial channels, vesiculo-vacuolar organelles, endothelial pockets and fenestrae, whose presence define several endothelial morphological types. A less explored observation is the differential expression of such structures in diverse settings of angiogenesis. This review will focus on the latest developments on the components, structure and function of these specific endothelial structures in normal endothelium as well as in diverse settings of angiogenesis. [Abstract/Link to Full Text]

Collas P, Noer A, Timoskainen S
Programming the genome in embryonic and somatic stem cells.
J Cell Mol Med. 2007 Jul-Aug;11(4):602-20.
In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state. [Abstract/Link to Full Text]

Cagalinec M, Chorvat D, Mateasik A, Bacharova L
Sustained spiral calcium wave patterns in rat ventricular myocytes.
J Cell Mol Med. 2007 May-Jun;11(3):598-9. [Abstract/Link to Full Text]

Suciu L, Popescu LM, Gherghiceanu M
Human placenta: de visu demonstration of interstitial Cajal-like cells.
J Cell Mol Med. 2007 May-Jun;11(3):590-7.
Traditional interstitial cells of Cajal (ICC) are present in the digestive tube and are supposed to act as pacemakers and neuromodulators. However, interstitial Cajal-like cells (ICLCs) were found outside the gastrointestinal tract, in various organs (e.g. ureter, bladder, fallopian tube, uterus, pancreas, mammary gland, myocardium etc.) and looking for such ICLC is a priority in our laboratories.We report here unequivocal visual evidence that ICLCs are present in the mesenchymal tissue of the villi from human term placenta.The following methods were used: a. vital staining with methylene blue (cryosections); b. silver impregnation (paraffin sections); c. Epon-embedded sections (approximately 1 microm) of glutaraldehyde/osmium fixed tissue, stained with toluidine blue; d. primary cell cultures (or second-passage cells) to reveal the characteristic, very long, moniliform cell processes and mitochondrial localization at dilations (molecular fluorescence probe: Mito Tracker Green); e. immunofluorescence for c-kit/CD117 marker or other characteristic proteins; f. transmission electron microscopy to establish the identity of ICLC. [Abstract/Link to Full Text]

Ribatti D, Nico B, Maxia C, Longo V, Murtas D, Mangieri D, Perra MT, De Giorgis M, Piras F, Crivellato E, Sirigu P
Neovascularization and mast cells with tryptase activity increase simultaneously in human pterygium.
J Cell Mol Med. 2007 May-Jun;11(3):585-9.
Mast cells (MC) have been implicated in both normal and pathological angiogenesis, such as that in chronic inflammatory diseases and tumors. This assumption is partially supported by the close structural association between MC and blood vessels and the recruitment of these cells during tumor growth. MC release a number of angiogenic factors among which tryptase, a serine protease stored in MC granules, is one of the most active. In this study, we correlate the extent of angiogenesis with the number of tryptase-reactive MC in tissue fragments from pterygium and normal bulbar conjunctiva investigated by immunohistochemistry, using two murine monoclonal antibodies against the endothelial cell marker CD31 and the MC marker tryptase. Angiogenesis, measured as microvessel density, was highly correlated with MC tryptase-positive cell count in pterygium tissues. These results suggest that the characteristic neovascularization observed in pterygium may be sustained, at least in part, by MC angiogenic mediators, in particular tryptase. [Abstract/Link to Full Text]

Perkins D, Chong H, Irvine B, Domagalski J
Genital co-infection with herpes simplex viruses type 1 and 2: comparison of real-time PCR assay and traditional viral isolation methods.
J Cell Mol Med. 2007 May-Jun;11(3):581-4.
We report the clinical case of a genital outbreak with both Herpes Simplex Type 1 (HSV-1) and Herpes Simplex Type 2 (HSV-2) during pregnancy. Herpes was presumptively identified by clinical presentation of lesion and Tzanck smear while serotypes were identified by cell culture and polymerase chain reaction (PCR). This case report highlights the need for increased surveillance of both serotypes in genital infection of pregnant women for effective disease management and reduced risk of transmission. Increasing rates of genital infection with HSV-1, the possibility of genital co-infection with HSV-1 and HSV-2 and the non-specificity and lack of sensitivity of traditional viral isolation methods may lead to under-diagnosis of genital HSV-1 infections unless molecular diagnostic methods, such as polymerase chain reaction (PCR) are routinely deployed in the clinical setting. [Abstract/Link to Full Text]

Romanitan MO, Popescu BO, Winblad B, Bajenaru OA, Bogdanovic N
Occludin is overexpressed in Alzheimer's disease and vascular dementia.
J Cell Mol Med. 2007 May-Jun;11(3):569-79.
The tight junctions (TJs) are key players in the control of blood-brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimer's disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin-expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin-expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs. [Abstract/Link to Full Text]

Klerk CP, Smorenburg SM, Spek CA, Van Noorden CJ
Colon cancer metastasis in mouse liver is not affected by hypercoagulability due to Factor V Leiden mutation.
J Cell Mol Med. 2007 May-Jun;11(3):561-8.
Clinical trials have shown life-prolonging effects of antithrombotics in cancer patients, but the molecular mechanisms remain unknown due to the multitude of their effects. We investigated in a mouse model whether one of the targets of antithrombotic therapy, fibrin deposition, stimulates tumour development. Fibrin may provide either protection of cancer cells in the circulation against mechanical stress and the immune system, or form a matrix for tumours and/or angiogenesis in tumours to develop. Mice homozygous for Factor V Leiden (FVL), a mutation in one of the coagulation factors that facilitates fibrin formation, were used to investigate whether hypercoagulability affects tumour development in an experimental metastasis model. Liver metastases of colon cancer were induced in mice with the FVL mutation and wild-type littermates. At day 21, number and size of tumours at the liver surface, fibrin/fibrinogen distribution, vessel density and the presence of newly formed vessels in tumours were analysed. Number and size of tumours did not differ between mice with and without the FVL mutation. Fibrin/fibrinogen was found in the cytoplasm of hepatocytes and cancer cells, in blood vessels in liver and tumour tissue and diffusely distributed outside vessels in tumours, indicating leaky vessels. Vessel density and angiogenesis varied widely between tumours, but a pre-dominance for vessel-rich or vessel-poor tumours or vessel formation could not be found in either genotype. In conclusion, the FVL mutation has no effect on the development of secondary tumours of colon cancer in livers of mice. Fibrin deposition and thus inhibition of fibrin formation by anticoagulants do not seem to affect tumour development in this model. [Abstract/Link to Full Text]

Zhang F, Pasumarthi KB
Ultrastructural and immunocharacterization of undifferentiated myocardial cells in the developing mouse heart.
J Cell Mol Med. 2007 May-Jun;11(3):552-60.
The recent discovery of several myogenic cardiac progenitor cells in the post-natal heart suggests that some myocardial cells may remain undifferentiated during embryonic development. In this study, we examined the subcellular characteristics of the embryonic (E) mouse ventricular myocardial cells using transmission electron microscopy (TEM). At the ultrastructural level, we identified three different cell populations within the myocardial layer of the E11.5 heart. These cells were designated as undifferentiated cells (43 +/- 6%), moderately differentiated cells (43 +/- 2%) and mature cardiomyocytes (14 +/- 4%). Undifferentiated cells contained a large nucleus and sparse cytoplasm with no myofibrillar bundles. Moderately differentiated cells contained randomly arranged myofilaments in the cytoplasm. In contrast, mature cardiomyocytes contained well-developed sarcomere structures. We also confirmed the presence of similar undifferentiated cells albeit at low levels in the E16.5 ( approximately 20%) and E18.5 ( approximately 7%) myocardium. Further we used immunogold labeling technique to test whether these distinct cell populations were also positive for markers such as Nkx2.5, ISL1 and ANF. A preponderance of anti-Nkx2.5 label was found in the undifferentiated and moderately differentiated cell types. Anti-ANF label was found only in the cytoplasmic compartment of moderately differentiated and mature myocardial cells. All of the undifferentiated cells were negative for anti-ANF labeling. We did not find immuno-gold labeling with ISL1 in any of the three myocardial cell types. Based on these results, we suggest that embryonic myocardial cell differentiation is a gradual process and undifferentiated cells expressing Nkx2.5 in post-chamber myocardium may represent a progenitor cell population while cells expressing Nkx2.5 and ANF represent differentiating myocytes. [Abstract/Link to Full Text]

Andryushkova AA, Kuznetsova IA, Bineva VN, Toporkova LB, Sakhno LV, Tikhonova MA, Chernykh ER, Orlovskaya IA, Nevinsky GA
Formation of different abzymes in autoimmune-prone MRL-lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors.
J Cell Mol Med. 2007 May-Jun;11(3):531-51.
It was shown that IgGs from the sera of 2-7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2-3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs.The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti-DNA Abs and urine protein. However, development of autoimmune (AI)-reactions and the increase in the sera anti-DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre-disease, disease, pregnancy and lactation leading to production of different auto-antibodies and abzymes are discussed. [Abstract/Link to Full Text]

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Recent Articles in Molecules and Cells

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Hur J, Buckley K, Lee S, Davis K
Transcriptional activator elements for curtovirus C1 expression reside in the 3' coding region of ORF C1.
Mol Cells. 2007 Feb 28;23(1):80-7.
Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV), members of curtoviruses, encode seven open reading frames (ORFs) within a approximately 3 kb genome. One of these viral ORFs, C1, is known to play an important role in the early stage of viral infection in plants during initiation of viral DNA replication. We used promoter:: reporter (beta-glucuronidase) gene fusions in transgenic Ara-bidopsis to identify the putative promoter region of BCTV ORF C1. Unlike other geminiviruses, the intergenic region of BCTV was not sufficient to promote C1 expression in transgenic plants. When sequences extending into the coding region of C1 were tested, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This expression was not dependent on the presence of the intergenic regions or proximal 5' portions of the C1 coding region. Transgenic plants expressing a reporter gene under control of the putative complete C1 promoter were inoculated with virus to determine if any viral transcript affected C1 expression. Virus inoculated plants did not show any altered pattern or change in of reporter gene expression level. These results suggest that (1) important transcriptional activator elements for C1 expression reside in the 3' portion of C1 coding area itself, (2) C1 protein does not auto-regulate its own expression and (3) C1 expression of two curtoviruses is controlled differently compared to other geminiviruses. [Abstract/Link to Full Text]

Cho YI, Jiang W, Chin JH, Piao Z, Cho YG, McCouch S, Koh HJ
Identification of QTLs associated with physiological nitrogen use efficiency in rice.
Mol Cells. 2007 Feb 28;23(1):72-9.
Demand for low-input sustainable crop cultivation is increasing to meet the need for environment-friendly agriculture. Consequently, developing genotypes with high nutrient use efficiency is one of the major objectives of crop breeding programs. This study was conducted to identify QTLs for traits associated with physiological nitrogen use efficiency (PNUE). A recombinant inbred population (DT-RILs) between Dasanbyeo (a tongil type rice, derived from an indica x japonica cross and similar to indica in its genetic make-up) and TR22183 (a Chinese japonica variety) consisting of 166 F8 lines was developed and used for mapping. A frame map of 1,409 cM containing 113 SSR and 103 STS markers with an average interval of 6.5 cM between adjacent marker loci was constructed using the DT-RILs. The RILs were cultivated in ordinary-N (N-P2O5-K2O = 100-80-80 kg/ha) and low-N (N-P2O5-K2O= 50-80-80 kg/ha) (100 kg/ha) conditions. PNUE was positively correlated with the harvest index and grain yield in both conditions. Twenty single QTLs (S-QTLs) and 58 pairs of epistatic loci (E-QTLs) were identified for the nitrogen concentration of grain, nitrogen concentration of straw, nitrogen content of shoot, harvest index, grain yield, straw yield and PNUE in both conditions. The phenotypic variance explained by these S-QTLs and E-QTLs ranged from 11.1 to 44.3% and from 16.0% to 63.6% , respectively. The total phenotypic variance explained by all the QTLs for each trait ranged from 35.8% to 71.3%, showing that the expression of PNUE and related characters depends significantly upon genetic factors. Both S-QTLs and E-QTLs may be useful for marker-assisted selection (MAS) to develop higher PNUE genotypes. [Abstract/Link to Full Text]

Yoon SH, Kim W
18S ribosomal DNA sequences provide insight into the phylogeny of patellogastropod limpets (Mollusca: Gastropoda).
Mol Cells. 2007 Feb 28;23(1):64-71.
To investigate the phylogeny of Patellogastropoda, the complete 18S rDNA sequences of nine patellogastropod limpets Cymbula canescens (Gmelin, 1791), Helcion dunkeri (Krauss, 1848), Patella rustica Linnaeus, 1758, Cellana toreuma (Reeve, 1855), Cellana nigrolineata (Reeve, 1854), Nacella magellanica Gmelin, 1791, Nipponacmea concinna (Lischke, 1870), Niveotectura pallida (Gould, 1859), and Lottia dorsuosa Gould, 1859 were determined. These sequences were then analyzed along with the published 18S rDNA sequences of 35 gastropods, one bivalve, and one chiton species. Phylogenetic trees were constructed by maximum parsimony, maximum likelihood, and Bayesian inference. The results of our 18S rDNA sequence analysis strongly support the monophyly of Patellogastropoda and the existence of three subgroups. Of these, two subgroups, the Patelloidea and Acmaeoidea, are closely related, with branching patterns that can be summarized as [(Cymbula + Helcion) + Patella] and [(Nipponacmea + Lottia) + Niveotectura]. The remaining subgroup, Nacelloidea, emerges as basal and paraphyletic, while its genus Cellana is monophyletic. Our analysis also indicates that the Patellogastropoda have a sister relationship with the order Cocculiniformia within the Gastropoda. [Abstract/Link to Full Text]

You HJ, Seo JM, Moon JY, Han SS, Ko YG, Kim JH
Leukotriene synthesis in response to A23187 is inhibited by methyl- beta-cyclodextrin in RBL-2H3 cells.
Mol Cells. 2007 Feb 28;23(1):57-63.
Leukotrienes (LTs) are produced by several biosynthetic enzymes including cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase activating protein (FLAP) in the perinuclear area. In the present study, we showed that pretreatment with methyl-beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent, dramatically reduced the synthesis of LTs in response to A23187 in mast cells. A23187-induced LT synthesis was inhibited by pretreatment with MbetaCD, and this effect was reversed when cholesterol was added. In an approach to identifying the MbetaCD-sensitive protein(s), we observed that FLAP co-localized with flotillin-1, a lipid raft marker protein, in the lipid raft-rich low-density region of sucrose gradients. In addition, electron microscopic analysis revealed that FLAP co-localized with flotillin-1. Together, these results suggest that FLAP is present in cholesterol-rich lipid raft-like domains and that its localization in these domains is critical for LT synthesis. [Abstract/Link to Full Text]

Kim SE, Kim BK, Gil JE, Kim SK, Kim JH
Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro.
Mol Cells. 2007 Feb 28;23(1):49-56.
One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential. [Abstract/Link to Full Text]

Choi BO, Kim J, Lee KL, Yu JS, Hwang JH, Chung KW
Rapid diagnosis of CMT1A duplications and HNPP deletions by multiplex microsatellite PCR.
Mol Cells. 2007 Feb 28;23(1):39-48.
Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are frequent forms of genetically heterogeneous peripheral neuropathies. Reciprocal unequal crossover between flanking CMT1A-REPs on chromosome 17p11.2-p12 is a major cause of CMT type 1A (CMT1A) and HNPP. The importance of a sensitive and rapid method for identifying the CMT1A duplication and HNPP deletion is being emphasized. In the present study, we established a molecular diagnostic method for the CMT1A duplication and HNPP deletion based on hexaplex PCR of 6 microsatellite markers (D17S921, D17S9B, D17S9A, D17S918, D17S4A and D17S2230). The method is highly time-, cost- and sample-saving because the six markers are amplified by a single PCR reaction and resolved with a single capillary in 3 h. Several statistical and forensic estimates indicated that most of these markers are likely to be useful for diagnosing the peripheral neuropathies. Reproducibility, as determined by concordance between independent tests, was estimated to be 100%. The likelihood that genotypes of all six markers are homozygous in randomly selected individuals was calculated to be 1.6 x 10(-4) which indicates that the statistical error rate for this diagnosis of HNPP deletion is only 0.016%. [Abstract/Link to Full Text]

Fan Y, Chen H, Qiao B, Luo L, Ma H, Li H, Jiang J, Niu D, Yin Z
Opposing effects of ERK and p38 MAP kinases on HeLa cell apoptosis induced by dipyrithione.
Mol Cells. 2007 Feb 28;23(1):30-8.
Dipyrithione (2, 2'-dithiobispyridine-1, 1'-dioxide, PTS2), a pyrithione derivate, is highly bactericidal and fungicidal. In this study we examined its apoptotic effect on HeLa cells. PTS2 induced HeLa cell death in a dose and time dependent manner. ERK1/2 and p38 were markedly activated, but little JNK1/2 activation was detected. Suppression of p38 activation by SB203580 reduced the extent of apoptosis of the HeLa cells and also prevented induction of p21, release of cytochrome c, and cleavage of caspase-3 and PARP. Inhibition of ERK1/2 with PD98059 increased apoptosis, indicating that ERK1/2 activation has an anti-apoptotic effect on PTS2-induced HeLa cell apoptosis. PTS2 also inhibited murine sarcoma 180 and hepatoma 22 tumor growth in an animal tumor model. Our findings indicate that PTS2 possesses anti-tumor activity, that caspase-3 and poly (ADP-ribose) polymerase (PARP) are involved in PTS2-induced HeLa cell apoptosis and that ERK1/2 and p38 have opposing effects on this apoptosis. [Abstract/Link to Full Text]

Jeon SH, Chae BC, Kim HA, Seo GY, Seo DW, Chun GT, Yie SW, Eom SH, Kim PH
The PKA/CREB pathway is closely involved in VEGF expression in mouse macrophages.
Mol Cells. 2007 Feb 28;23(1):23-9.
Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-gamma, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at 399 approximately 388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays. [Abstract/Link to Full Text]

Kim MO, Kim SH, Shin MJ, Lee DB, Kim TW, Kim KS, Ha JH, Lee S, Park YB, Kim SJ, Ryoo ZY
Human erythropoietin induces lung failure and erythrocytosis in transgenic mice.
Mol Cells. 2007 Feb 28;23(1):17-22.
We have expressed human erythropoietin (EPO) in transgenic mice using a recombinant EPO cDNA combined with a partial TPO construct. The gene was microinjected using standard techniques and five mice were detected as transgenic by PCR and further used as founders. The life span of the transgenic founders was much shorter than that of their normal littermates. Most of the tissues of the transgenic founders contained human EPO transcripts as judged by RT-PCR. Especially high expression levels were seen in the liver and lung. EPO protein levels in serum were examined by ELISA and ranged from 266, 414 mIU/ml. The number of red blood cell, white blood cell and hemoglobin in the hEPO transgenic mice was higher than in normal mice. These results indicate that overexpression of hEPO is deleterious and can provoke lung failure and erythrocytosis. [Abstract/Link to Full Text]

Lee YK, Kim HL, Kim YL, Im DS
Multiple actions of dimethylsphingosine in 1321N1 astrocytes.
Mol Cells. 2007 Feb 28;23(1):11-6.
N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular Ca2+ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular Ca2+ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the Gi/o protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular Ca2+ with the Ca2+ chelator EGTA or depletion of intracellular Ca2+ stores with thapsigargin impeded the DMS-induced increase of intracellular Ca2+ concentration. Pretreatment of cells with NH4Cl or monensin reduced the DMS-induced Ca2+ increase. However, inhibition of the DMS-induced Ca2+ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular Ca2+ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain. [Abstract/Link to Full Text]

Lee MS, Kim YJ
Pattern-recognition receptor signaling initiated from extracellular, membrane, and cytoplasmic space.
Mol Cells. 2007 Feb 28;23(1):1-10.
Invading pathogens are recognized by diverse germline-encoded pattern-recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasmic. In mammals, the major extracellular PRRs such as complements may first encounter the invading pathogens and opsonize them for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors including complement receptors. The major membrane-associated PRRs, Toll-like receptors, recognize diverse pathogens and generate inflammatory signals to coordinate innate immune responses and shape adaptive immune responses. Furthemore, certain membrane-associated PRRs such as Dectin-1 can mediate phagocytosis and also induce inflammatory response. When these more forefront detection systems are avoided by the pathogens, cytoplasmic PRRs may play major roles. Cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid-inducible protein I (RIG-I)melanoma differentiation-associated gene 5 (MDA5), mediate antiviral immunity by inducing the production of type I interferons. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammasomes to induce inflammatory responses upon ligand recognition. Thus, diverse families of PRRs coordinately mediate immune responses against diverse types of pathogens. [Abstract/Link to Full Text]

Lee JW, Park E, Bang O, Eom SH, Cheong GW, Chung CH, Seol JH
Nucleotide triphosphates inhibit the degradation of unfolded proteins by HslV peptidase.
Mol Cells. 2007 Apr 30;23(2):252-7.
Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation. [Abstract/Link to Full Text]

Koh JM, Kim GS, Oh B, Lee JY, Park BL, Shin HD, Hong JM, Kim TH, Kim SY, Park EK
Microphthalmia-associated transcription factor polymorphisms and association with bone mineral density of the proximal femur in postmenopausal women.
Mol Cells. 2007 Apr 30;23(2):246-51.
Osteoporosis is a common metabolic bone disease characterized by low bone mineral density (BMD) with an increased risk of fracture. Low bone mass results from an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts. Microphthalmia-associated transcription factor (MITF) plays a critical role in osteoclast development and thus is an important candidate gene affecting bone turnover and BMD. In order to investigate the genetic effects of MITF variations on osteoporosis, we directly sequenced the MITF gene in 24 Koreans, and identified fifteen sequence variants. Two polymorphisms (+227719C > T and +228953A > G) were selected based on their allele frequencies, and then genotyped in a larger number of postmenopausal women (n = 560). Areal BMD (g/cm2) of the anterior-posterior lumbar spine and the non-dominant proximal femur was measured by dual-energy X-ray absorptiometry. We found that the MITF + 227719C > T polymorphism was significantly associated with low BMD of the trochanter (p = 0.005-0.006) and total femur (p = 0.02-0.03) (codominant and dominant models), while there was no association with BMD of the lumbar spine. The MITF+228953A > G polymorphism was also associated with low BMD of the femoral shaft (p = 0.05) in the recessive model. Haplotype analysis showed that haplotype 3 of the MITF gene (MITF-ht3) was associated with low BMD of the trochanter (p = 0.03-0.05) and total femur (p = 0.05) (dominant and codominant models). Our results suggest that MITF variants may play a role in the decreased BMD of the proximal femur in postmenopausal women. [Abstract/Link to Full Text]

Kim SW, Bae YG, Pyo SN, Rhee DK
Differential regulation of the genes of the Streptococcus pneumoniae dnaK operon by Ca++.
Mol Cells. 2007 Apr 30;23(2):239-45.
DnaK is a major antigen in Streptococcus pneumoniae, and is induced by a minor shift in temperature (30 to 37 degrees ) but not by ethanol shock. Although HrcA in the presence of Ca++ represses the expression of both groEL and hrcA, the control of transcription of the dnaK operon is not completely understood. In this study, the dnaK operon of S. pneumoniae (5' hrcA-grpE-dnaK-dnaJ) was cloned and analyzed. It contains large intergenic regions in grpE/dnaK and dnaK/dnaJ. Pulse labeling with [35S]-methionine and immunoblot analyses revealed the presence of higher levels of DnaK than of HrcA even in the presence of Ca++ after heat shock suggesting that Ca++ differentially regulates the heat shock responses of hrcA and dnaK. By blocking de novo mRNA synthesis with rifampin it was shown that neither the hrcA nor the groEL transcripts were stabilized by heat shock even though dnaK transcripts were stabilized. We conclude that S. pneumoniae uses fine regulation of the transcription of the individual genes of the tetracistronic dnaK operon to cope with the various stresses experienced during infections. [Abstract/Link to Full Text]

Ro-Choi TS, Choi YC
A modeling study of co-transcriptional metabolism of hnRNP using FMR1 gene.
Mol Cells. 2007 Apr 30;23(2):228-38.
Since molecular structure of hnRNP is not available in foreseeable future, it is best to construct a working model for hnRNP structure. A geometric problem, assembly of 700 +/- 20 nucleotides with 48 proteins, is visualized by a frame work in which all the proteins participate in primary binding, followed by secondary, tertiary and quaternary binding with neighboring proteins without additional import. Thus, 40S hnRNP contains crown-like secondary structure (48 stem-loops) and appearance of 6 petal (octamers) rose-like architectures. The proteins are wrapped by RNA. Co-transcriptional folding for RNP fibril of FMR1 gene can produce 2,571 stem-loops with frequency of 1 stem-loop/15.3 nucleotides and 53 40S hnRNP beaded structure. By spliceosome driven reactions, there occurs removal of 16 separate lariated RNPs, joining 17 separate beaded exonic structures and anchoring EJC on each exon junction. Skipping exon 12 has 5'GU, 3'AG and very compact folding pattern with frequency of 1 stem-loop per 12 nucleotides in short exon length (63 nucleotides). 5'end of exon 12 contains SS (Splicing Silencer) element of UAGGU. In exons 10, 15 and 17 where both regular and alternative splice sites exist, SS (hnRNP A1 binding site) is observed at the regular splicing site. End products are mature FMR-1 mRNP, 4 species of Pri-microRNAs derived from introns 7,9,15 and 3'UTR of exon17, respectively. There may also be some other regulatory RNAs containing ALU/Line elements as well. [Abstract/Link to Full Text]

Park MH, Sim CJ, Baek J, Min GS
Identification of genes suitable for DNA barcoding of morphologically indistinguishable Korean Halichondriidae sponges.
Mol Cells. 2007 Apr 30;23(2):220-7.
The development of suitable genetic markers would be useful for defining species and delineating the species boundaries of morphologically indistinguishable sponges. In this study, genetic variation in the sequences of nuclear rDNA and the mitochondrial cytochrome c oxidase subunit 1 and 3 (CO1 and CO3) regions were compared in morphologically indistinguishable Korean Halichondriidae sponges in order to determine the most suitable species-specific molecular marker region. The maximal congeneric nucleotide divergences of Halichondriidae sponges in CO1 and CO3 are similar to those found among anthozoan cnidarians, but they are 2- to 8-fold lower than those found among genera of other triploblastic metazoans. Ribosomal internal transcribed spacer regions (ITS: ITS1 + ITS2) showed higher congeneric variation (17.28% in ITS1 and 10.29% in ITS2) than those of CO1 and CO3. Use of the guidelines for species thresholds suggested in the recent literature indicates that the mtDNA regions are not appropriate for use as species-specific DNA markers for the Halichondriidae sponges, whereas the rDNA ITS regions are suitable because ITS exhibits a low level of intraspecific variation and a relatively high level of interspecific variation. In addition, to test the reliability of the ITS regions for identifying Halichondriidae sponges by PCR, a species-specific multiplex PCR primer set was developed. [Abstract/Link to Full Text]

Son YJ, Park JW, Lee BJ
TTF-1 expression in PACAP-expressing retinal ganglion cells.
Mol Cells. 2007 Apr 30;23(2):215-9.
In mammals light input resets the central clock of the suprachiasmatic nucleus by inducing secretion of pituitary adenylate cyclase-activating polypeptide (PACAP) from retinal ganglion cells (RGCs). We previously showed that thyroid transcription factor 1 (TTF-1), a homeodomain-containing transcription factor, specifically regulates PACAP gene expression in the rat hypothalamus. In the present study we examined the expression of TTF-1 in PACAP-synthesizing retinal cells. Fluorescence in situ hybridization (FISH) showed that it is abundantly expressed in RGCs of the superior region of the retina, but in only a small subset of RGCs in the inferior region. Double FISH experiments revealed that TTF-1 is exclusively expressed in PACAP-producing RGCs. These results suggest that TTF-1 plays a regulatory role in PACAP-expressing retinal ganglion cells. [Abstract/Link to Full Text]

Hwang du H, Kim ST, Kim SG, Kang KY
Comprehensive analysis of the expression of twenty-seven beta-1, 3-glucanase genes in rice (Oryza sativa L.).
Mol Cells. 2007 Apr 30;23(2):207-14.
Plant beta-1, 3-glucanases are involved in plant defense and in development. Very little data are available on the expression of rice glucanases both in developmental tissues and under various stresses. In this study, we cloned and characterized twenty-seven rice beta-1, 3-glucanases (OsGlu) from at total of 71 putative glucanases. The OsGlu genes were obtained by PCR from a cDNA library and were classified into seven groups (Group I to VII) according to their DNA or amino acid sequence homology. Analysis of the expression of the twenty-seven OsGlu genes by Northern blotting revealed that they were differentially expressed in different developmental tissues as well as in response to plant hormones, biotic stress, high salt etc. OsGlu11 and 27 in Group IV were clearly expressed only in stem and leaf and were also induced strongly by SA (5 mM), ABA (200 microM), and M. grisea. OsGlu1, 10, 11, and 14 were induced earlier and to higher levels in incompatible M. grisea interaction than in compatible one. Taken together, our findings suggest that the twenty-seven rice OsGlu gene products play diverse roles not only in plant defense but also in hormonal responses and in development. [Abstract/Link to Full Text]

Lee JY, Kim JY, Lee YG, Shin WC, Chun T, Rhee MH, Cho JY
Hydroquinone, a reactive metabolite of benzene, reduces macrophage-mediated immune responses.
Mol Cells. 2007 Apr 30;23(2):198-206.
Hydroquinone is a toxic compound and a major benzene metabolite. We report that it strongly inhibits the activation of macrophages and associated cells. Thus, it suppressed the production of proinflammatory cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-3, IL-6, IL-10, IL-12p40, IL-23], secretion of toxic molecules [nitric oxide (NO) and reactive oxygen species (ROS)] and the activation and expression of CD29 as judged by cell-cell adhesion and surface staining experiments. The inhibition was due to the induction of heme oxygenase (HO)-1 in LPS-activated macrophages, since blocking HO-1 activity with ZnPP, an HO-1 specific inhibitor, abolished hydroquinone's NO inhibitory activity. In addition, hydroquinone and inhibitors (wortmannin and LY294002) of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway had very similar inhibitory effects on LPS-induced and CD29-mediated macrophage responses, including the phosphorylation of Akt. Therefore, our data suggest that hydroquinone inhibits macrophage-mediated immune responses by modulating intracellular signaling and protective mechanisms. [Abstract/Link to Full Text]

Kim S, Bang H, Yoo KS, Pike L
Marker-assisted genotype analysis of bulb colors in segregating populations of onions (Allium cepa).
Mol Cells. 2007 Apr 30;23(2):192-7.
Bulb color in onions (Allium cepa) is an important trait whose complex inheritance mechanism involves epistatic interactions among major color-related loci. Recent studies revealed that inactivation of dihydroflavonol 4-reductase (DFR) in the anthocyanin synthesis pathway was responsible for the color differences between yellow and red onions, and two recessive alleles of the anthocyanidin synthase (ANS) gene were responsible for a pink bulb color. Based on mutations in the recessive alleles of these two genes, PCR-based markers for allelic selection were developed. In this study, genotype analysis of onions from segregating populations was carried out using these PCR-based markers. Segregating populations were derived from the cross between yellow and red onions. Five yellow and thirteen pink bulbs from one segregating breeding line were genotyped for the two genes. Four pink bulbs were heterozygous for the DFR gene, which explains the continuous segregation of yellow and pink colors in this line. Most pink onions were homozygous recessive for the ANS gene, except for two heterozygotes. This finding indicated that the homozygous recessive ANS gene was primarily responsible for the pink color in this line. The two pink onions, heterozygous for the ANS gene, were also heterozygous for the DFR gene, which indicated that the pink color was produced by incomplete dominance of a red color gene over that of yellow. One pink line and six other segregating breeding lines were also analyzed. The genotyping results matched perfectly with phenotypic color segregation. [Abstract/Link to Full Text]

Woo HJ, Lee YS, Park SJ, Lim JT, Jang KH, Choi EH, Choi YG, Hwang UW
Complete mitochondrial genome of a troglobite millipede Antrokoreana gracilipes (Diplopoda, Juliformia, Julida), and juliformian phylogeny.
Mol Cells. 2007 Apr 30;23(2):182-91.
The complete mitochondrial genome of a troglobite millipede Antrokoreana gracilipes (Verhoeff, 1938) (Dipolopoda, Juliformia, Julida) was sequenced and characterized. The genome (14,747 bp) contains 37 genes (2 ribosomal RNA genes, 22 transfer RNA genes and 13 protein-encoding genes) and two large non-coding regions (225 bp and 31 bp), as previously reported for two diplopods, Narceus annularus (order Spirobolida) and Thyropygus sp. (order Spirostreptida). The A + T content of the genome is 62.1% and four tRNAs (tRNA(Ser(AGN)), tRNA(Cys), tRNA(Ile) and tRNA(Met)) have unusual and unstable secondary structures. Whereas Narceus and Thyropygus have identical gene arrangements, the tRNA(Thr) and tRNA(Trp) of Antrokoreana differ from them in their orientations and/or positions. This suggests that the Spirobolida and Spirostreptida are more closely related to each other than to the Dipolopoda. Three scenarios are proposed to account for the unique gene arrangement of Antrokoreana. The data also imply that the Duplication and Nonrandom Loss (DNL) model is applicable to the order Julida. Bayesian inference (BI) and maximum likelihood (ML) analyses using amino acid sequences deduced from the 12 mitochondrial protein-encoding genes (excluding ATP8) support the view that the three juliformian members are monophyletic (BI 100%; ML 100%), that Thyropygus (Spirostreptida) and Narceus (Spirobolida) are clustered together (BI 100%; ML 83%), and that Antrokoreana (Julida) is a sister of the two. However, due to conflict with previous reports using cladistic approaches based on morphological characteristics, further studies are needed to confirm the close relationship between Spirostreptida and Spirobolida. [Abstract/Link to Full Text]

Je HD, Sohn UD
SM22alpha is required for agonist-induced regulation of contractility: evidence from SM22alpha knockout mice.
Mol Cells. 2007 Apr 30;23(2):175-81.
The present study was undertaken to determine whether SM22alpha participates in the regulation of vascular smooth muscle contractility using SM22alpha knockout mice and, if so, to investigate the mechanisms involved. Aortic ring preparations were mounted and equilibrated in organ baths for 60 min before observing contractile responses to 50 mM KCl, and then exposed to contractile agents such as phenylephrine and phorbol ester. Measurement of isometric contractions using a computerized data acquisition system was combined with molecular or cellular experiments. Interestingly, the aortas from SM22alpha-deficient mice (SM22(-/-LacZ)) displayed an almost three-fold increase in the level of SM22beta protein compared to wild-type mice, but no change in the levels of caldesmon, actin, desmin or calponin. Ca2+-independent contraction in response to phenylephrine or phorbol ester was significantly decreased in the SM22alpha-deficient mice, whereas in the presence of Ca2+ neither contraction nor subcellular translocation of myosin light chain kinase (MLCK) in response to phenylephrine or 50 mM KCl was significantly affected. A decrease in phosphorylation of extracellular signal regulated kinase (ERK) 1/2 was observed in the SM22alpha-deficient mice and this may be related to the decreased vascular contractility. Taken together, this study provides evidence for a pivotal role of SM22alpha in the regulation of Ca2+-independent vascular contractility. [Abstract/Link to Full Text]

Park KM, Kang E, Jeon YJ, Kim N, Kim NS, Yoo HS, Yeom YI, Kim SJ
Identification of novel regulators of apoptosis using a high-throughput cell-based screen.
Mol Cells. 2007 Apr 30;23(2):170-4.
High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity. [Abstract/Link to Full Text]

Lee JR, Jung JH, Kang JS, Kim JC, Jung IJ, Seok MS, Kim JH, Kim WY, Kim MG, Kim JY, Lim CO, Lee KO, Lee SY
Molecular and functional characterization of monocot-specific Pex5p splicing variants, using OsPex5pL and OsPex5pS from rice (Oryza sativa).
Mol Cells. 2007 Apr 30;23(2):161-9.
We identified two alternatively spliced variants of the peroxisomal targeting signal 1 (PTS1) receptor protein Pex5ps in monocot (rice, wheat, and barley) but not in dicot (Arabidopsis and tobacco) plants. We characterized the molecular and functional differences between the rice (Oryza sativa) Pex5 splicing variants OsPex5pL and OsPex5pS. There is only a single-copy of OsPEX5 in the rice genome and RT-PCR analysis points to alternative splicing of the transcripts. Putative light-responsive cis-elements were identified in the 5' region flanking OsPEX5L and Northern blot analysis demonstrated that this region affected light-dependent expression of OsPEX5 transcription. Using the pex5-deficient yeast mutant Scpex5, we showed that OsPex5pL and OsPex5pS are able to restore translocation of a model PTS1 protein (GFP-SKL) into peroxisomes. OsPex5pL and OsPex5pS formed homo-complexes via specific interaction domains, and interacted with each other and OsPex14p to form hetero-complexes. Although overexpression of OsPex5pL in the Arabidopsis pex5 mutant (Atpex5) rescued the mutant phenotype, overexpression of OsPex5pS only resulted in partial recovery. [Abstract/Link to Full Text]

Phee BK, Park S, Cho JH, Jeon JS, Bhoo SH, Hahn TR
Comparative proteomic analysis of blue light signaling components in the Arabidopsis cryptochrome 1 mutant.
Mol Cells. 2007 Apr 30;23(2):154-60.
An Arabidopsis hy4 mutant that is specifically impaired in its ability to undergo blue light dependent photomorphogenesis was used to identify cryptochrome 1 signaling-related components. Proteomic analysis revealed about 205 differentially expressed protein spots in the blue light-irradiated hy4 mutant compared to the wild-type. The proteins corresponding to 28 up-regulated and 33 down-regulated spots were identified. Obvious morphological changes in the hy4 mutant were closely related to the expression of various transcription factors. Our findings suggest that blue light signals may be involved in many cellular processes including disease resistance and stress responses. [Abstract/Link to Full Text]

Kim JA, Yang TJ, Kim JS, Park JY, Kwon SJ, Lim MH, Jin M, Lee SC, Lee SI, Choi BS, Um SH, Kim HI, Chun C, Park BS
Isolation of circadian-associated genes in Brassica rapa by comparative genomics with Arabidopsis thaliana.
Mol Cells. 2007 Apr 30;23(2):145-53.
Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosensory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years. [Abstract/Link to Full Text]

Choi HJ, Park YG, Kim CH
Lactosylceramide alpha2,3-sialyltransferase is induced via a PKC/ERK/CREB-dependent pathway in K562 human leukemia cells.
Mol Cells. 2007 Apr 30;23(2):138-44.
Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with Gö6976, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with Gö6976 and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors Gö6976 and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway. [Abstract/Link to Full Text]

Kim D, Hong KS, Song J
The present status of cell tracking methods in animal models using magnetic resonance imaging technology.
Mol Cells. 2007 Apr 30;23(2):132-7.
With the advance of stem cell transplantation research, in vivo cell tracking techniques have become increasingly important in recent years. Magnetic resonance imaging (MRI) may provide a unique tool for non-invasive tracking of transplanted cells. Since the initial findings on the stem cell migration by MRI several years ago, there have been numerous studies using various animal models, notably in heart or brain disease models. In order to develop more reliable and clinically applicable methodologies, multiple aspects should be taken into consideration. In this review, we will summarize the current status and future perspectives of in vivo cell tracking technologies using MRI. In particular, use of different MR contrast agents and their detection methods using MRI will be described in much detail. In addition, various cell labeling methods to increase the sensitivity of signals will be extensively discussed. We will also review several key experiments, in which MRI techniques were utilized to detect the presence and/or migration of transplanted stem cells in various animal models. Finally, we will discuss the current problems and future directions of cell tracking methods using MRI. [Abstract/Link to Full Text]

Kim HJ, Hwang NR, Lee KJ
Heat shock responses for understanding diseases of protein denaturation.
Mol Cells. 2007 Apr 30;23(2):123-31.
Extracellular stresses induce heat shock response and render cells resistant to lethal stresses. Heat shock response involves induction of heat shock proteins (Hsps). Recently the roles of Hsps in neurodegenerative diseases and cancer are attracting increasing attention and have accelerated the study of heat shock response mechanism. This review focuses on the stress sensing steps, molecules involved in Hsps production, diseases related to Hsp malfunctions, and the potential of proteomics as a tool for understanding the complex signaling pathways relevant to these events. [Abstract/Link to Full Text]

Huh JW, Kim DS, Ha HS, Kim TH, Kim W, Kim HS
Formation of a new solo-LTR of the human endogenous retrovirus H family in human chromosome 21.
Mol Cells. 2006 Dec 31;22(3):360-3.
Human endogenous retroviruses (HERVs) contribute to various kinds of genomic instability via rearrangement and retrotransposition events. In the present study the formation of a new human-specific solo-LTR belonging to the HERV-H family (AP001667; chromosome 21q21) was detected by a comparative analysis of human chromosome 21 and chimpanzee chromosome 22. The solo-LTR was formed as a result of an equal homologous recombination excision event. Several evolutionary processes have occurred at this locus during primate evolution, indicating that mammalian-wide interspersed repeat (MIR) and full-length HERV-H elements integrated into hominoid genomes after the divergence of Old World monkeys and hominoids, and that the solo-LTR element was created by recombination excision of the HERV-H only in the human genome. [Abstract/Link to Full Text]

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Recent Articles in Cell Research

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Zhang WY, Wan Y, Li DG, Tang Y, Zhou W
A mimotope of pre-S2 region of surface antigen of viral hepatitis B screened by phage display.
Cell Res. 2001 Sep;11(3):203-8.
To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design. [Abstract/Link to Full Text]

Zhang X, Miao YC, An GY, Zhou Y, Shangguan ZP, Gao JF, Song CP
K+ channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells.
Cell Res. 2001 Sep;11(3):195-202.
A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA-inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPI), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane. [Abstract/Link to Full Text]

Meng YL, Wang YM, Zhang B, Nii N
Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions.
Cell Res. 2001 Sep;11(3):187-93.
Plants synthesize the osmoprotectant glycine betaine (GB) via choline-->betaine aldehyde-->glycine betaine[1]. Two enzymes are involved in the pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spinacia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42 degrees C), as determined by immunoblot analysis, but did not respond to cold stress (4 degrees C), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents. [Abstract/Link to Full Text]

Tang W, Luo XY, Sanmuels V
Gene silencing: double-stranded RNA mediated mRNA degradation and gene inactivation.
Cell Res. 2001 Sep;11(3):181-6.
The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double-stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants. [Abstract/Link to Full Text]

Guo DF, Sun YL, Hamet P, Inagami T
The angiotensin II type 1 receptor and receptor-associated proteins.
Cell Res. 2001 Sep;11(3):165-80.
The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors. [Abstract/Link to Full Text]

Gao CF, Kong XT, Gressner AM, Weiskirchen R
The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria.
Cell Res. 2001 Jun;11(2):95-100.
In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies. [Abstract/Link to Full Text]

Liao JH, Chen JS, Chai MQ, Zhao S, Song JG
The involvement of p38 MAPK in transforming growth factor beta1-induced apoptosis in murine hepatocytes.
Cell Res. 2001 Jun;11(2):89-94.
We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis. [Abstract/Link to Full Text]

Geng JG
Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies.
Cell Res. 2001 Jun;11(2):85-8.
Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines. [Abstract/Link to Full Text]

Jin XP, Huang F, Yang N, Lu BF, Fei J, Guo LH
GABA transporter 1 transcriptional starting site exhibiting tissue specific difference.
Cell Res. 2001 Jun;11(2):161-3.
GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5' Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5'RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5' direction. [Abstract/Link to Full Text]

Deng XY, Wei ZM, An HL
Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.
Cell Res. 2001 Jun;11(2):156-60.
After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us. [Abstract/Link to Full Text]

Huang JQ, Wel ZM, An HL, Zhu YX
Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer.
Cell Res. 2001 Jun;11(2):149-55.
Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant calli were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests. [Abstract/Link to Full Text]

Yang SX, Zhao YX, Zhang Q, He YK, Zhang H, Luo
HAL1 mediate salt adaptation in Arabidopsis thaliana.
Cell Res. 2001 Jun;11(2):142-8.
The yeast HAL1 gene was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation with vacuum infiltration under the control of CaMV 35S promoter. Thirty-three individual kanamycin resistant plants were obtained from 75,000 seeds. Southern blotting analysis indicated that HAL1 gene had been integrated into all of the transgenic plants' genomes. The copy number of HAL1 gene in transgenic plants was mostly 1 to 3 by Southern analysis. Phenotypes of transgenic plants have no differences with wild type plants. Several samples of transformants were self-pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance and gene expression. Measurement of concentrations of intracellular K+ and Na+ showed that transgenic lines were able to retain less Na+ than that of the control under salt stress. Results from different tests indicated the expression of HAL1 gene promotes a higher level of salt tolerance in vivo in the transgenic Arabidopsis plants. [Abstract/Link to Full Text]

FD, Bian W, Kong LW, Zhao FJ, Guo JS, Jing NH
Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice.
Cell Res. 2001 Jun;11(2):135-41.
Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 30 mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10.5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults. [Abstract/Link to Full Text]

Jin ML, Zhang P, Ding MX, Yun JP, Chen PF, Chen YH, Chew YQ
Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells.
Cell Res. 2001 Jun;11(2):125-34.
The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process. [Abstract/Link to Full Text]

Xing L, Xia GH, Fei J, Huang F, Guo LH
Adenovirus-mediated expression of pig alpha(1, 3) galactosyltransferase reconstructs Gal alpha(1, 3) gal epitope on the surface of human tumor cells.
Cell Res. 2001 Jun;11(2):116-24.
Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay. [Abstract/Link to Full Text]

Sun HZ, Wu SF, Tu ZH
Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity.
Cell Res. 2001 Jun;11(2):107-15.
A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy. [Abstract/Link to Full Text]

Guo BC, Xu YH
Bcl-2 over-expression and activation of protein kinase C suppress the trail-induced apoptosis in Jurkat T cells.
Cell Res. 2001 Jun;11(2):101-6.
Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell. [Abstract/Link to Full Text]

Peng S, Lu XM, Luo HR, Xiang-Yu JG, Zhang YP
Melanocortin-1 receptor gene variants in four Chinese ethnic populations.
Cell Res. 2001 Mar;11(1):81-4.
There is strong relationship between melanocortin-1 receptor (MC1R) gene variants and human hair color and skin type. Based on a sequencing study of MC1R gene in 50 individuals from the Uygur, Tibetan, Wa and Dai ethnic populations, we discuss the occurrence of 7 mc1r variants consisting of 5 nonsynonymous sites (Val60Leu, Arg67Gln, Val92Met, Arg163Gln and Ala299Val) and 2 synonymous sites (C414T and A942G), among which C414T and Ala299Val were reported for the first time. Confirmation and analysis were also made of 122 individuals at three common point mutations (Val92Met, Arg163Gln, A942G) using PCR-SSCP. The frequency of Arg163Gln variant varies in the four ethnic populations, with percentage of 40%, 85.0%, 66.2% and 72.7%, respectively, while those of Val92Met and A942G are roughly similar in these four populations. The different environments, migration and admixture of various ethnic groups in China might have impact on the observed frequency of Arg163Gln. [Abstract/Link to Full Text]

Hsia SC, Wang H, Shi YB
Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor.
Cell Res. 2001 Mar;11(1):8-16.
The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFbB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner. [Abstract/Link to Full Text]

Yang ZX, An GY, Zhu ZP
Rice bicoid-related cDNA sequence and its expression during early embryogenesis.
Cell Res. 2001 Mar;11(1):74-80.
Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice. [Abstract/Link to Full Text]

Tao W, Yan CH, Cai T, Hao S, Zhai ZH
Structural components of the nuclear body in nuclei of Allium cepa cells.
Cell Res. 2001 Mar;11(1):68-73.
Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs. [Abstract/Link to Full Text]

Ma Y, Hu JH, Zhao WJ, Fei J, Yu Y, Zhou XG, Mei ZT, Guo LH
Overexpression of gamma-aminobutyric acid transporter subtype I leads to susceptibility to kainic acid-induced seizure in transgenic mice.
Cell Res. 2001 Mar;11(1):61-7.
Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters. [Abstract/Link to Full Text]

Takano S, Wadhwa R, Mitsui Y, Kaul SC
p53-independent upregulation of p21WAF1 in NIH 3T3 cells malignantly transformed by mot-2.
Cell Res. 2001 Mar;11(1):55-60.
Mot-2 protein is shown to interact with p53 and inhibit its transcriptional activation function. Mot-2 overexpressing stable clones of NIH 3T3 cells were malignantly transformed, however, they had a high level of expression of a p53 downstream gene, p21WAF1. The present study was undertaken to elucidate possible molecular mechanism(s) of such upregulation. An increased level of p21WAF1 expression was detected in stable transfectants although an exogenous reporter gene driven by p21WAF1 promoter exhibited lower activity in these cells suggesting that some post-transcriptional mechanism contributes to upregulation. Western analyses of transient and stable clones revealed that upregulation of p21WAF1 in stable NIH 3T3/mot-2 cells may be mediated by cyclin D1 and cdk-2. [Abstract/Link to Full Text]

Zamorano J, Kelly AE, Austrian J, Wang HY, Keegan AD
Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation.
Cell Res. 2001 Mar;11(1):44-54.
IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway. [Abstract/Link to Full Text]

Zhang X, Dong FC, Gao JF, Song CP
Hydrogen peroxide-induced changes in intracellular pH of guard cells precede stomatal closure.
Cell Res. 2001 Mar;11(1):37-43.
Epidermal bioassay demonstrated that benzylamine, a membrane-permeable weak base, can mimick hydrogen peroxide (H2O2) to induce stomatal closure, and butyric acid, a membrane-permeable weak acid, can partly abolish the H2O2-induced stomatal closure. Confocal pH mapping with the probe 5-(and-6)-carboxy seminaphthorhodafluor-1-acetoxymethylester (SNARF-1-AM) revealed that H2O2 leads to rapid changes in cytoplasmic and vacuolar pH in guard cells of Vicia faba L, i. e. alkalinization of cytoplasmic areas occur red in parallel with a decrease of the vacuolar pH, and that butyric acid pretreatment can abolish alkalinization of cytoplasmic areas and acidification of vacuolar areas of guard cells challenged with H2O2. These results imply that the alkalinization of cytoplasm via efflux of cytosol protons into the vacuole in guard cells challenged with H2O2 is important at an early stage in the signal cascade leading to stomatal closure. [Abstract/Link to Full Text]

Aeed PA, Geng JG, Asa D, Raycroft L, Ma L, Elhammer AP
Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1).
Cell Res. 2001 Mar;11(1):28-36.
PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding. [Abstract/Link to Full Text]

Fan LC, Yang ST, Gui JF
Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp.
Cell Res. 2001 Mar;11(1):17-27.
Gynogenetic silver crucian carp, Carassius auratus gibelio, is an intriguing model system. In the present work, a systemic study has been initiated by introducing suppression subtractive hybridization technique into this model system to identify the differentially expressed genes in oocytes between gynogenetic silver crucian carp and its closely related gonochoristic color crucian carp. Five differential cDNA fragments were identified from the preliminary screening, and two of them are ZP3 homologues. Moreover, the full length ZP3 cDNAs were cloned from their oocyte cDNA libraries. The length of ZP3 cDNAs were 1378 bp for gyno-carp and 1367 bp for gono-carp, and they can be translated into proteins with 435 amino acids. Obvious differences are not only in the composition of amino acids, but also in the number of potential O-linked oligosaccharide sites. In addition, gyno-carp ZP3 amino acid sequence has an unexpected higher identity value with common carp (83.5%) than that with the closely related gono-carp (74.7%). The unique homology may be originated from the ancient hybridization. Northern blot analysis confirmed that expression of the ZP3 gene occurred exclusively in the oocytes. Because O-linked oligosaccharides on ZP3 have been demonstrated to play very important roles in fertilization, it is suggested that the extra O-linked glycosylation sites may be related to the unique sperm-egg recognition mechanism in gynogenesis. [Abstract/Link to Full Text]

Fan XQ, Guo YJ
Apoptosis in oncology.
Cell Res. 2001 Mar;11(1):1-7.
Apoptosis is a complex process involving a large array of genes and mutation of any of these genes may lead to malignancy formation. Re-acquirement of FasL by tumor cells may enable them to evade the surveillance of immune system and thus contributes to the growth of tumor. Apart from traditional therapies, inducing apoptosis of tumor cell by new methods employing death receptor ligands and making use of Fas counterattack is also being developed. [Abstract/Link to Full Text]

Yue S, Zhang W, Li FL, Guo YL, Liu TL, Huang H
Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis.
Cell Res. 2000 Dec;10(4):325-35.
Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana. However, the leaf development that is specific to plants has been little studied. With the aim of contributing to the genetic dissection of leaf development, we have performed a large-scare screening for mutants with abnormal leaves. Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethyl-methae sulfonate (EMS) mutagenesis, four independent mutant lines have been identified and studied genetically. Phenotypes of these mutant lines represent the defects of four novel nuclear genes designated LL1 (LOTUS LEAF 1), LL2 (LOTUS LEAF 2), URO (UPRIGHT ROSETTE). and EIL (ENVIRONMENT CONDITION INDUCED LESION). The phenotypic analysis indicates that these genes play important roles during leaf development. For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2, we have mapped these genes to chromosome regions with an efficient and rapid mapping method. [Abstract/Link to Full Text]

Wang G, Huang CH, Zhao Y, Cai L, Wang Y, Xiu SJ, Jiang ZW, Yang S, Zhao T, Huang W, Gu JR
Genetic aberration in primary hepatocellular carcinoma: correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21-q23 and 9p21-p23.
Cell Res. 2000 Dec;10(4):311-23.
To elucidate the molecular pathology underlying the development of hepatocellular carcinoma (HCC), we used 41 highly polymorphic microsatellite markers to examine 55 HCC and corresponding non-tumor liver tissues on chromosome 9, 16 and 17. Loss-of-heterozygosity (LOH) is observed with high frequency on chromosomal region 17p13 (36/55, 65%), 9p21-p23 (28/55, 51%), 16q21-q23 (27/55, 49%) in tumors. Meanwhile, microsatellite instability is rarely found in these microsatellite loci. Direct sequencing was performed to detect the tentative mutation of tumor suppressor genes in these regions: p53, MTS1/p16, and CDH1/E-cadherin. Within exon 5-9 of p53 gene, 14 out of 55 HCC specimens (24%) have somatic mutations, and nucleotide deletion of this gene is reported in HCC for the first time. Mutation in MTS1/p16 is found only in one tumor case. We do not find mutations in CDH1/E-cadherin. Furthermore, a statistically significant correlation is present between p53 gene mutation and loss of chromosome region 16q21-q23 and 9p21-p23, which indicates that synergism between p53 inactivation and deletion of 16q21-q23 and 9p21-p23 may play a role in the pathogenesis of HCC. [Abstract/Link to Full Text]

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Recent Articles in Cellular & Molecular Biology Letters

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Przybecki Z, Kowalczyk ME, Witkowicz J, Filipecki M, Siedlecka E
Polymorphom of sexually different cucumber (Cucumis sativus L.) NIL lines.
Cell Mol Biol Lett. 2004;9(4B):919-33.
Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.), which differ with respect to sex, were carried out using the subtraction hybridization methods of DSC (Differential Subtraction Chain) and GDDSC (Genetically Directed DSC). 266 DSC tags were isolated from the entire genome region, and 38 GDDSC tags were isolated from the region containing the sex genes. Based on the obtained results, the methods used may be considered highly effective. The attained sequences, like 11 AFLP clones obtained earlier [Witkowicz, J. et al. Cell. Mol. Biol. Lett. 8 (2003) 375-381], were characterized by analyzing their hybridization with differential (dhaom) and subtractive cDNA libraries (cDNAsubtractom) from 1- to 2- mm floral buds of the same lines, and by the sequencing of 28 tags. A high average degree of homology was found to exist in the genpolom to dhom and cDNAsubtractom, particularly in the case of "dominant" (when the tester used was a line in which the sex of the plants was dependent upon the dominant allele). This indicates a significant share of coding sequences in the polymorphic genomic tags as well as their share in flower formation. Many of these sequences originate from the sex gene region. Analysis of the sequenced tags showed their interesting composition, including many organelle sequences which transferred into the nucleus, and coding. [Abstract/Link to Full Text]

Przetakiewicz A, Kara? A, Orczyk W, Nadolska-Orczyk A
Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat.
Cell Mol Biol Lett. 2004;9(4B):903-17.
Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper. [Abstract/Link to Full Text]

Yin Z, Paw?owicz I, Bartoszewski G, Malinowski R, Malepszy S, Rorat T
Transcriptional expression of a Solanum sogarandium pGT::Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings.
Cell Mol Biol Lett. 2004;9(4B):891-902.
The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6 degrees C for 24 h. This was confirmed by RT-PCR. However, no DHN10 protein was detected by the alkaline phosphatase-conjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6 degrees C, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2 degrees C for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandnium GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level. [Abstract/Link to Full Text]

Tyrka M, B?aszczyk L, Che?kowski J, Lind V, Kramer I, Weilepp M, Wi?niewska H, Ordon F
Development of the single nucleotide polymorphism marker of the wheat Lr1 leaf rust resistance gene.
Cell Mol Biol Lett. 2004;9(4B):879-89.
The range of publicly available data on plant nucleotide sequences opens a new possibility in the design of SNP assays. The purpose of this study was to identify point mutations in genomic sequences closely linked to the Lr1 leaf rust resistance gene, and to develop SNP markers based on primer extension (SNuPE) facilitating efficient marker-based selection procedures, e.g. the pyramiding of resistance genes. Studies were performed on the panel of 37 wheat cultivars, the set of 41 Thatcher near-isogenic lines of spring wheat and on the 21 individuals derived from doubled-haploid (DH) lines derived from 'Henika' (Lr1) x 'IPG-SW-14'. A minisequencing reaction run with Lr1_98F primer detected four genotypes (T, C+T, C and "null") in the set of all Triticum aestivum varieties tested. In this study, it turned out that the T allele is associated with the Lr1 gene in a wide genetic background. [Abstract/Link to Full Text]

B?aszczyk L, Goyeau H, Huang XQ, Röder M, Stepie? L, Che?kowski J
Identifying leaf rust resistance genes and mapping gene Lr37 on the microsatellite map of wheat.
Cell Mol Biol Lett. 2004;9(4B):869-78.
Based on seedling resistance tests, five resistance genes (Lr10, Lr3, Lr13, Lr14a and Lr37) against leaf rust (Puccinia triticina) were identified in 16 cultivars of European winter wheat. STS and SCAR markers were used to verify the presence of the resistance genes Lr37 and Lr10 against leaf rust in cultivars, near-isogenic lines and segregating populations. The Lr37 gene is present in a small translocation from Triticum ventricosum Ces. (Aegilops ventricosa Tausch) and is tightly linked with resistance genes Yr17 and Sr38. The Lr37 gene was identified in the cultivars Kris, Clever, Slade, Apache, Caphorn, Lorraine, Balthasar, Renan and confirmed by two PCR markers. The F3 progenies of the crosses Kris (Lr37) X Nutka (Lr37 not present) were used for map construction. Two STS/SCAR markers specific for Lr37 were mapped in relation to nine polymorphic microsatellites on chromosome 2AS. The microsatellite marker Xgwm1176 mapped relatively close to the STS and SCAR markers for Lr37 with a linkage distance of 4.1 cM. [Abstract/Link to Full Text]

Sliwka J
Genetic factors encoding resistance to late blight caused by Phytophthora infestans (Mont.) de Bary on the potato genetic map.
Cell Mol Biol Lett. 2004;9(4B):855-67.
Late blight, a potato disease caused by Phytophthora infestans (Mont.) de Bary, is of great economic significance, and has been the subject of numerous research projects aimed at both introducing resistance to the disease into potato cultivars, and at unravelling the mechanisms and genes underlying this resistance. This report is on publications about mapping the resistance to P. infestans encoded by major resistance genes or polygenes, introduced into the potato from different sources. Applied methods for resistance evaluation, methods for revealing DNA polymorphisms and for the construction of genetic maps are described and compared, as are results obtained by independent authors working in this field. [Abstract/Link to Full Text]

Mac A, Krzymowska M, Barabasz A, Hennig J
Transcriptional regulation of the gluB promoter during plant response to infection.
Cell Mol Biol Lett. 2004;9(4B):843-53.
Several studies suggest that plant hydrolytic enzymes, such as 1,3-beta-glucanases, may be components of a general defense system against pathogen invasion in several different plant species. We isolated and characterized a genomic sequence coding for a new acidic 1,3-beta-glucanase (gluB) from Solanum tuberosum. The 5' flanking region of the gluB gene was also characterized. A chimeric gene composed of 2998 bp of the promoter sequence from the gluB gene was fused to the beta-glucuronidase (GUS) coding region and used to transform potato and tobacco plants. Transcriptional activation of the gluB promoter was investigated in response to inoculation with Phytophthora infestans (Pi) or tobacco mosaic virus (TMV). In pathogen inoculated transgenic plants, GUS activity was strongly induced locally around necrotic lesions. [Abstract/Link to Full Text]

Ludwikow A, Gallois P, Sadowski J
Ozone-induced oxidative stress response in Arabidopsis: transcription profiling by microarray approach.
Cell Mol Biol Lett. 2004;9(4B):829-42.
High ozone concentration generates oxidative stress in plants. To investigate the detailed transcriptional regulation of Arabidopsis thaliana genes encoding antioxidant enzymes upon ozone stress, we performed a microarray analysis using Affymetrix GeneChip technology. Our transcription profiling revealed a differential expression equal or greater than 2-fold change for 2385 genes (at confidence 99%) in response to 350 ppb ozone dose after 3 and 6 hours of treatment. Among these, we chose 38 genes to be oxidative stress related in ozone treatment: 29 of them were 2 times up-regulated and 9 were shown to be down-regulated in at least one of the time points. Our study revealed a new transcription pattern for catalase genes and showed the first detailed transcriptional analysis of phenylopropanoid-related genes in ozone stress conditions. [Abstract/Link to Full Text]

Stepie? L, Che?kowski J, Wenzel G, Mohler V
Combined use of linked markers for genotyping the Pm1 locus in common wheat.
Cell Mol Biol Lett. 2004;9(4B):819-27.
Genotyping of 98 wheat cultivars/lines was carried out with molecular markers that are linked to the Pm1 locus: two bi-allelic (dominant) markers: the sequence-tagged site Xsts638-7A and the amplified fragment length polymorphism XE39M58-77-7A; and the multi-allelic simple sequence repeat marker Xgwm344-7A. Employing segregation data recorded in the population Chinese Spring x Virest (Pm1e), genetic mapping revealed that Xgwm344-7A and XE39M58-77-7A were distally linked to Pm1e in the repulsion phase with respective linkage distances of 0.9 cM and 4.8 cM, while Xsts638-7A was found to co-segregate with Pm1e in the coupling phase. The genotyping results of Xsts638-7A and XE39M58-77-7A confirmed disease scoring, except for the accessions of cultivars Omega, Remus and Weihenstephan Stamm M1N. The SSR marker Xgwm344 amplified 15 different fragments ranging from 102 bp to 147 bp, with 15 entries being null-allelic at the 7A and 7B homoeoloci. It was found that wheat lines having resistance alleles at the Pm1 locus mainly show the null allele at the Xgwm344-7A locus. Due to their fast-evolving nature, the use of multi-allelic SSRs for genotype determination may be complicated. However, the combined use of multiple linked marker alleles seems to be a promising approach for genotyping a broad range of plant materials. [Abstract/Link to Full Text]

B?aszczyk L, Che?kowski J, Korzun V, Kraic J, Ordon F, Ovesná J, Purnhauser L, Tar M, Vida G
Verification of STS markers for leaf rust resistance genes of wheat by seven European laboratories.
Cell Mol Biol Lett. 2004;9(4B):805-17.
A set of Thatcher near-isogenic lines and two breeding lines were used to examine sequence tagged site (STS) markers linked to leaf rust resistance genes Lr9, Lr10, Lr19, Lr24, Lr28, Lr29, Lr35, and a simple sequenced repeat (SSR) marker for Lr39. The selected STS markers for resistance genes Lr9, Lr10, Lr19, Lr24 and Lr28 were identified in seven accessions by seven European laboratories. Near-isogenic lines of the spring wheat Thatcher were used as positive controls. Markers for resistance genes Lr9, Lr10, Lr19, Lr24 were identified in all seven laboratories as amplification products of 1100 bp, 310 bp, 130 bp and 310 bp, respectively. The STS markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr29, Lr35 and the SSR marker for Lr39 were robust and highly specific for these genes and will be useful in marker-assisted selection in wheat. However, the amplification product of 378 bp that corresponded with resistance gene Lr28 was detected in all accessions including genotypes lacking this gene in all seven laboratories. This marker needs to be improved. [Abstract/Link to Full Text]

Linkiewicz A, Filipecki M, Tomczak A, Grabowska A, Malepszy S
The cloning of sequences differentially transcribed during the induction of somatic embryogenesis in cucumber (Cucumis sativus L.).
Cell Mol Biol Lett. 2004;9(4B):795-804.
Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process. In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development. We identified and cloned 120 candidate cDNA fragments from differential display gels. The selected cDNAs were confirmed by reverse northern, and 83 were sequenced. The obtained sequences represent 64 independent transcripts. The search for similarities in the databases gave a significant result in 16 cases. The potential involvement of these sequences in somatic embryogenesis is discussed. [Abstract/Link to Full Text]

Ob?ak E, Lachowicz TM, Luczy?ski J, Witek S
The aminoesters as inhibitors of plasma membrane H+-ATPase in the yeast Saccharomyces cerevisiae.
Cell Mol Biol Lett. 2004;9(4A):755-63.
A set of oxalates of alpha-dimethylamino fatty acids n-alkyl esters (MEM-ns and n-MEM-8s) and n-dodecyl-N,N-dimethylalaninate (DMAL-12s) were synthesized. Their activities on the growth, transport, and ATPases from the yeast Saccharomyces cerevisiae were compared. The compounds differ in the number of carbon atoms in their aliphatic chain and in the position of that chain in their molecular structure. The tested aminoesters with twelve carbon atoms (MEM-10s and DMAL-12s) appeared to have the highest level of activity. These drugs inhibited plasma membrane H+-ATPase, but no inhibition of mitochondrial ATPase was observed. Under nitrogen-derepressed conditions, the aminoesters inhibited amino acid uptake by yeast cells. [Abstract/Link to Full Text]

Cottage A, Mullan L, Portela MB, Hellen E, Carver T, Patel S, Vavouri T, Elgar G, Edwards YJ
Molecular characterisation of the SAND protein family: a study based on comparative genomics, structural bioinformatics and phylogeny.
Cell Mol Biol Lett. 2004;9(4A):739-53.
The activities of vertebrate lysosomes are critical to many essential cellular processes. The yeast vacuole is analogous to the mammalian lysosome and is used as a tool to gain insights into vesicle mediated vacuolar/lysosome transport. The protein SAND, which does not contain a SAND domain (PFAM accession number PF01342), has recently been shown to function at the tethering/docking stage of vacuole fusion as a critical component of the vacuole SNARE complex. In this publication we have identified SAND in diverse eukaryotes, from single celled organisms such as the yeasts to complex multi-cellular chordates such as mammals. We have demonstrated subfamily divisions in the SAND proteins and show that in vertebrates, a duplication event gave rise to two SAND sequences. This duplication appears to have occurred during early vertebrate evolution and conceivably with the evolution of lysosomes. Using bioinformatics we predict a secondary structure, solvent accessibility profile and protein fold for the SAND proteins and determine conserved sequence motifs, present in all SAND proteins and those that are specific to subsets. A comprehensive evaluation of yeast and human functional studies in conjunction with our in silico analysis has identified potential roles for some of these motifs. [Abstract/Link to Full Text]

Brzóska E, Wróbel E, Grabowska I, Moraczewski J
Talin distribution during the differentiation of satellite cells isolated from rat skeletal muscle.
Cell Mol Biol Lett. 2004;9(4A):723-37.
Satellite cells (myogenic stem cells) dissociated from adult muscle tissue proliferate, fuse and form multinucleate myotubes when placed in culture. This study focused on the role of talin distribution during the differentiation of satellite cells. Talin plays a key role in anchoring actin filaments to integrins as well as to the plasma membrane in focal contacts. We demonstrated that there is a colocalization of talin and phosphoserine residues during the differentiation of satellite cells, and that it changes after TPA (a protein kinase C activator) treatment, and showed that talin existing in the cell-extracellular matrix and cell-cell contact area was not phosphorylated. In the presence of TPA (24 and 48 h exposure) the level of colocalization of both talin and phosphoserine residues was the same in the treated cells and in the control cells, but the level of talin phosphorylation was higher in the treated cells. We found that in myotubes from TPA treated cultures (144 h exposure to TPA), talin had localized near the cell membrane in the absence of phosphoserine residues, and that the level of talin phosphorylation was lower than in the control cells. We also demonstrated that the expression of talin during satellite cell differentiation was constant in both the control and TPA-treated cells. [Abstract/Link to Full Text]

Dobrzy?ska I, Snieci?ska A, Skrzydlewska E, Figaszewski Z
Green tea modulation of the biochemical and electric properties of rat liver cells that were affected by ethanol and aging.
Cell Mol Biol Lett. 2004;9(4A):709-21.
The oxidative stress induced by chronic ethanol consumption, particularly in concert with the aging process, has been implicated in changes in the structure and functions of liver cell components including membrane phospholipids. To counteract such changes, particularly those resulting from lipid peroxidation, antioxidants may be applied. Green tea contains large amounts of polyphenols, mainly catechins, which possess antioxidant properties. The aim of this study was to estimate the efficacy of green tea's influence on the physicochemical and biochemical properties of the rat liver as affected by the aging process and/or chronic ethanol intoxication. Several methods were used to evaluate this effect. Antioxidant properties were evaluated by vitamin E and antioxidant status determination. The liver trigliceride and cholesterol levels were also estimated. The extent of lipid peroxidation was determined by measuring the level of lipid peroxidation products as thiobarbituric reactive substances (TBARS). The surface charge density of the rat liver cells was measured using electrophoresis. The concentration of the marker enzymes of liver damage (alanine aminotransferase and aspartate aminotransferase) in the blood serum was also evaluated. Relative to the controls, aging was found to cause a decrease in the liver's antioxidant abilities and provoke an increase in the level of lipid peroxidation; it also increased the surface charge density of the rat liver cell membrane. Ethanol significantly aggravated these changes. This might have resulted in the liver cell membrane damage visible as a leak of alanine aminotransferase and aspartate aminotransferase into the blood. The ingestion of green tea with ethanol partially prevented these aging and/or ethanol-induced changes. Long-term drinking of green tea partially prevents the changes in the structure and function of the cell membrane caused by chronic ethanol intoxication. [Abstract/Link to Full Text]

Manoharan S, Kolanjiappan K, Kayalvizhi M
Enhanced lipid peroxidation and impaired enzymic antioxidant activities in the erythrocytes of patients with cervical carcinoma.
Cell Mol Biol Lett. 2004;9(4A):699-707.
This study examined the relationship between oxidative stress and enzymic antioxidant status in the erythrocytes of thirty-two adult cervical cancer patients and an equal number of age-matched cervicitis patients and healthy subjects. Lipid peroxidation was significantly increased, while the activities of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and glucose-6-phosphate dehydrogenase were decreased in the erythrocytes of cervical cancer patients as compared to healthy subjects and cervicitis patients. Thus, this study has demonstrated elevated lipid peroxidation and impaired enzymic antioxidant activities in the erythrocytes of cervical cancer patients. [Abstract/Link to Full Text]

Krishna GK, Zhang J, Burow M, Pittman RN, Delikostadinov SG, Lu Y, Puppala N
Genetic diversity analysis in valencia peanut (Arachis hypogaea L.) using microsatellite markers.
Cell Mol Biol Lett. 2004;9(4A):685-97.
Cultivated peanut or groundnut (Arachis hypogaea L) is an important source of oil and protein. Considerable variation has been recorded for morphological, physiological and agronomic traits, whereas few molecular variations have been recorded for this crop. The identification and understanding of molecular genetic diversity in cultivated peanut types will help in effective genetic conservation along with efficient breeding programs in this crop. The New Mexico breeding program has embarked upon a program of improvement of Valencia peanut (belonging to the sub species fastigiata), because efforts to improve the yield potential are lacking due to lack of identified divergent exotic types. For the first time, this study has shown molecular diversity using microsatellite markers in the cultivated Valencia peanut (sub spp. fastigiata) from around the globe. In this investigation, 48 cultivated Valencia peanut genotypes have been selected and analyzed using 18 fluorescently labeled SSR (f-SSR) primer pairs. These primer pairs amplified 120 polymorphic loci among the genotypes screened and amplified from 3 to 19 alleles with an average of 6.9 allele per primer pair. The f-SSR marker data was further analyzed using cluster algorithms and principal component analysis. The results indicated that (1) considerable genetic variations were discovered among the analyzed genotypes; (2) The f-SSR based clustering could identify the putative pedigree types of the present Valencia types of diverse origins, and (3) The f-SSR in general is sufficient to obtain estimates of genetic divergence for the material in study. The results are being utilized in our breeding program for parental selection and linkage map construction. [Abstract/Link to Full Text]

Krasowska A, Chmielewska L, Adamski R, Luczy?ski J, Witek S, Sigler K
The sensitivity of yeast and yeast-like cells to new lysosomotropic agents.
Cell Mol Biol Lett. 2004;9(4A):675-83.
The lysosomotropic action of the compounds DM-11 and DMAL-12s against Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans is species- and pH-dependent. At pH 6.0, DMAL-12s is less effective against S. cerevisiae and S. pombe but more effective against C. albicans than DM-11. At pH 8.0, DMAL-12s strongly inhibits the growth of S. cerevisiae but has only a marginal effect on the resistant C. albicans. S. pombe did not grow at pH 8.0. As shown by quinacrine accumulation, DM-11 causes a general intracellular acidification in all three species, while with DMAL-12s, the acidification is marginal. Morphological changes caused by DMAL-12s in S. cerevisiae affect the cell interior but not surface structures, while S. pombe cells exhibit a thickened and wrinkled cell wall, shrunken protoplast and "grainy" plasma membrane. A large number of blisters resembling lipid droplets were observed inside S. cerevisiae and S. pombe vacuoles. The high susceptibility of S. pombe cells to the action of DM-11 and DMAL-12s contrasts with the low sensitivity of S. pombe H+-ATPase to the agents. In our C. albicans isolate, DMAL 12s did not have an effect on cell morphology and appeared to be unable to penetrate the cells, especially at pH 8.0. [Abstract/Link to Full Text]

Baskar AA, Manoharan S, Manivasagam T, Subramanian P
Temporal patterns of lipid peroxidation product formation and antioxidants activity in oral cancer patients.
Cell Mol Biol Lett. 2004;9(4A):665-73.
The aim of this study was to assess the temporal patterns of (the formation of) thiobarbituric acid reactive substances and the activities of antioxidant enzymes in the erythrocytes of ten healthy adult subjects and ten oral cancer patients of comparable age. The levels of thiobarbituric acid reactive substances and the activities of antioxidant enzymes were assayed at 6-hour intervals using colorimetric methods. The Cosinorwin computer software program was used to analyse the characteristics of biochemical rhythms, such as acrophase, amplitude, and mesor (rhythms: acrophase, amplitude, mesor, etc.). There is a phase delay in erythrocyte thiobarbituric acid reactive substance levels and enzymatic antioxidant activities in oral cancer patients as compared to healthy subjects. The desynchronisation of thiobarbituric acid reactive substance production and enzymatic antioxidant rhythms reflected an alteration of circadian clock function in oral cancer patients and may require specific measures for chronotherapy. [Abstract/Link to Full Text]

Starzy?ski RR, Gralak MA, Smuda E, Lipi?ski P
A characterization of the activities of iron regulatory protein 1 in various farm animal species.
Cell Mol Biol Lett. 2004;9(4A):651-64.
Iron regulatory protein 1 (IRP1) post-transcriptionally regulates the expression of proteins involved in the iron metabolism of mammals. IRP1 is a bifunctional cytosolic protein which can exhibit aconitase activity or bind to iron responsive element (IREs) in the untranslated regions of specific mRNAs. The modulation of IRP1 activities and its consequence for intracellular iron homeostasis is best characterized in rodents and humans. Little is known about IRP1 in farm animals. In this study, we analyzed the two activities of IRP1 in the livers of four farm animal species (cattle, goat, pig and rabbit) and their relationship to hepatic iron content. We found an inverse correlation between spontaneous IRP1 IRE binding activity and non-haem iron content in the liver. Using the electrophoretic mobility shift assay, we showed differential mobility of IRE/IRP1 complexes formed with hepatic cytosolic extracts from various farm animal species. We discuss this observation in relation to a comparative analysis of mammalian IRP1 amino acid sequences. [Abstract/Link to Full Text]

Sienkiewicz P, Pa?ka M, Pa?ka J
Oxidative stress induces IGF-I receptor signaling disturbances in cultured human dermal fibroblasts. A possible mechanism for collagen biosynthesis inhibition.
Cell Mol Biol Lett. 2004;9(4A):643-50.
The effects of oxidative stress on collagen and DNA biosynthesis, beta-galactosidase and prolidase activities, and the expression of prolidase, beta1-integrin receptor, FAK, IGF-IR and MAP-kinases (ERK1, ERK2) were evaluated in human dermal fibroblasts. Subconfluent cells were subjected to repetitive stresses with 30 microM t-BHP for 1 hour per day over the course of 5 days. It was found that oxidative stress induced the inhibition of collagen biosynthesis in these cells in a time-dependent manner. Exposure of the cells to 5 stresses contributed to a decrease in collagen and DNA biosynthesis to about 30% and 50% of the control values, respectively. Prolidase activity and expression were only suppressed in fibroblasts subjected to 1 and 3 stresses. In these cells prolidase activity was decreased by about 20%. As a result of 5 stresses, no further inhibition of prolidase activity occurred; however, expression of the enzyme was slightly increased, as demonstrated by Western blot analysis. It was found that these phenomena were neither related to the expression of beta1-integrin receptor nor to that of FAK. However, the exposure of the cells to 3 and 5 stresses contributed to a distinct decrease in IGF-IR and MAP-kinases (ERK1, ERK2) expression, which is probably responsible for the collagen biosynthesis inhibition. [Abstract/Link to Full Text]

Kedziora-Kornatowska K, Czuczejko J, Pawluk H, Kornatowski T, Motyl J, Szadujkis-Szadurski L, Szewczyk-Golec K, Kedziora J
The markers of oxidative stress and activity of the antioxidant system in the blood of elderly patients with essential arterial hypertension.
Cell Mol Biol Lett. 2004;9(4A):635-41.
We estimated the nitrate/nitrite, carbonyl groups, reduced glutathione (GSH) and malondialdehyde (MDA) concentrations and Cu,Zn superoxide dismutase (SOD-1), catalase (CAT), glutathione peroxidase (cGSH-Px) and glutathione S-transferase (GST) activities in the blood of 17 normotensive young subjects (mean age 39+/-7.0 years), 21 normotensive elderly subjects (mean age 82+/-8.2 years) and 38 patients with essential arterial hypertension (mean age 73+/-8.0 years). Our examinations showed that hypertension in the elderly is associated with greater than normal levels of protein and lipid oxidation, decreased nitric oxide concentration and an imbalance in antioxidant status (decreased GSH concentration and SOD-1 activity). The increased activity of GST compensated the decreased activity of cGSH-Px in the blood of hypertensive patients. Our study confirms that the degree of oxidative stress in elderly patients intensifies, especially if said patients have associated essential arterial hypertension. [Abstract/Link to Full Text]

Benito C, Gallego A
A simple method for the estimation of recombination frequencies and genetic distances.
Cell Mol Biol Lett. 2004;9(4A):617-34.
We have developed an alternative approach to the maximum likelihood method for calculating recombination values and linkage intensities. This new method is simpler than those in current use in that it obviates the need for formulae and tables. Maximum likelihood methods imply the use of iterative procedures over highly complicated estimation equations (at least second degree polynomials), whereas simplified methods use single-step procedures involving simple linear equations. The proposed method uses the frequencies of the distinguishable phenotypes that came from the union of at least one recombinant gamete with another gamete. We performed Monte-Carlo simulations for various combinations of genetic distance and offspring size. The recombination values obtained using the new method were compared with those derived by the maximum likelihood method on both simulated and experimental data. They were found to be nearly identical. The observed distribution of the recombination frequency values does not differ significantly from the Normal distribution, except for extreme values of the mean, as the Skewedness and kurtosis coefficients do not differ from zero. Our method has a similar accuracy to the maximum likelihood methods for recombination frequencies smaller than 25 cM and the difference does not increase greatly for greater frequencies. [Abstract/Link to Full Text]

Zawada Z
A single-step method of liposome preparation.
Cell Mol Biol Lett. 2004;9(4A):603-15.
All the liposome preparation protocols, which involve drug encapsulation are multi-step processes, i.e. they consist of one or several steps of preparation and homogenization. The conditions of converting all lipids into vesicles smaller than 200 nm were determined by replacing ultrasonication with mechanical stirring of the buffer and solution of lipids in a low-boiling point organic solvent or solvents in a simple preparator. Preferably, the process should be carried out at a temperature higher than the temperature of the gel/fluid phase transition (T(m)), and higher than the boiling point of the organic solvent(s) used to obtain the lipid solution. For many lipid membrane compositions, the products of preparation are as follows: a dominant fraction of unilamellar vesicles (vesicle of diameter smaller than 200 nm) and a fraction of much larger multivesicular or multilamellar vesicles, easily separated by simple centrifugation at 15000xg. If PEG-phosphatidylethanolamine or cholesteryl palmitate are additional membrane components, multivescular or multilamellar vescicles are virtually absent in the final product, of a single-step process and all the used lipids were quantitatively converted into vesicles smaller than 200 nm in diameter. [Abstract/Link to Full Text]

Zawada ZH
Vesicles with a double bilayer.
Cell Mol Biol Lett. 2004;9(4A):589-602.
A modified reverse phase evaporation method was used to prepare intermediate unilamellar vesicles coated with an additional membrane, or large vesicles in which several vesicles were coated with a common membrane. In both kinds of vesicle, the outer and inner membranes are usually of different phospholipid composition. The preparation involves the formation of a double emulsion: vesicles in a buffer are emerged in a low-boiling point organic solution of phospholipids. Then the organic solvent is evaporated during the heating and mixing process. As result large unilamellar vesicles (LUVs), about 100 nm in diameter, were coated with an additional membrane from egg lecithin or dipalmitoylphosphatidylcholine and cholesterol. The highest yield of the coating was about 50%. When DPPC was used for coating above the phase transition temperature Tm, the data suggested the formation of vesicles that were slightly larger than the starting LUVs. It might be concluded that many of these had a double bilayer. If the coating was done below Tm, the micrographs suggested the formation of structures resembling multi-vesicular vesicles. They looked like LUV clusters coated with a common membrane. [Abstract/Link to Full Text]

Olas B, Nowak P, Wachowicz B
Resveratrol protects against peroxynitrite-induced thiol oxidation in blood platelets.
Cell Mol Biol Lett. 2004;9(4A):577-87.
The peroxynitrite anion (ONOO-) is a reactive species produced in the reaction between the superoxide anion (O2*-) and nitric oxide (*NO). ONOO- is involved in several pathological conditions such as inflammation, arteriosclerosis, and neurodegenerative and cardiovascular disorders. Our earlier results showed that ONOO- inhibits different steps of blood platelet activation and causes the depletion of platelet thiols. In this study, we investigated the effects of resveratrol (3, 4', 5-trihydroxystilbene) and other antioxidants (uric acid and deferoxamine (DFO)) on the level of low molecular thiols such as glutathione, cysteine and cysteinylglycine (in reduced and oxidized form) in blood platelets treated with ONOO-. Our results showed that ONOO- (100 microM, 2 min) induces changes in these thiols (measured by HPLC method); these changes are diminished in the presence of resveratrol. Preincubation of human platelets with resveratrol at a concentration of 100 microM (30 min) has a protective effect against the oxidation of platelet thiols induced by ONOO- or its intermediate. The other tested antioxidants also have a protectory action. In conclusion, we suggest that the resveratrol present in the human diet may partially protect -SH groups from oxidation and may be responsible for redox regulation and control in platelets. [Abstract/Link to Full Text]

Sakowicz T, Bowater R, Parniewski P
Isolation, cloning and characterisation of motifs containing (GA/TC)n repeats isolated from vetch, Vicia bithynica.
Cell Mol Biol Lett. 2004;9(3):557-66.
Microsatellites are widely distributed in plant genomes and comprise unstable regions that undergo mutational changes at rates much greater than that observed for non-repetitive sequences. They demonstrate intrinsic genetic instability, manifested as frequent length changes due to insertions or deletions of repeat units. Detailed analysis of 1600 clones containing genomic sequences of Vicia bithynica revealed the presence of microsatellite repeats in its genome. Based on the screening of a partial DNA library of plasmids, 13 clones harbouring (GA/TC)n tracts of various lengths of repeated motif were identified for further analysis of their internal sequence organization. Sequence analyses revealed the precise length, number of repeats, interruptions within tracts, as well as sequence composition flanking the repeat motifs. Representative plasmids containing different lengths of (GA/TC)n embedded in their original flanking sequence were used to investigate the genetic stability of the repeats. In the study presented herein, we employed a well characterised and tractable bacterial genetic system. Recultivations of Escherichia coli harbouring plasmids containing (GA/TC)n inserts demonstrated that the genetic instability of (GA/TC)n microsatellites depends highly on their length (number of repeats). These observations are in agreement with similar studies performed on repetitive sequences from humans and other organisms. [Abstract/Link to Full Text]

Wang Y, Lin X, Dong B, Wang Y, Liu B
DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.
Cell Mol Biol Lett. 2004;9(3):543-56.
RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns. [Abstract/Link to Full Text]

Blanco A, Simeone R, Cenci A, Gadaleta A, Tanzarella OA, Porceddu E, Salvi S, Tuberosa R, Figliuolo G, Spagnoletti P, Röder MS, Korzun V
Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell.
Cell Mol Biol Lett. 2004;9(3):529-41.
A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci. [Abstract/Link to Full Text]

Ruci?ska R, Sobkowiak R, Gwó?d? EA
Genotoxicity of lead in lupin root cells as evaluated by the comet assay.
Cell Mol Biol Lett. 2004;9(3):519-28.
This paper presents the results of a study on the influence of lead (Pb(+2)) on DNA integrity on plant cells. The study was performed on the root tips of lupin (Lupinus luteus cv. Juno) seedlings treated with two selected concentrations of Pb(NO3)2: 150 and 350 mg l(-1), which were found to inhibit root growth by 50% and 70%, respectively [Ruci?ska et al. Plant Physiol. Biochem. 37 (1999) 37187-37194]. Roots exposed to those external lead concentrations took up about 50 and 70 mg l(-1) Pb(+2) g(-1) fresh weight (FW) over 48 h of incubation. A dose-dependent increase in the degree of root injury was observed in the presence of both tested concentrations. The genotoxicity of lead in lupin root cells was analysed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. The quantity of the DNA fragments migrating away from the nuclear remnant (tail area) increased proportionally to the lead content inside the roots, and was positively correlated with the degree of root injury. At 150 mg l(-1) Pb(+2), a high frequency distribution of nuclei having large values of tail lengths and moments was observed. By contrast, the number of nuclei with minimum values of these parameters increased at 350 mg l(-1) Pb(+2). This data suggests that lead at low concentrations induces the formation of short, rapidly migrating DNA fragments, whereas at higher concentrations, lead probably causes other changes to DNA that result in slower DNA migration in the electric field. [Abstract/Link to Full Text]

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Recent Articles in BMC Cell Biology

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Liu X, Luo F, Pan K, Wu W, Chen H
High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells.
BMC Cell Biol. 2007;81.
BACKGROUND: Connective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated. RESULTS: Expression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFbeta-dependent since anti-TGFbeta antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration. CONCLUSION: Our data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications. [Abstract/Link to Full Text]

Lahousse SA, Carter JJ, Xu XJ, Wands JR, de la Monte SM
Differential growth factor regulation of aspartyl-(asparaginyl)-beta-hydroxylase family genes in SH-Sy5y human neuroblastoma cells.
BMC Cell Biol. 2006;741.
BACKGROUND: Aspartyl (asparaginyl)-beta-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells. RESULTS: Real time quantitative RT-PCR demonstrated 8- or 9-fold higher levels of Humbug than AAH and Junctin, and lower levels of all 3 transcripts in normal human brains compared with neuroblastic tumor cells. AAH and Humbug expression were significantly increased in response to insulin and IGF-I stimulation, and these effects were associated with increased directional motility. However, over-expression of AAH and not Humbug significantly increased motility. Treatment with chemical inhibitors of Akt, Erk MAPK, or cyclin-dependent kinase 5 (Cdk-5) significantly reduced IGF-I stimulated AAH and Humbug expression and motility relative to vehicle-treated control cells. In addition, significantly increased AAH and Humbug expression and directional motility were observed in cells co-transfected with Cdk-5 plus its p35 or p25 regulatory partner. Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase. CONCLUSION: AAH and Humbug are over-expressed in SH-Sy5y neuroblastoma cells, and their mRNAs are regulated by insulin/IGF-1 signaling through Erk MAPK, PI3 kinase-Akt, and Cdk-5, which are known mediators of cell migration. Although AAH and Humbug share regulatory signaling pathways, AAH and not Humbug mediates directional motility in SH-Sy5y neuroblastoma cells. [Abstract/Link to Full Text]

Singiser RH, McCann RO
Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion.
BMC Cell Biol. 2006;740.
BACKGROUND: Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene. RESULTS: We show here that, based on their distribution in cDNA libraries, Talin-a and Talin-b are differentially expressed during C. intestinalis development. The I/LWEQ modules of the two proteins also have different affinities for F-actin. Consistent with the hypothesis that Talin-a and Talin-b have different roles in cell adhesion, the distinct I/LWEQ modules of Talin-a and Talin-b possess different subcellular targeting determinants. The I/LWEQ module of Talin-a is targeted to focal adhesions, where it most likely serves as the link between integrin and the actin cytoskeleton. The Talin-b I/LWEQ module is not targeted to focal adhesions, but instead preferentially labels F-actin stress fibers. These different properties of C. intestinalis the Talin-a and Talin-b I/LWEQ modules mimic the differences between mammalian Talin1 and Talin2. CONCLUSION: Vertebrates and D. discoideum contain two talin genes that encode proteins with different functions. The urochordate C. intestinalis has a single talin gene but produces two separate talins by alternative splicing that vary in a domain crucial for talin function. This suggests that multicellular organisms require multiple talins as components of adhesion complexes. In C. intestinalis, alternative splicing, rather than gene duplication followed by neo-functionalization, accounts for the presence of multiple talins with different properties. Given that C. intestinalis is an excellent model system for chordate biology, the study of Talin-a and Talin-b will lead to a deeper understanding of cell adhesion in the chordate lineage and how talin functions have been parceled out to multiple proteins during metazoan evolution. [Abstract/Link to Full Text]

Qui M, Paromov VM, Yang H, Smith M, Stone WL
Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages.
BMC Cell Biol. 2006;739.
BACKGROUND: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. RESULTS: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity CONCLUSION: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-kappaB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters. [Abstract/Link to Full Text]

Chen IH, Huber M, Guan T, Bubeck A, Gerace L
Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis.
BMC Cell Biol. 2006;738.
BACKGROUND: The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation. RESULTS: Using transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling. CONCLUSION: This work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies. [Abstract/Link to Full Text]

Vernier PT, Sun Y, Gundersen MA
Nanoelectropulse-driven membrane perturbation and small molecule permeabilization.
BMC Cell Biol. 2006;737.
BACKGROUND: Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization. RESULTS: In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X7 receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts. CONCLUSION: Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane. [Abstract/Link to Full Text]

Lynen Jansen P, Rosch R, Rezvani M, Mertens PR, Junge K, Jansen M, Klinge U
Hernia fibroblasts lack beta-estradiol-induced alterations of collagen gene expression.
BMC Cell Biol. 2006;736.
BACKGROUND: Estrogens are reported to increase type I and type III collagen deposition and to regulate Metalloproteinase 2 (MMP-2) expression. These proteins are reported to be dysregulated in incisional hernia formation resulting in a significantly decreased type I to III ratio. We aimed to evaluate the beta-estradiol mediated regulation of type I and type III collagen genes as well as MMP-2 gene expression in fibroblasts derived from patients with or without history of recurrent incisional hernia disease. We compared primary fibroblast cultures from male/female subjects without/without incisional hernia disease. RESULTS: Incisional hernia fibroblasts (IHFs) revealed a decreased type I/III collagen mRNA ratio. Whereas fibroblasts from healthy female donors responded to beta-estradiol, type I and type III gene transcription is not affected in fibroblasts from males or affected females. Furthermore beta-estradiol had no influence on the impaired type I to III collagen ratio in fibroblasts from recurrent hernia patients. CONCLUSION: Our results suggest that beta-estradiol does not restore the imbaired balance of type I/III collagen in incisional hernia fibroblasts. Furthermore, the individual was identified as an independent factor for the beta-estradiol induced alterations of collagen gene expression. The observation of gender specific beta-estradiol-dependent changes of collagen gene expression in vitro is of significance for future studies of cellular response. [Abstract/Link to Full Text]

Bergman LM, Morris L, Darley M, Mirnezami AH, Gunatilake SC, Blaydes JP
Role of the unique N-terminal domain of CtBP2 in determining the subcellular localisation of CtBP family proteins.
BMC Cell Biol. 2006;735.
BACKGROUND: CtBP1 and CtBP2 are transcriptional co-repressors that modulate the activity of a large number of transcriptional repressors via the recruitment of chromatin modifiers. Many CtBP-regulated proteins are involved in pathways associated with tumorigenesis, including TGF-beta and Wnt signalling pathways and cell cycle regulators such as RB/p130 and HDM2, as well as adenovirus E1A. CtBP1 and CtBP2 are highly similar proteins, although evidence is emerging that their activity can be differentially regulated, particularly through the control of their subcellular localisation. CtBP2s from diverse species contain a unique N-terminus, absent in CtBP1 that plays a key role in controlling the nuclear-cytoplasmic distribution of the protein. RESULTS: Here we show that amino acids (a.a.) 4-14 of CtBP2 direct CtBP2 into an almost exclusively nuclear distribution in cell lines of diverse origins. Whilst this sequence contains similarity to known nuclear localisation motifs, it cannot drive nuclear localisation of a heterologous protein, but rather has been shown to function as a p300 acetyltransferase-dependent nuclear retention sequence. Here we define the region of CtBP2 required to co-operate with a.a. 4-14 to promote CtBP2 nuclear accumulation as being within a.a. 1-119. In addition, we show that a.a. 120-445 of CtBP2 can also promote CtBP2 nuclear accumulation, independently of a.a. 4-14. Finally, CtBP1 and CtBP2 can form heterodimers, and we show that the interaction with CtBP2 is one mechanism whereby CtBP1 can be recruited to the nucleus. CONCLUSION: Together, these findings represent key distinctions in the regulation of the functions of CtBP family members that may have important implications as to their roles in development, and cell differentiation and survival. [Abstract/Link to Full Text]

Galbaugh T, Cerrito MG, Jose CC, Cutler ML
EGF-induced activation of Akt results in mTOR-dependent p70S6 kinase phosphorylation and inhibition of HC11 cell lactogenic differentiation.
BMC Cell Biol. 2006;734.
BACKGROUND: HC11 mouse mammary epithelial cells differentiate in response to lactogenic hormone resulting in expression of milk proteins including beta-casein. Previous studies have shown that epidermal growth factor (EGF) blocks differentiation not only through activation of the Ras/Mek/Erk pathway but also implicated phosphatidylinositol-3-kinase (PI-3-kinase) signaling. The current study analyzes the mechanism of the PI-3-kinase pathway in an EGF-induced block of HC11 lactogenic differentiation. RESULTS: HC11 and HC11-luci cells, which contain luciferase gene under the control of a beta-casein promotor, were treated with specific chemical inhibitors of signal transduction pathways or transiently infected/transfected with vectors encoding dominant negative-Akt (DN-Akt) or conditionally active-Akt (CA-Akt). The expression of CA-Akt inhibited lactogenic differentiation of HC11 cells, and the infection with DN-Akt adenovirus enhanced beta-casein transcription and rescued beta-casein promotor-regulated luciferase activity in the presence of EGF. Treatment of cells with Rapamycin, an inhibitor of mTOR, blocked the effects of EGF on beta-casein promotor driven luciferase activity as effectively as PI-3-kinase inhibitors. While expression of CA-Akt caused a constitutive activation of p70S6 kinase (p70S6K) in HC11 cells, the inhibition of either PI-3-kinase or mTOR abolished the activation of p70S6K by EGF. The activation of p70S6K by insulin or EGF resulted in the phosphorylation of ribosomal protein S6 (RPS6), elongation initiation factor 4E (elF4E) and 4E binding protein1 (4E-BP1). But lower levels of PI-3-K and mTOR inhibitors were required to block insulin-induced phosphorylation of RPS6 than EGF-induced phosphorylation, and insulin-induced phosphorylation of elF4E and 4E-BP1 was not completely mTOR dependent suggesting some diversity of signaling for EGF and insulin. In HC11 cells undergoing lactogenic differentiation the phosphorylation of p70S6K completely diminished by 12 hours, and this was partly attributable to dexamethasone, a component of lactogenic hormone mix. However, p70S6K phosphorylation persisted in the presence of lactogenic hormone and EGF, but the activation could be blocked by a PI-3-kinase inhibitor. CONCLUSION: PI-3-kinase signaling contributes to the EGF block of lactogenic differentiation via Akt and p70S6K. The EGF-induced activation of PI-3-kinase-Akt-mTOR regulates phosphorylation of molecules including ribosomal protein S6, eIF4E and 4E-BP1 that influence translational control in HC11 cells undergoing lactogenic differentiation. [Abstract/Link to Full Text]

Yang K, Guo Y, Stacey WC, Harwalkar J, Fretthold J, Hitomi M, Stacey DW
Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels.
BMC Cell Biol. 2006;733.
BACKGROUND: The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. RESULTS: We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. CONCLUSION: Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth. [Abstract/Link to Full Text]

Baschal EE, Chen KJ, Elliott LG, Herring MJ, Verde SC, Wolkow TD
The fission yeast DNA structure checkpoint protein Rad26ATRIP/LCD1/UVSD accumulates in the cytoplasm following microtubule destabilization.
BMC Cell Biol. 2006;732.
BACKGROUND: DNA structure checkpoints are conserved eukaryotic signal transduction pathways that help preserve genomic integrity. Upon detecting checkpoint signals such as stalled replication forks or double-stranded DNA breaks, these pathways coordinate appropriate stress responses. Members of the PI-3 kinase related kinase (PIKK) family are essential elements of DNA structure checkpoints. In fission yeast, the Rad3 PIKK and its regulatory subunit Rad26 coordinate the detection of checkpoint signals with pathway outputs. RESULTS: We found that untreated rad26Delta cells were defective for two microtubule-dependent processes: chromosome segregation and morphogenesis. Interestingly, cytoplasmic accumulation of Rad26-GFP occurred following treatment with microtubule destabilizing drugs, but not during treatment with the genotoxic agent Phleomycin. Cytoplasmic accumulation of Rad26-GFP depended on Rad24, a 14-3-3 protein also required for DNA structure checkpoints and morphogenesis. Results of over expression and epistasis experiments confirm that Rad26 and Rad24 define a response to microtubule destabilizing conditions. CONCLUSION: Two DNA structure checkpoint proteins with roles in morphogenesis define a response to microtubule destabilizing conditions. [Abstract/Link to Full Text]

Zhang XM, Chang Q, Zeng L, Gu J, Brown S, Basch RS
TBLR1 regulates the expression of nuclear hormone receptor co-repressors.
BMC Cell Biol. 2006;731.
BACKGROUND: Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. RESULTS: TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies approximately 200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest. CONCLUSION: TBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation. [Abstract/Link to Full Text]

Kekarainen T, Mannelin S, Laine J, Jaatinen T
Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells.
BMC Cell Biol. 2006;730.
BACKGROUND: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. RESULTS: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. CONCLUSION: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood. [Abstract/Link to Full Text]

Tafuri LS, Rocha GF, Gobbi H
Cell cycle related proteins in hyperplasia of usual type in breast specimens of patients with and without breast cancer.
BMC Cell Biol. 2006;729.
BACKGROUND: Hyperplasia of usual type (HUT) is a common proliferative lesion associated with a slight elevated risk for subsequent development of breast cancer. Cell cycle-related proteins would be helpful to determine the putative role of these markers in the process of mammary carcinogenesis. The aim of this study was to analyze the expression of cell cycle related proteins in HUT of breast specimens of patients with and without breast cancer, and compare this expression with areas of invasive carcinomas. RESULTS: Immunohistochemical evaluation was performed using antibodies against cell cycle related proteins ER, PR, p53, p21, p63, and Ki-67 in hyperplasia of usual type (HUT) in specimens of aesthetic reduction mammaplasty (ARM), in specimens of mammaplasty contralateral to breast cancer (MCC), and in specimens of invasive mammary carcinomas (IMC) presenting HUT in the adjacent parenchyma. The results showed that the immunoexpression of ER, PR, p21, p53, p63, and KI-67 was similar in HUT from the three different groups. The p63 expression in myoepithelial cells showed discontinuous pattern in the majority of HUT, different from continuous expression in normal lobules. Nuclear expression of p53 and p21 was frequently higher expressed in IMC and very rare in HUT. We also found cytoplasmic expression of p21 in benign hyperplastic lesions and in neoplastic cells of IMC. CONCLUSION: Our data failed to demonstrate different expression of cell cycle related proteins in HUT from patients with and without breast cancer. However, we found discontinuous expression of p63 in myoepithelial cells around HUT adjacent to carcinomas and cytoplasmic expression of p21 in epithelial cells of hyperplastic foci. Further studies are needed to determine how these subgroups relate to molecular abnormalities and cancer risk. [Abstract/Link to Full Text]

Blitzer JT, Nusse R
A critical role for endocytosis in Wnt signaling.
BMC Cell Biol. 2006;728.
BACKGROUND: The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation. RESULTS: We show here that Wnt proteins are rapidly endocytosed by a clathrin- and dynamin-mediated process. While endocytosis has traditionally been considered a principal mechanism for receptor down-regulation and termination of signaling pathways, we demonstrate that interfering with clathrin-mediated endocytosis actually blocks Wnt signaling at the level of beta-catenin accumulation and target gene expression. CONCLUSION: A necessary component of Wnt signaling occurs in a subcellular compartment distinct from the plasma membrane. Moreover, as internalized Wnts transit partially through the transferrin recycling pathway, it is possible that a "signaling endosome" serves as a nexus for activated Wnt pathway components. [Abstract/Link to Full Text]

Christman SA, Kong BW, Landry MM, Kim H, Foster DN
Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line.
BMC Cell Biol. 2006;727.
BACKGROUND: The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years. RESULTS: The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells. CONCLUSION: The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21WAF1, may have contributed to the immortalization of the SC-2 CEF cell line. [Abstract/Link to Full Text]

Stout JR, Rizk RS, Kline SL, Walczak CE
Deciphering protein function during mitosis in PtK cells using RNAi.
BMC Cell Biol. 2006;726.
BACKGROUND: Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed. RESULTS: By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency approximately 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. P-MCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest. CONCLUSION: This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells. [Abstract/Link to Full Text]

Ohnuma K, Yomo T, Asashima M, Kaneko K
Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.
BMC Cell Biol. 2006;725.
BACKGROUND: Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. RESULTS: We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells. CONCLUSION: We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses. [Abstract/Link to Full Text]

Mould AP, McLeish JA, Huxley-Jones J, Goonesinghe AC, Hurlstone AF, Boot-Handford RP, Humphries MJ
Identification of multiple integrin beta1 homologs in zebrafish (Danio rerio).
BMC Cell Biol. 2006;724.
BACKGROUND: Integrins comprise a large family of alpha,beta heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single beta1 gene, and the beta1 subunit associates with a large number of alpha subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about beta1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish beta1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two beta1 paralogs (beta1-1 and beta1-2) that have a high degree of identity to other vertebrate beta1 subunits. In addition, a third, more divergent, beta1 paralog is present (beta1-3), which may have altered ligand-binding properties. Zebrafish also have other divergent beta1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with beta1-3 these truncated forms comprise a novel group of beta1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to beta1-1 and beta1-2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of beta1-2 may have given rise to beta1-3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated beta1 paralogs appears to have taken place. The different zebrafish beta1 paralogs have varied patterns of temporal expression during development. Beta1-1 and beta1-2 are ubiquitously expressed in adult tissues, whereas the other beta1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin beta1 paralogs. beta1-1 and beta1-2 may share the roles of the solitary beta1 subunit found in other vertebrates, whereas beta1-3 and the truncated beta1 paralogs may have acquired novel functions. [Abstract/Link to Full Text]

Münter S, Enninga J, Vazquez-Martinez R, Delbarre E, David-Watine B, Nehrbass U, Shorte SL
Actin polymerisation at the cytoplasmic face of eukaryotic nuclei.
BMC Cell Biol. 2006;723.
BACKGROUND: There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE) membrane system. However, this interaction has yet to be characterised in living interphase cells. RESULTS: Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY) we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies). CONCLUSION: We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes. [Abstract/Link to Full Text]

Wang H, Zheng Y, He S
Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases.
BMC Cell Biol. 2006;722.
BACKGROUND: Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined. RESULTS: A549 cells express all four PARs at both protein and mRNA levels as assessed by flow cytometry, immunofluorescence microscopy and reverse transcription polymerase chain reaction (PCR). Thrombin, tryptase, elastase and trypsin induce a up to 8, 4.3, 4.4 and 5.1 fold increase in IL-8 release from A549 cells, respectively following 16 h incubation period. The thrombin, elastase and trypsin induced secretion of IL-8 can be abolished by their specific inhibitors. Agonist peptides of PAR-1, PAR-2 and PAR-4 stimulate up to 15.6, 6.6 and 3.5 fold increase in IL-8 secretion, respectively. Real time PCR shows that IL-8 mRNA is up-regulated by the serine proteinases tested and by agonist peptides of PAR-1 and PAR-2. CONCLUSION: The proteinases, possibly through activation of PARs can stimulate IL-8 release from A549 cells, suggesting that they are likely to contribute to IL-8 related airway inflammatory disorders in man. [Abstract/Link to Full Text]

Aho TL, Sandholm J, Peltola KJ, Ito Y, Koskinen PJ
Pim-1 kinase phosphorylates RUNX family transcription factors and enhances their activity.
BMC Cell Biol. 2006;721.
BACKGROUND: The pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode alpha-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression. RESULTS: Here we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim-1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion. CONCLUSION: Altogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis. [Abstract/Link to Full Text]

Nollevaux G, Devillé C, El Moualij B, Zorzi W, Deloyer P, Schneider YJ, Peulen O, Dandrifosse G
Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21).
BMC Cell Biol. 2006;720.
BACKGROUND: The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. RESULTS: In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. CONCLUSION: The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. [Abstract/Link to Full Text]

Drubin DA, Garakani AM, Silver PA
Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics.
BMC Cell Biol. 2006;719.
BACKGROUND: Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. RESULTS: We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. CONCLUSION: Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery. [Abstract/Link to Full Text]

Hagens O, Ballabio A, Kalscheuer V, Kraehenbuhl JP, Schiaffino MV, Smith P, Staub O, Hildebrand J, Wallingford JB
A new standard nomenclature for proteins related to Apx and Shroom.
BMC Cell Biol. 2006;718.
Shroom is a recently-described regulator of cell shape changes in the developing nervous system. This protein is a member of a small family of related proteins that are defined by sequence similarity and in most cases by some link to the actin cytoskeleton. At present these proteins are named Shroom, APX, APXL, and KIAA1202. In light of the growing interest in this family of proteins, we propose here a new standard nomenclature. [Abstract/Link to Full Text]

Tamilarasan KP, Kolluru GK, Rajaram M, Indhumathy M, Saranya R, Chatterjee S
Thalidomide attenuates nitric oxide mediated angiogenesis by blocking migration of endothelial cells.
BMC Cell Biol. 2006;717.
BACKGROUND: Thalidomide is an immunomodulatory agent, which arrests angiogenesis. The mechanism of anti-angiogenic activity of thalidomide is not fully understood. As nitric oxide is involved in angiogenesis, we speculate a cross-talk between thalidomide and nitric oxide signaling pathway to define angiogenesis. The aim of present study is to understand the mechanistic aspects of thalidomide-mediated attenuation of angiogenesis induced by nitric oxide at the cellular level. METHODS: To study the cellular mechanism of thalidomide-mediated blocking of angiogenesis triggered by nitric oxide, we used two endothelial cell based models: 1) wound healing and 2) tube formation using ECV 304, an endothelial cell line. These cell-based models reflect pro-angiogenic events in vivo. We also studied the effects of thalidomide on nitric oxide mediated egg yolk angiogenesis. Thalidomide could block the formation of blood vessels both in absence and presence of nitric oxide. Thalidomide effects on migration of, and actin polymerization in, ECV 304 cells were studied at the single cell level using live cell imaging techniques and probes to detect nitric oxide. RESULTS: Results demonstrate that thalidomide blocks nitric oxide-mediated angiogenesis in egg yolk model and also reduces the number of tubes formed in endothelial cell monolayers. We also observed that thalidomide arrests wound healing in presence and absence of nitric oxide in a dose-dependent fashion. Additionally, thalidomide promotes actin polymerization and antagonizes the formation of membrane extensions triggered by nitric oxide in endothelial cells. Experiments targeting single tube structure with thalidomide, followed by nitric oxide treatment, show that the tube structures are insensitive to thalidomide and nitric oxide. These observations suggest that thalidomide interferes with nitric oxide-induced migration of endothelial cells at the initial phase of angiogenesis before cells co-ordinate themselves to form organized tubes in endothelial cells and thereby inhibits angiogenesis. CONCLUSION: Thalidomide exerts inhibitory effects on nitric oxide-mediated angiogenesis by altering sub-cellular actin polymerization pattern, which leads to inhibition of endothelial cell migration. [Abstract/Link to Full Text]

Valdimarsdottir G, Goumans MJ, Itoh F, Itoh S, Heldin CH, ten Dijke P
Smad7 and protein phosphatase 1alpha are critical determinants in the duration of TGF-beta/ALK1 signaling in endothelial cells.
BMC Cell Biol. 2006;716.
BACKGROUND: In endothelial cells (EC), transforming growth factor-beta (TGF-beta) can bind to and transduce signals through ALK1 and ALK5. The TGF-beta/ALK5 and TGF-beta/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-beta signaling is an important specificity determinant for signaling responses. TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently. RESULTS: The temporal activation of TGF-beta-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-beta/ALK1 signaling pathway. Smad7 and protein phosphatase 1alpha (PP1alpha) mRNA expression levels were found to be specifically upregulated by TGF-beta/ALK1. Ectopic expression of Smad7 or PP1alpha potently inhibited TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1alpha enhanced TGF-beta/ALK1-induced signaling responses. PP1alpha interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor. CONCLUSION: Our results suggest that upon its induction by the TGF-beta/ALK1 pathway, Smad7 may recruit PP1alpha to ALK1, and thereby control TGF-beta/ALK1-induced Smad1/5 phosphorylation. [Abstract/Link to Full Text]

Tesseur I, Zou K, Berber E, Zhang H, Wyss-Coray T
Highly sensitive and specific bioassay for measuring bioactive TGF-beta.
BMC Cell Biol. 2006;715.
BACKGROUND: Transforming Growth Factor-beta (TGF-beta) regulates key biological processes during development and in adult tissues and has been implicated in many diseases. To study the biological functions of TGF-beta, sensitive, specific, and convenient bioassays are necessary. Here we describe a new cell-based bioassay that fulfills these requirements. RESULTS: Embryonic fibroblasts from Tgfb1-/- mice were stably transfected with a reporter plasmid consisting of TGF-beta responsive Smad-binding elements coupled to a secreted alkaline phosphatase reporter gene (SBE-SEAP). Clone MFB-F11 showed more than 1000-fold induction after stimulation with 1 ng/ml TGF-beta1, and detected as little as 1 pg/ml TGF-beta1. MFB-F11 cells were highly induced by TGF-beta1, TGF-beta2 and TGF-beta3, but did not show induction with related family members activin, nodal, BMP-2 and BMP-6 or with trophic factors bFGF and BDNF. MFB-F11 cells can detect and quantify TGF-beta in biological samples without prior enrichment of TGF-betas, and can detect biologically activated TGF-beta in a cell co-culture system. CONCLUSION: MFB-F11 cells can be used to rapidly and specifically measure TGF-beta with high sensitivity. [Abstract/Link to Full Text]

Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B
Aging of mesenchymal stem cell in vitro.
BMC Cell Biol. 2006;714.
BACKGROUND: A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells. RESULTS: The mean long term culture was 118 days and the mean passage number was 9. The average number of PD decreased from 7.7 to 1.2 in the 10th passage. The mean telomere length decreased from 9.19 Kbp to 8.7 kbp in the 9th passage. Differentiation potential dropped from the 6th passage on. The culture's morphological abnormalities were typical of the Hayflick model of cellular aging. CONCLUSION: We believe that MSC enter senescence almost undetectably from the moment of in vitro culturing. Simultaneously these cells are losing their stem cell characteristics. Therefore, it is much better to consider them for cell and gene therapy early on. [Abstract/Link to Full Text]

Holy J, Lamont G, Perkins E
Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine.
BMC Cell Biol. 2006;713.
BACKGROUND: The quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells. RESULTS: 5-10 microM sanguinarine effectively inhibits MCF-7 proliferation after a single application of drug. This growth inhibition is accompanied by a striking relocalization of cyclin D1 and topoisomerase II from the nucleus to the cytoplasm, and this effect persists for at least three days after drug addition. DNA synthesis is transiently inhibited by sanguinarine, but cells recover their ability to synthesize DNA within 24 hours. Taking advantage of the fluorescence characteristics of sanguinarine to follow its uptake and distribution suggests that these effects arise from a window of activity of a few hours immediately after drug addition, when sanguinarine is concentrated in the nucleus. These effects occur in morphologically healthy-looking cells, and thus do not simply represent part of an apoptotic response. CONCLUSION: It appears that sub-apoptotic concentrations of sanguinarine can suppress breast cancer cell proliferation for extended lengths of time, and that this effect results from a relatively brief period of activity when the drug is concentrated in the nucleus. Sanguinarine transiently inhibits DNA synthesis, but a novel mechanism of action appears to involve disrupting the trafficking of a number of molecules involved in cell cycle regulation and progression. The ability of sub-apoptotic concentrations of sanguinarine to inhibit cell growth may be a useful feature for potential chemotherapeutic applications; however, a narrow effective range for these effects may exist. [Abstract/Link to Full Text]

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Recent Articles in Eukaryotic Cell

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Metcalf T, van der Wel H, Escalante R, Sastre L, West CM
Role of SP65 in assembly of the Dictyostelium discoideum spore coat.
Eukaryot Cell. 2007 Jul;6(7):1137-49.
Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function. [Abstract/Link to Full Text]

Baker LG, Specht CA, Donlin MJ, Lodge JK
Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans.
Eukaryot Cell. 2007 May;6(5):855-67.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a "leaky melanin" phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets. [Abstract/Link to Full Text]

Rydholm C, Dyer PS, Lutzoni F
DNA sequence characterization and molecular evolution of MAT1 and MAT2 mating-type loci of the self-compatible ascomycete mold Neosartorya fischeri.
Eukaryot Cell. 2007 May;6(5):868-74.
Degenerate PCR and chromosome-walking approaches were used to identify mating-type (MAT) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus Neosartorya fischeri, a close relative of the opportunistic human pathogen Aspergillus fumigatus. Both putative alpha- and high-mobility-group-domain MAT genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed MAT1 and MAT2 to recognize the presence of distinct loci. Complete copies of putative APN1 (DNA lyase) and SLA2 (cytoskeleton assembly control) genes were found bordering the MAT1 locus. Partial copies of APN1 and SLA2 were also found bordering the MAT2 locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the N. fischeri MAT1 region and the A. fumigatus MAT locus region, but no such long-range conservation in the N. fischeri MAT2 region was evident. The sequence upstream of MAT2 contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the Aspergillus section Fumigati. [Abstract/Link to Full Text]

Beaudoin J, Labbé S
Crm1-mediated nuclear export of the Schizosaccharomyces pombe transcription factor Cuf1 during a shift from low to high copper concentrations.
Eukaryot Cell. 2007 May;6(5):764-75.
In this study, we examine the fate of the nuclear pool of the Schizosaccharomyces pombe transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional cuf1(+)-GFP allele in the presence of a copper chelator. We then extinguished cuf1(+)-GFP expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES), (349)LAALNHISAL(358), within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in crm1(+) exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of ctr4(+) gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like ctr4(+). In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration. [Abstract/Link to Full Text]

Dabas N, Morschhäuser J
Control of ammonium permease expression and filamentous growth by the GATA transcription factors GLN3 and GAT1 in Candida albicans.
Eukaryot Cell. 2007 May;6(5):875-88.
In response to nitrogen starvation, the human fungal pathogen Candida albicans switches from yeast to filamentous growth. This morphogenetic switch is controlled by the ammonium permease Mep2p, whose expression is induced under limiting nitrogen conditions. In order to understand in more detail how nitrogen starvation-induced filamentous growth is regulated in C. albicans, we identified the cis-acting sequences in the MEP2 promoter that mediate its induction in response to nitrogen limitation. We found that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To investigate whether the GATA transcription factors GLN3 and GAT1 regulate MEP2 expression, we constructed mutants of the C. albicans wild-type strain SC5314 lacking one or both of these transcription factors. Expression of Mep2p was strongly reduced in gln3Delta and gat1Delta single mutants and abolished in gln3Delta gat1Delta double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, but the filamentation defect of gln3Delta mutants could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation, and we found that filamentation became independent of the presence of a functional MEP2 gene in the gat1Delta mutants, indicating that the loss of GAT1 function results in the activation of other pathways inducing filamentous growth. These results demonstrate that the GATA transcription factors GLN3 and GAT1 control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. [Abstract/Link to Full Text]

Alvarez FJ, Douglas LM, Konopka JB
Sterol-rich plasma membrane domains in fungi.
Eukaryot Cell. 2007 May;6(5):755-63. [Abstract/Link to Full Text]

Dünkler A, Wendland J
Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation.
Eukaryot Cell. 2007 May;6(5):844-54.
Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum. [Abstract/Link to Full Text]

Cottrell TR, Griffith CL, Liu H, Nenninger AA, Doering TL
The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis.
Eukaryot Cell. 2007 May;6(5):776-85.
Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted. [Abstract/Link to Full Text]

Engh I, Würtz C, Witzel-Schlömp K, Zhang HY, Hoff B, Nowrousian M, Rottensteiner H, Kück U
The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies.
Eukaryot Cell. 2007 May;6(5):831-43.
Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation. [Abstract/Link to Full Text]

Ohnuki S, Nogami S, Kanai H, Hirata D, Nakatani Y, Morishita S, Ohya Y
Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae.
Eukaryot Cell. 2007 May;6(5):817-30.
Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca(2+)-induced morphological changes in Ca(2+)-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca(2+)-induced morphological changes in the Ca(2+)-sensitive mutant zds1 reflect changes in the Ca(2+) signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca(2+) in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca(2+) exerts a wide variety of effects on yeast and that there are multiple Ca(2+)-regulatory pathways that are distinct from the Zds1p-related pathway. [Abstract/Link to Full Text]

Chen M, Lopes JM
Multiple basic helix-loop-helix proteins regulate expression of the ENO1 gene of Saccharomyces cerevisiae.
Eukaryot Cell. 2007 May;6(5):786-96.
The basic helix-loop-helix (bHLH) eukaryotic transcription factors have the ability to form multiple dimer combinations. This property, together with limited DNA-binding specificity for the E box (CANNTG), makes them ideally suited for combinatorial control of gene expression. We tested the ability of all nine Saccharomyces cerevisiae bHLH proteins to regulate the enolase-encoding gene ENO1. ENO1 was known to be activated by the bHLH protein Sgc1p. Here we show that expression of an ENO1-lacZ reporter was also regulated by the other eight bHLH proteins, namely, Ino2p, Ino4p, Cbf1p, Rtg1p, Rtg3p, Pho4p, Hms1p, and Ygr290wp. ENO1-lacZ expression was also repressed by growth in inositol-choline-containing medium. Epistatic analysis and chromatin immunoprecipitation experiments showed that regulation by Sgc1p, Ino2p, Ino4p, and Cbf1p and repression by inositol-choline required three distal E boxes, E1, E2, and E3. The pattern of bHLH binding to the three E boxes and experiments with two dominant-negative mutant alleles of INO4 and INO2 support the model that bHLH dimer selection affects ENO1-lacZ expression. These results support the general model that bHLH proteins can coordinate different biological pathways via multiple mechanisms. [Abstract/Link to Full Text]

Bohse ML, Woods JP
Expression and interstrain variability of the YPS3 gene of Histoplasma capsulatum.
Eukaryot Cell. 2007 Apr;6(4):609-15.
The YPS3 locus of the dimorphic fungus Histoplasma capsulatum encodes a secreted and surface-localized protein specific to the pathogenic yeast phase. In this study we examined this locus in 32 H. capsulatum strains and variants. Although protein production is limited to a select group of strains, the North American restriction fragment length polymorphism class 2/NAm 2 isolates, the locus was present in all the strains we examined. The YPS3 gene is well conserved in its 5' and 3' regions but displays an intragenic hypervariable region of tandem repeats that fluctuates in size between strains. This feature is similar to that seen with genes encoding several cell surface proteins in other fungi. [Abstract/Link to Full Text]

Liu OW, Kelly MJ, Chow ED, Madhani HD
Parallel beta-helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans.
Eukaryot Cell. 2007 Apr;6(4):630-40.
The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered. [Abstract/Link to Full Text]

Wiesenberger G, Steinleitner K, Malli R, Graier WF, Vormann J, Schweyen RJ, Stadler JA
Mg2+ deprivation elicits rapid Ca2+ uptake and activates Ca2+/calcineurin signaling in Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Apr;6(4):592-9.
To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress. [Abstract/Link to Full Text]

Fraser JA, Stajich JE, Tarcha EJ, Cole GT, Inglis DO, Sil A, Heitman J
Evolution of the mating type locus: insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii.
Eukaryot Cell. 2007 Apr;6(4):622-9.
Sexual reproduction of fungi is governed by the mating type (MAT) locus, a specialized region of the genome encoding key transcriptional regulators that direct regulatory networks to specify cell identity and fate. Knowledge of MAT locus structure and evolution has been considerably advanced in recent years as a result of genomic analyses that enable the definition of MAT locus sequences in many species as well as provide an understanding of the evolutionary plasticity of this unique region of the genome. Here, we extend this analysis to define the mating type locus of three dimorphic primary human fungal pathogens, Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii, using genomic analysis, direct sequencing, and bioinformatics. These studies provide evidence that all three species possess heterothallic bipolar mating type systems, with isolates encoding either a high-mobility-group (HMG) domain or an alpha-box transcriptional regulator. These genes are intact in all loci examined and have not been subject to loss or decay, providing evidence that the loss of fertility upon passage in H. capsulatum is not attributable to mutations at the MAT locus. These findings also suggest that an extant sexual cycle remains to be defined in both Coccidioides species, in accord with population genetic evidence. Based on these MAT sequences, a facile PCR test was developed that allows the mating type to be rapidly ascertained. Finally, these studies highlight the evolutionary forces shaping the MAT locus, revealing examples in which flanking genes have been inverted or subsumed and incorporated into an expanding MAT locus, allowing us to propose an expanded model for the evolution of the MAT locus in the phylum Ascomycota. [Abstract/Link to Full Text]

Rubenstein EM, Schmidt MC
Mechanisms regulating the protein kinases of Saccharomyces cerevisiae.
Eukaryot Cell. 2007 Apr;6(4):571-83. [Abstract/Link to Full Text]

Wang C, St Leger RJ
The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants.
Eukaryot Cell. 2007 May;6(5):808-16.
Metarhizium anisopliae is a fungus of considerable metabolic and ecological versatility, being a potent insect pathogen that can also colonize plant roots. The mechanistic details of these interactions are unresolved. We provide evidence that M. anisopliae adheres to insects and plants using two different proteins, MAD1 and MAD2, that are differentially induced in insect hemolymph and plant root exudates, respectively, and produce regional localization of adhesive conidial surfaces. Expression of Mad1 in Saccharomyces cerevisiae allowed this yeast to adhere to insect cuticle. Expression of Mad2 caused yeast cells to adhere to a plant surface. Our study demonstrated that as well as allowing adhesion to insects, MAD1 at the surface of M. anisopliae conidia or blastospores is required to orientate the cytoskeleton and stimulate the expression of genes involved in the cell cycle. Consequently, the disruption of Mad1 in M. anisopliae delayed germination, suppressed blastospore formation, and greatly reduced virulence to caterpillars. The disruption of Mad2 blocked the adhesion of M. anisopliae to plant epidermis but had no effects on fungal differentiation and entomopathogenicity. Thus, regulation, localization, and specificity control the functional distinction between Mad1 and Mad2 and enable M. anisopliae cells to adapt their adhesive properties to different habitats. [Abstract/Link to Full Text]

Bubnick M, Smulian AG
The MAT1 locus of Histoplasma capsulatum is responsive in a mating type-specific manner.
Eukaryot Cell. 2007 Apr;6(4):616-21.
Recombination events associated with sexual replication in pathogens may generate new strains with altered virulence. Histoplasma capsulatum is a mating-competent, pathogenic fungus with two described phenotypic mating types, + and -. The mating (MAT) locus of H. capsulatum was identified to facilitate molecular studies of mating in this organism. Through syntenic analysis of the H. capsulatum genomic sequence databases, a MAT1-1 idiomorph region was identified in H. capsulatum strains G217B and WU24, and a MAT1-2 idiomorph region was identified in the strain G186AR. A mating type-specific PCR assay was developed, and two clinical isolates of opposite genotypic mating type, UH1 and VA1, were identified. A known--mating type strain, T-3-1 (ATCC 22635), was demonstrated to be of MAT1-2 genotypic mating type. The clinical isolates UH1 and VA1 were found to be mating compatible and also displayed mating-type-dependent regulation of the MAT transcription factors in response to extracts predicted to contain mating pheromones. These studies support a role for the identified MAT1 locus in determining mating type in H. capsulatum. [Abstract/Link to Full Text]

Endoh-Yamagami S, Hirakawa K, Morioka D, Fukuda R, Ohta A
Basic helix-loop-helix transcription factor heterocomplex of Yas1p and Yas2p regulates cytochrome P450 expression in response to alkanes in the yeast Yarrowia lipolytica.
Eukaryot Cell. 2007 Apr;6(4):734-43.
The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica, is highly regulated and can be induced by alkanes. Previously, we identified a cis-acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes. [Abstract/Link to Full Text]

Sen A, Chatterjee NS, Akbar MA, Nandi N, Das P
The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress.
Eukaryot Cell. 2007 Apr;6(4):664-73.
The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of Entamoeba histolytica exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of eh29 gene expression by antisense RNA technology has shown approximately 55% inhibition in eh29 expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of E. histolytica has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis. [Abstract/Link to Full Text]

Takebe S, Witola WH, Schimanski B, Günzl A, Ben Mamoun C
Purification of components of the translation elongation factor complex of Plasmodium falciparum by tandem affinity purification.
Eukaryot Cell. 2007 Apr;6(4):584-91.
Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1beta harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1beta-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1beta, the G-protein PfEF-1alpha, and two new proteins that we named PfEF-1gamma and PfEF-1delta based on their homology to other eukaryotic gamma and delta translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1beta. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen. [Abstract/Link to Full Text]

Goosen C, Yuan XL, van Munster JM, Ram AF, van der Maarel MJ, Dijkhuizen L
Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity.
Eukaryot Cell. 2007 Apr;6(4):674-81.
A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides. [Abstract/Link to Full Text]

Truman AW, Millson SH, Nuttall JM, Mollapour M, Prodromou C, Piper PW
In the yeast heat shock response, Hsf1-directed induction of Hsp90 facilitates the activation of the Slt2 (Mpk1) mitogen-activated protein kinase required for cell integrity.
Eukaryot Cell. 2007 Apr;6(4):744-52.
Yeast is rendered temperature sensitive with loss of the C-terminal (CT) domain of heat shock transcription factor (Hsf1). This domain loss was found to abrogate heat stimulation of Slt2 (Mpk1), the mitogen-activated protein kinase that directs the reinforced cell integrity gene expression needed for high-temperature growth. In Hsf1 CT domain-deficient cells, Slt2 still undergoes Mkk1/2-directed dual-Thr/Tyr phosphorylation in response to the heat stimulation of cell integrity pathway signaling, but the low Hsp90 expression level suppresses any corresponding increase in Slt2 kinase activity due to Slt2 being a "client" of the Hsp90 chaperone. A non-Hsf1-directed Hsp90 overexpression restored the heat induction of Slt2 activity in these cells, as well as both Slt2-dependent (Rlm1, Swi4) and Slt2-independent (MBF) transcriptional activities. Their high-temperature growth was also rescued, not just by this Hsp90 overexpression but by osmotic stabilization, by the expression of a Slt2-independent form of the Rlm1 transcriptional regulator of cell integrity genes, and by a multicopy SLT2 gene vector. In providing the elevated Hsp90 needed for an efficient activation of Slt2, heat activation of Hsf1 indirectly facilitates (Slt2-directed) heat activation of yet another transcription factor (Rlm1). This provides an explanation as to why, in earlier transcript analysis compared to chromatin immunoprecipitation studies, many more genes of yeast displayed an Hsf1-dependent transcriptional activation by heat than bound Hsf1 directly. The levels of Hsp90 expression affecting transcription factor regulation by Hsp90 client protein kinases also provides a mechanistic model for how heat shock factor can influence the expression of several non-hsp genes in higher organisms.