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Light Chain (light + chain)
Kinds of Light Chain Terms modified by Light Chain Selected AbstractsGeneration of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Sylvia Niebruegge Abstract The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called "embryoid bodies" (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493,507. © 2008 Wiley Periodicals, Inc. [source] Unsupervised immunophenotypic profiling of chronic lymphocytic leukemiaCYTOMETRY, Issue 3 2006Luzette K. Habib Abstract Background Proteomics and functional genomics have revolutionized approaches to disease classification. Like proteomics, flow cytometry (FCM) assesses concurrent expression of many proteins, with the advantage of using intact cells that may be differentially selected during analysis. However, FCM has generally been used for incremental marker validation or construction of predictive models based on known patterns, rather than as a tool for unsupervised class discovery. We undertook a retrospective analysis of clinical FCM data to assess the feasibility of a cell-based proteomic approach to FCM by unsupervised cluster analysis. Methods Multicolor FCM data on peripheral blood (PB) and bone marrow (BM) lymphocytes from 140 consecutive patients with B-cell chronic lymphoproliferative disorders (LPDs), including 81 chronic lymphocytic leukemia (CLLs), were studied. Expression was normalized for CD19 totals, and recorded for 10 additional B-cell markers. Data were subjected to hierarchical cluster analysis using complete linkage by Pearson's correlation. Analysis of CLL in PB samples (n = 63) discovered three major clusters. One cluster (14 patients) was skewed toward "atypical" CLL and was characterized by high CD20, CD22, FMC7, and light chain, and low CD23. The remaining two clusters consisted almost entirely (48/49) of cases recorded as typical BCLL. The smaller "typical" BCLL cluster differed from the larger cluster by high CD38 (P = 0.001), low CD20 (P = 0.001), and low CD23 (P = 0.016). These two typical BCLL clusters showed a trend toward a difference in survival (P = 0.1090). Statistically significant cluster stability was demonstrated by expanding the dataset to include BM samples, and by using a method of random sampling with replacement. Conclusions This study supports the concept that unsupervised immunophenotypic profiling of FCM data can yield reproducible subtypes of lymphoma/chronic leukemia. Expanded studies are warranted in the use of FCM as an unsupervised class discovery tool, akin to other proteomic methods, rather than as a validation tool. © 2006 International Society for Analytical Cytology [source] Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus,CYTOSKELETON, Issue 1 2010Brian A. Young Abstract Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested,actin, myosin heavy and light chain, and IQGAP,as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis. © 2009 Wiley-Liss, Inc. [source] In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesisCYTOSKELETON, Issue 2 2008Ryota Uehara Abstract Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomasCYTOSKELETON, Issue 1 2008Bodour Salhia Abstract The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Native nonmuscle myosin II stability and light chain binding in Drosophila melanogasterCYTOSKELETON, Issue 10 2006Josef D. Franke Abstract Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo,thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in divisionCYTOSKELETON, Issue 2 2003Rosalind V. Silverman-Gavrila Abstract We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597,609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180,197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182,195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed. Cell Motil. Cytoskeleton 55:97,113, 2003. © 2003 Wiley-Liss, Inc. [source] Calyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggsCYTOSKELETON, Issue 4 2001Yukako Asano Abstract When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a 32P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. Cell Motil. Cytoskeleton 48:245,261, 2001. © 2001 Wiley-Liss, Inc. [source] Conditional expression of a myocardium-specific transgene in zebrafish transgenic linesDEVELOPMENTAL DYNAMICS, Issue 4 2005Chiu-Ju Huang Abstract To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage. Developmental Dynamics 233:1294,1303, 2005. © 2005 Wiley-Liss, Inc. [source] Muscle fiber differentiation in fish embryos as shown by in situ hybridization of a large repertoire of muscle-specific transcriptsDEVELOPMENTAL DYNAMICS, Issue 2 2005F. Chauvigné Abstract Skeletal muscles are composed of different fiber types, largely defined by differential expression of protein isoforms involved in myofibrillogenesis or metabolism. To learn more about the gene activations that underlie the differentiation and the diversification of embryonic fish myotomal fibers, we investigated the developmental expression of 25 muscle genes in trout embryos by in situ hybridization of muscle-specific transcripts. The earliest event of muscle differentiation, at approximately the 25-somite stage, was the expression of a variety of muscle-specific genes, including slow-twitch and fast-twitch muscle isoforms. The activation of these muscle genes started in the deep somitic domain, where the slow muscle precursors (the adaxial cells) were initially located, and progressively spread laterally throughout the width of the myotome. This mediolateral progression of gene expression was coordinated with the lateral migration of slow adaxial cells, which specifically expressed the slow myosin light chain 1 and the SLIM1/FHL1 genes. Subsequently, the fast and slow skeletal muscle isoforms precociously expressed in the course of the mediolateral wave of muscle gene activation became down-regulated in the superficial slow fibers and the deep fast fibers, respectively. Finally, several muscle-specific genes, including troponins, a slow myosin-binding protein C, tropomodulins, and parvalbumin started their transcription only in late embryos. Taken together, these findings show in fish embryos that a common myogenic program is triggered in a mediolateral progression in all muscle cells. The acquisition of the slow phenotype involves the additional activation of several slow-specific genes in migrating adaxial muscle cells. These events are followed by sequential gene activations and repressions in fast and slow muscle cells. Developmental Dynamics 233:659,666, 2005. © 2005 Wiley-Liss, Inc. [source] Age-dependent variations of cell response to oxidative stress: Proteomic approach to protein expression and phosphorylationELECTROPHORESIS, Issue 14 2005Yuri Miura Dr. Abstract We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2 -exposure of cells at 100,µM for 30,min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2 -exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S -adenosyl- L -homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2 -exposure in astrocytes from variously aged rats. Using the Pro-Q® Diamond staining, heat shock protein 60,kDa (Hsp 60) and ,-tubulin were observed to be phosphorylated upon H2O2 -exposure. While phosphorylation of ,-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules. [source] Lymphocyte-expressed BILL-cadherin/cadherin-17 contributes to the development of B cells at two stagesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2005Kazuo Ohnishi Abstract The gene encoding BILL-cadherin/cadherin-17, a nonclassical cadherin expressed on B lymphocytes in a stage-and-site-specific manner, was inactivated by targeted disruption of its transmembrane/cytoplasmic portion-encoding parts. BILL-cadherin deficiency caused a threefold proB cell accumulation, as well as a reduction to half of the numbers of immature B cells in bone marrow. In spleen, CD21hiCD23lo marginal zone B cells were found reduced and the structure of the marginal zone was impaired. In addition, the size and number of germinal center as well as the number of PNA+ cells were significantly reduced in BILL-cadherin-deficient mice. In the peritoneal cavity of mutant mice IgM+Mac-1+CD5+ B1a cell, that express high BILL-cadherin in wild-type mice, was also reduced in number. The IgG1 and IgG3 antibody response to the T-independent antigen, TNP-Ficoll, was impaired in the mutant mice. These results indicate that BILL-cadherin participates in B lymphocyte development at least at two stages, first at the transition of pro/preB-I cells to preB-II cells possibly in association with surrogate light chain in bone marrow, and later at the point of development, accumulation and reactiveness of immature B cells in spleen. [source] Expression of muscle-related genes and two MyoD genes during amphioxus notochord developmentEVOLUTION AND DEVELOPMENT, Issue 5 2003Aki Urano Summary The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expre-ssion patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropo-myosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), Amphi-nTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expres-sed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle. [source] Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thalianaFEBS JOURNAL, Issue 15 2001Koen Peeters To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fab fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3, regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3, regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fab fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of Fab fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants. [source] Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approachFEBS JOURNAL, Issue 20 2000Sophie Quevillon-Chéruel The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain. [source] Ca2+ -independent phospholipase A2-dependent sustained Rho-kinase activation exhibits all-or-none responseGENES TO CELLS, Issue 9 2006Akio Maeda Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+ -independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response. [source] The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8GENES TO CELLS, Issue 7 2004Kenji Sugawara Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 Å resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABAA receptor-associated protein (GABARAP), contains a ubiquitin core with two , helices, ,1 and ,2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among ,1, ,2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins. [source] Roles of the novel interleukin-12-associated cytokine, interleukin-23, in the regulation of T-cell-mediated immunityHEPATOLOGY RESEARCH, Issue 2007Masanori Matsui Interleukin (IL)-12 is a heterodimeric proinflammatory cytokine formed by a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40). This cytokine is a key regulator of cell-mediated immunity, and therefore should have therapeutic potential in infectious diseases and tumors. Recently, a novel IL-12-associated cytokine, IL-23 has been discovered. IL-23 is also a heterodimer that consists of the p40 subunit of IL-12 and a novel subunit, p19. Several studies have shown that IL-23 possesses immunoadjuvant activity against tumor and infectious diseases as well as IL-12. On the other hand, there is increasing evidence that IL-12 and IL-23 have discrete roles in the regulation of T-cell-mediated immunity despite their structural similarities. IL-12 leads to the development ofinterferon-,-producing T-helper type 1 (Th1) cells, whereas IL-23 amplifies and stabilizes a new CD4+ T-cell subset, Th17 producing IL-17. The IL-23/Th17 axis rather than the IL-12/Th1 axis contributes to several immune-mediated inflammatory autoimmune diseases. Furthermore, IL-23/IL-17 promotes tumor incidence and growth. Therefore, IL-23 and Th17 are attracting considerable attention at present. Taken together, these findings suggest that IL-23 may be an immunoadjuvant against infectious diseases and tumors, and a viable target for the treatment of inflammatory diseases. [source] Unusual presentation of large B cell lymphoma: a case report and review of literatureINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 5 2006L. AIRAGHI Summary Diffuse large B cell lymphoma (DLBCL) is the largest subtype of non-Hodgkin's lymphomas (NHLs) and is characterized by relatively frequent extranodal presentation. In these cases, the most common extranodal localizations are stomach, CNS, bone, testis and liver. Simultaneous detection of multiple extranodal involvement at presentation is quite uncommon, with the majority of these cases characterized by gastric or intestinal disease localization. Retrospective analysis concerning multifocal extranodal NHLs never pointed out disease features such as those described here. We report a patient with an unusual presentation of DLBCL, characterized by adrenal and renal involvement, associated with symptoms and signs of the cold agglutinin disease and a hypercoagulable state. Subsequently, computed tomography (CT) and fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning disclosed a rapidly extensive spread to nodes and bones. Cytofluorimetric analysis of a renal specimen showed medium-to-large lympho-monocytoid elements positive for CD20 with monoclonal expression of immunoglobulin kappa light chain. Histopathological examination confirmed a renal CD20 positive DLBCL localization. [source] Myosin Light Chain Kinase as a Multifunctional Regulatory Protein of Smooth Muscle ContractionIUBMB LIFE, Issue 6 2001Ying Gao Abstract Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction. [source] The biological activity of ubiquitinated BoNT/B light chain in vitro and in human SHSY-5Y neuronal cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2009Xuerong Shi Abstract BoNT/B light chain is a zinc-dependent endopeptidase. After entering its target, the neuronal cell, BoNT/B is responsible for synaptobrevin-2 (VAMP-2) cleavage. This results in reduced neurotransmitter (acetylcholine) release from synaptic vesicles, yielding muscular paralysis. Since the toxin persists in neuronal cells for an extended period, regeneration of VAMP-2 is prevented. We evaluated therapeutic targets to overcome botulinum persistence because early removal would rescue the neuronal cell. The ubiquitination/proteasome cellular pathway is responsible for removing "old" or undesirable proteins. Therefore, we assessed ubiquitination of BoNT/B light chain in vitro, and characterized the effects of ubiquitination modulating drugs, PMA (phorbol 12-myristate 13-acetate) and expoxomicin, on ubiquitination of BoNT/B light chain in neuronal cells. Both drugs altered BoNT/B light chain ubiquitination. Ubiquitination in vitro and in cells decreased the biological activity of BoNT/B light chain. These results further elucidate BoNT protein degradation pathways in intoxicated neuronal cells and mechanisms to enhance toxin removal. J. Cell. Biochem. 108: 660,667, 2009. Published 2009 Wiley-Liss, Inc. [source] Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Takeomi Mizutani Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild-type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical-scanning probe microscope (M-SPM). The LPA treatment increased cellular stiffness in the wild-type MRLC expressing cells, while it had no effect on the mutated cells. Immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers. J. Cell. Physiol. 209: 726,731, 2006. © 2006 Wiley-Liss, Inc. [source] Qualification and Quantification of Fish Protein in Prepared Surimi CrabstickJOURNAL OF FOOD SCIENCE, Issue 5 2008Z.H. Reed ABSTRACT:, Species identification and protein quantification in surimi crabstick were achieved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the Lowry and Kjeldahl protein determination methods were compared, the former showed more consistent results. Densitometric scanning of the gels was used for quantification of total fish protein as well as total egg white protein. The lower molecular weight proteins, 30 kDa and lower, proved to be the most useful in fish species identification as well as egg white protein addition. Using a combination of the myosin heavy chain band and the species-specific myosin light chain (Alaska pollock: 22.5 kDa; Pacific whiting: 24.4 kDa) proved the most accurate in calculating fish protein content of the crabstick sample, while for those samples that contained egg white, quantification was accomplished from the densitometric analysis of the overlapping bands of actin (45 kDa) from fish and ovalbumin from egg white. Lysozyme (14.3 kDa) proved to be a unique protein band in determining the presence of egg white when the content of dried egg white was equal to or exceeded 0.5% of the total weight of the final crabstick. [source] Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replicationJOURNAL OF MEDICAL VIROLOGY, Issue 7 2009Shu-Chen Huang Abstract Enterovirus 71 (EV71) is an important pathogen causing death in children under 5 years old worldwide. However, the underlying pathogenesis remains unclear. This study reveals that EV71 infection in rhabdomyosarcoma (RD) and neuroblastoma (SK-N-SH) cells stimulated the autophagic process, which was demonstrated by an increase of punctate GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3), the level of autophagosome-bound LC3-II protein and double-membrane autophagosome formation. EV71-induced autophagy benefited EV71 replication, which was confirmed by the autophagic inducer rapamycin and the inhibitor 3-methyladenine. Signaling pathway investigation revealed that the decreased expression of phosphorylated mTOR and phosphorylated p70S6K is involved in EV71-induced autophagy in a cell-specific manner. The expression of phosphorylated extracellular signal-regulated kinase (Erk) was suppressed consistently in EV71-infected cells. However it did not participate in the autophagic response of the cell. Other signaling pathway molecules, such as Erk, PI3K/Akt, Bcl-2, BNIP3, and Beclin-1 were not affected by infection with EV71. Electron microscopy showed co-localization of autophagosome-like vesicles with either EV71-VP1 or LC3 protein in neurons of the cervical spinal cord in ICR mice infected with EV71. In conclusion, EV71 infection triggered autophagic flux and induced autophagosome formation both in vitro and in vivo. Autophagy induced by EV71 is beneficial for viral replication. Understanding the role of autophagy induced by EV71 in vitro and the formation of autophagosome-like vesicle in vivo provide new insights into the pathogenesis of EV71 infection. J. Med. Virol. 81:1241,1252, 2009. © 2009 Wiley-Liss, Inc. [source] Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance moleculeJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Takekazu Kubo Abstract Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin , a specific myosin II inhibitor , and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa. [source] Tubulin and CRMP-2 complex is transported via Kinesin-1JOURNAL OF NEUROCHEMISTRY, Issue 6 2005Nariko, Toshihide, Yuko Kimura Arimura Fukata Abstract The transport of tubulin and microtubules in a growing axon is essential for axonal growth and maintenance. However, the molecular mechanism underlying the linkage of tubulin and microtubules to motor proteins is not yet clear. Collapsin response mediator protein-2 (CRMP-2) is enriched at the distal part of growing axons in primary hippocampal neurons and plays a critical role in axon differentiation through its interaction with tubulin dimer and Numb. In this study, we show that CRMP-2 regulates tubulin transport by linking tubulin and Kinesin-1. The C-terminal region of CRMP-2 directly binds to the tetratricopeptide repeat domain of kinesin light chain 1 (KLC1). Soluble tubulin binds to the middle of CRMP-2 and forms a trimeric complex with CRMP-2/KLC1. Furthermore, the movement of GFP,tubulin in the photobleached area is weakened by knockdown of KLCs or CRMP-2. These results indicate that the CRMP-2/Kinesin-1 complex regulates soluble tubulin transport to the distal part of the growing axon. [source] Antiapoptotic and antiautophagic effects of glial cell line-derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2010Jingwei Shang Abstract Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser2448 (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to ,-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways. © 2010 Wiley-Liss, Inc. [source] Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Kenji Nakao Abstract Activated astrocytes show various patterns of Ca2+ mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca2+ dynamics through investigation of thrombin-induced unique Ca2+ entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03,3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca2+ concentration ([Ca2+]i) showed that thrombin induced heterogeneous Ca2+ responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca2+ release from intracellular stores, followed by refilling of Ca2+ from the extracellular region without a direct [Ca2+]i increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca2+ mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca2+ entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca2+ responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca2+ influx. Moreover, TRPC3-mediated Ca2+ dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca2+ dynamics, including Ca2+ entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca2+ channel and might modulate astrocytic activation in response to brain hemorrhaging. © 2008 Wiley-Liss, Inc. [source] Regulation of oxidative-stress responsive genes by arecoline in human keratinocytesJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2009G. S. Thangjam Background and Objective:, Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular basis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes. Material and Methods:, Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription,polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation. Results:, Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase. Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:, Our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes. [source] Melatonin suppresses cyclosporine A-induced autophagy in rat pituitary GH3 cellsJOURNAL OF PINEAL RESEARCH, Issue 3 2010Yeong-Min Yoo Abstract:, Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0,100 ,m) decreased cell survival of rat pituitary GH3 cells in a dose-dependent manner. At concentrations ranging from 1.0 to 10 ,m, CsA induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-I and LC3-II. Cells treated with 2.5 ,m CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0,10 ,m CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol-requiring enzyme 1 alpha (IRE1,), increased as compared those levels in untreated cells. These results suggested that CsA-induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA-induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 ,m) suppressed autophagy induced by 2.5 and 5 ,m CsA. Furthermore, co-treatment with 100 ,m melatonin inhibited LC3-II expression, and increased catalase and phosphorylated p-ERK levels in the presence of 2.5 and 5 ,m CsA. BiP and IRE1, expression in melatonin-co-treated cells was superior to that in cells treated with 2.5 and 5 ,m CsA alone. Thus, melatonin suppresses CsA-mediated autophagy in rat pituitary GH3 cells. [source] | |||||||||||||||||||||||||||||||||||||