965 resultados para Heavy-chain Gene


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Impaired function of shoulder muscles, resulting from rotator cuff tears, is associated with abnormal deposition of fat in muscle tissue, but corresponding cellular and molecular mechanisms, likely reflected by altered gene expression profiles, are largely unknown. Here, an analysis of muscle gene expression was carried out by semiquantitative RT-PCR in total RNA extracts of supraspinatus biopsies collected from 60 patients prior to shoulder surgery. A significant increase of alpha-skeletal muscle actin (p = 0.0115) and of myosin heavy polypeptide 1 (p = 0.0147) gene transcripts was observed in parallel with progressive fat deposition in the muscle, assessed on parasagittal T1-weighted turbo-spin-echo magnetic resonance images according to Goutallier. Upregulation of alpha-skeletal muscle actin and of myosin heavy polypeptide-1 has been reported to be associated with increased muscle tissue metabolism and oxidative stress. The findings of the present study, therefore, challenge the hypothesis that increased fat deposition in rotator cuff muscle after injury reflects muscle degeneration.

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Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-Ddependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart fromaffecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional,more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize oskmRNA correctly.Moreover,we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes.

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The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

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Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

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Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.

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Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

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Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

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The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.

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The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

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Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

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Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.

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Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-ζ blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-ζ (on residue Thr410). These findings demonstrate for the first time that PKC-ζ and Sp1 phosphorylation mediate the inducible expression of this growth factor.

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Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

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The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.