共查询到20条相似文献,搜索用时 15 毫秒
1.
alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments. 相似文献
2.
The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin. 相似文献
3.
Shenhav Cohen Jeffrey J. Brault Steven P. Gygi David J. Glass David M. Valenzuela Carlos Gartner Esther Latres Alfred L. Goldberg 《The Journal of cell biology》2009,185(6):1083-1095
Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1. 相似文献
4.
Sandrine Arbogast Jacqueline Smith Yves Matuszczak Brian J Hardin Jennifer S Moylan Jeffrey D Smith Jeffrey Ware Ann R Kennedy Michael B Reid 《Journal of applied physiology》2007,102(3):956-964
Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2',7'-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness. 相似文献
5.
A fundamental concept in development is that secreted molecules such as Wingless (Wg) and Hedgehog (Hh) generate pattern by inducing cell fate. By following markers of cellular identity posterior to the Wg- and Hh-expressing cells in the Drosophila dorsal embryonic epidermis, we provide evidence that neither Wg nor Hh specifies the identity of the cell types they pattern. Rather, they maintain pre-existing cellular identities that are otherwise unstable and progress stepwise towards a default fate. Wg and Hh therefore generate pattern by inhibiting specific switches in cell identity, showing that the specification and the patterning of a given cell are uncoupled. Sequential binary decisions without induction of cell identity give rise to both the groove cells and their posterior neighbors. The combination of independent progression of cell identity and arrest of progression by signals facilitates accurate patterning of an extremely plastic developing epidermis. 相似文献
6.
Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells 总被引:24,自引:30,他引:24
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E Lazarides 《The Journal of cell biology》1976,68(2):202-219
During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells. 相似文献
7.
Barend M deC Bronsvoort Alfons Renz Virginia Tchakouté Vincent N Tanya David Ekale Alexander J Trees 《Filaria journal》2005,4(1):1-8
Background
Ivermectin (Mectizan?, Merck and CO. Inc.) is being widely used in the control of human onchocerciasis (Onchoverca volvulus) because of its potent effect on microfilariae. Human studies have suggested that, at the standard dose of 150 μg/kg an annual treatment schedule of ivermectin reversibly interferes with female worm fertility but is not macrofilaricidal. Because of the importance of determining whether ivermectin could be macrofilaricidal, the efficacy of high and prolonged doses of ivermectin and a related avermectin, doramectin, were investigated in cattle infected with O. ochengi.Methods
Drugs with potential macrofilaricidal activity, were screened for the treatment of human onchocerciasis, using natural infections of O. ochengi in African cattle. Three groups of 3 cows were either treated at monthly intervals (7 treatments) with ivermectin (Ivomec®, Merck and Co. Inc.) at 500 μg/kg or doramectin (Dectamax®, Pfizer) at 500 μg/kg or not treated as controls. Intradermal nodules were removed at 6 monthly intervals and adult worms were examined for signs of drug activity.Results
There was no significant decline in nodule diameter, the motility of male and female worms, nor in male and female viability as determined by the ability to reduce tetrazolium, compared with controls, at any time up to 24 months from the start of treatments (mpt). Embryogenesis, however, was abrogated by treatment, which was seen as an accumulation of dead and dying intra-uterine microfilariae (mf) persisting for up to 18 mpt. Skin mf densities in treated animals had fallen to zero by <3 mpt, but by 18 mpt small numbers of mf were found in the skin of some treated animals and a few female worms were starting to produce multi-cellular embryonic stages. Follow-up of the doramectin treated group at 36 mpt showed that mf densities had still only regained a small proportion of their pre-treatment levels.Conclusion
These results have important implications for onchocerciasis control in the field. They suggest that ivermectin given at repeated high does may sterilise O. volvulus female worms for prolonged periods but is unlikely to kill them. This supports the view that control programmes may need to continue treatments with ivermectin for a period of decades and highlights the need to urgently identify new marcofiliaricidal compounds. 相似文献8.
Molecular genetics of Drosophila alpha-actinin: mutant alleles disrupt Z disc integrity and muscle insertions 总被引:4,自引:8,他引:4
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We have isolated a Drosophila melanogaster alpha-actinin gene and partially characterized several mutant alleles. The Drosophila protein sequence is very similar (68% identity) to those of chicken alpha-actinin isoforms, but less closely related (30% identity) to Dictyostelium alpha-actinin. The gene is within subdivision 2C of the X chromosome, coincident with 15 lethal (1)2Cb mutations. At least four alleles, l(1)2Cb1, l(1)2Cb2, l(1)2Cb4, and l(1)2Cb5 are interrupted by rearrangement breakpoints and must be null. In all four cases, hemizygous mutants complete embryogenesis and do not die until the second day of larval growth, signifying that either the role of alpha-actinin in nonmuscle cells is redundant or that a distinct and only distantly related gene encodes the non-muscle isoform. Allelic but less severely affected fliA mutants are apparently due to point mutations, and develop into adults having thoracic muscle abnormalities. EM of mutant muscles reveals that Z discs and myofibrillar attachments are disrupted, whereas epithelial "tendon" cells are less affected. We discuss these phenotypes in the light of presumed in vivo alpha-actinin functions. 相似文献
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The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death 总被引:2,自引:0,他引:2
BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation. 相似文献
11.
Eccentric contractions leading to DOMS do not cause loss of desmin nor fibre necrosis in human muscle 总被引:15,自引:4,他引:15
High force eccentric muscle contractions can result in delayed onset muscle soreness (DOMS), prolonged loss of muscle strength, decreased range of motion, muscle swelling and an increase of muscle proteins in the blood. At the ultrastructural level Z-line streaming and myofibrillar disruptions have been taken as evidence for muscle damage. In animal models of eccentric exercise-induced injury, disruption of the cytoskeleton and the sarcolemma of muscle fibres occurs within the first hour after the exercise, since a rapid loss of staining of desmin, a cytoskeletal protein, and the presence of fibronectin, a plasma and extracellular protein, are observed within the muscle fibres. In the present study, biopsies from subjects who had performed different eccentric exercises and had developed DOMS were examined. Our aim was to determine whether eccentric exercise leading to DOMS causes sarcolemmal disruption and loss of desmin in humans. Our study shows that even though the subjects had DOMS, muscle fibres had neither lost staining for desmin nor contained plasma fibronectin. This study therefore does not support previous conclusions that there is muscle fibre degeneration and necrosis in human skeletal muscle after eccentric exercise leading to DOMS. Our data are in agreement with the recent findings that there is no inflammatory response in skeletal muscle following eccentric exercise in humans. In combination, these findings should stimulate the search for other mechanisms explaining the functional and structural alterations in human skeletal muscle after eccentric exercise. 相似文献
12.
Bekheirnia MR Zhang W Eble T Willis A Shaibani A Wong LJ Scaglia F Dhar SU 《Gene》2012,499(1):209-212
Mutations in POLG account for one of the most frequent nuclear encoded causes of mitochondrial disorders to date. Individuals harboring POLG mutations exhibit fairly heterogeneous clinical presentations leading to increasing difficulties in classifying these patients into defined clinical phenotypes. This study aims to investigate the molecular basis of a mitochondrial cytopathy in a patient with 3-methylglutaconic aciduria and to expand the clinical phenotype associated with POLG mutations. Clinical, molecular and genetic analyses as well as neurophysiological examinations were carried out for a 23-year-old woman of mixed Caucasian and Latin American ancestry with a history of cataracts diagnosed at age 1 year, she had onset of distal muscle weakness at age 2 years progressing to atrophy and ovarian dysgenesis at puberty. The patient was found to have 3-methylglutaconic acid with normal 3 hydroxyisovaleric acid on urine organic acid analysis. POLG sequencing was done and a heterozygous variant, c.2851T>A (p.Y951N) was found which is predicted to be deleterious. There are limited reports of POLG mutations in individuals with 3-methylglutaconic aciduria. This case report of a young woman with a heterozygous mutation in POLG, presenting with muscle weakness and atrophy at a young age aims to aid clinicians in similar challenging diagnostic situations as well as enhances our understanding of POLG-related disease phenotypes. 相似文献
13.
Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cowpea leaves 总被引:10,自引:0,他引:10
Fabio Rossi Cavalcanti José Tadeu Abreu Oliveira Aparecida Simone Martins-Miranda Ricardo Almeida Viégas Joaquim Albenísio Gomes Silveira 《The New phytologist》2004,163(3):563-571
14.
Park SK Sedore SA Cronmiller C Hirsh J 《The Journal of biological chemistry》2000,275(27):20588-20596
We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol. 相似文献
15.
Houngbédji GM Bouchard P Frenette J 《American journal of physiology. Regulatory, integrative and comparative physiology》2011,300(3):R724-R732
Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans. These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients. 相似文献
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Small decreases in SBPase cause a linear decline in the apparent RuBP regeneration rate, but do not affect Rubisco carboxylation capacity 总被引:7,自引:0,他引:7
Harrison EP Olcer H Lloyd JC Long SP Raines CA 《Journal of experimental botany》2001,52(362):1779-1784
The response of net photosynthetic CO(2) uptake (A) to increasing leaf intercellular CO(2) concentration (c(i)) was determined in antisense Nicotiana tabacum plants, derived from six independent transformation lines, displaying a range of sedoheptulose-1, 7-bisphosphatase (SBPase) activities. The maximum in vivo ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V(c,max)) and RuBP regeneration (J(max)) rates were calculated from the steady-state measurements of the A to c(i) response curves. In plants with reductions in SBPase activity of between 9% and 60%, maximum RuBP regeneration capacity declined linearly (r(2)=0.79) and no significant change in apparent in vivo Rubisco activity (V(c,max)) was observed in these plants. No correlation between V(c,max) and a decrease in capacity for RuBP regeneration was observed (r(2)=0.14) in the SBPase antisense plants. These data demonstrate that small decreases in SBPase activity limit photosynthetic carbon assimilation by reducing the capacity for RuBP regeneration. 相似文献
19.
We report here the characterization of slamdance (sda), a Drosophila melanogaster "bang-sensitive" (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical "bang" or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations. 相似文献
20.
Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, we purified the Drosophila myosin-II tail expressed in Escherichia coli and showed that it was phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, we show that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, we generated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way. 相似文献