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In an attempt to understand why muscle recovery is limited following atrophy due to limb immobilization, satellite cell activity and muscle fiber regeneration were analyzed in rat soleus muscles. Adult rat hindlimbs were immobilized in plaster casts for a period of two to ten weeks. Soleus muscles were examined by electron microscopy for evidence of fiber degeneration or regeneration, and to quantify satellite cell nuclei. Immunocytochemical localization of embryonic myosin was used to identify regenerating myofibers. Soleus muscle wet weight to body weight ratios for the casted muscles significantly decreased over the 10‐week immobilization period. The casted muscles displayed ultrastructural evidence of minor fiber damage, including myofibrillar atrophy, Z‐disc disruption, and abnormal triadic junctions. No ultrastructural evidence of regeneration was seen in the casted animals. The number of satellite cells in the casted muscles significantly decreased from 6.4% to 3.3% by eight to 10 weeks of immobilization. Approximately 1.0% of extrafusal fibers in the control soleus muscles appeared to be regenerating since they expressed embryonic myosin and were of a small diameter, while in casted muscles, only 0.1% of the fibers were embryonic myosin‐positive. Following release from immobilization, a reappearance of embryonic myosin‐positive fibers was noted within four days of renewed activity. In contrast to control muscles, embryonic myosin‐positive fibers in the recovery muscles included both small and large diameter fibers. Subtle changes in functional activity influence muscle damage and subsequent myofiber regeneration. Reduced activity reduces muscle fiber regeneration, while increased activity, as seen by increased hindlimb weight bearing and return to normal activity following immobilization, increase regenerating fibers and also the expression of embryonic myosin in adult fibers. Anat Rec 258:176–185, 2000. © 2000 Wiley‐Liss, Inc.  相似文献   

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Background: Tropomodulins are actin‐capping proteins that regulate the stability of the slow‐growing, minus‐ends of actin filaments. The C. elegans tropomodulin homolog, UNC‐94, has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC‐94 in C. elegans intestinal morphogenesis. Results: In the embryonic C. elegans intestine, UNC‐94 localizes to the terminal web, an actin‐ and intermediate filament‐rich structure that underlies the apical membrane. Loss of UNC‐94 function results in areas of flattened intestinal lumen. In worms homozygous for the strong loss‐of‐function allele, unc‐94(tm724), the terminal web is thinner and the amount of F‐actin is reduced, pointing to a role for UNC‐94 in regulating the structure of the terminal web. The non‐muscle myosin, NMY‐1, also localizes to the terminal web, and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel‐11, can rescue the flattened lumen phenotype of unc‐94 mutants. Conclusions: The data support a model in which minus‐end actin capping by UNC‐94 promotes proper F‐actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis. Developmental Dynamics 243:753–764, 2014. © 2014 Wiley Periodicals, Inc.  相似文献   

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Animal cells contain two populations of microtubules: one radiating from the centrosome and the other growing from non‐centrosomal sites. Whether or not they have differing roles in cellular architecture and function remains not fully understood. The cytoplasmic protein Nezha (also known as CAMSAP3) stabilizes non‐centrosomal microtubules by attaching to their minus ends. Here, we found that depletion of CAMSAP3 in HeLa cells resulted in a relative increase in centrosomal microtubules, and this change was accompanied by accelerated actin stress fiber formation. In these cells, RhoA activity was upregulated, and the soluble fraction of GEF‐H1, a RhoGEF whose activity is inhibited by binding to microtubules, increased, explaining why stress fiber formation was promoted. We further found that CAMSAP3 depletion led to an increase in detyrosinated microtubules, and these microtubules did not interact with GEF‐H1. These findings suggest that CAMSAP3‐anchored non‐centrosomal microtubules capture GEF‐H1 more efficiently than other microtubules do and that a balance between these microtubules is important to maintain proper actin organization.  相似文献   

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During cytokinesis in many eukaryotic cells, myosin‐II concentrates at the equatorial cortex with actin filaments (F‐actin) and is supposed to generate forces to divide the cell into two, which is called the contractile ring (CR) hypothesis. Several lines of evidence indicate that the myosin‐II is recruited independently of F‐actin and interacts specifically with the equatorial F‐actin. Molecular details of these mechanisms are still unknown. We used the fission yeast Schizosaccharomyces pombe to investigate the regulation of myosin‐II localization. We demonstrate that the CR myosin‐II was composed of F‐actin‐dependent and ‐independent fractions by simultaneously observing F‐actin and myosin. The F‐actin‐independent fraction was visualized as cortical dots in the absence of F‐actin. IQGAP Rng2, an indispensable element of CR, was implicated in maintenance of the F‐actin‐independent fraction of myosin‐II, whereas anillin Mid1 was required for assembly but not for maintenance of the fraction. In the CR of the rng2 mutant, myosin‐II was less concentrated, unstable, and nonhomogeneous, which often resulted in cytokinesis failure. These results suggest that Rng2 tethers myosin‐II to the cortex along the CR independently of F‐actin to provide a sufficient concentration. The robust localization of myosin‐II would ensure successful cytokinesis.  相似文献   

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Rab proteins play a critical role in intracellular vesicle trafficking and require post‐translational modification by adding lipids at the C‐terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue‐specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep‐1 gene and found that a rep‐1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab‐3 or rab‐27 suggested that rep‐1 functions only in the RAB‐27 pathway, and not in the RAB‐3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP‐1 did not cause defecation defects compared to severe defects in either RAB‐27 or RabGGTase disruption, suggesting that REP‐1 is not essential for RAB‐27 signaling in defection. Some Rab proteins did not physically interact with REP‐1, and localization of these Rab proteins was not severely affected by REP‐1 disruption. These findings suggest that REP‐1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP‐1.  相似文献   

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大鼠拟“血管性痴呆”模型的改进   总被引:120,自引:4,他引:120       下载免费PDF全文
随着社会进入老龄化 ,作为老年病的脑血管病 ,已成为人类三大死亡原因之一。近年来由于医疗水平的提高和防治知识的深入 ,脑血管病死亡率有所下降 ,而脑血管病造成的各种功能和智力障碍却有增高的趋势。血管性痴呆的防治日益受到人们的关注。目前虽建立了数种脑缺血动物模型 ,但脑缺血晚期学习记忆功能障碍的动物模型较少并不够完善 ,在一定程度上影响了对其机理及治疗的研究。本工作在前人实验的基础上加以改进 ,建立了一种拟“血管性痴呆”的动物模型 ,为研究脑缺血后学习、记忆功能障碍的发生机理及防治提供实验基础。材 料 和 方 法…  相似文献   

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We have previously shown that SGIP1α is an endocytic protein specifically expressed in neural tissues. SGIP1α has a lipid‐binding domain called the MP domain, which shows no significant homology to any other domains. In this study, we characterized FCHO2, a protein with a high level of homology to SGIP1α. FCHO2 lacks the MP domain but has another lipid‐binding domain, the EFC/F‐BAR domain. FCHO2 was ubiquitously expressed. The FCHO2 EFC domain bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubes. FCHO2 localized to clathrin‐coated pits at the plasma membrane and bound to Eps15, an important adaptor protein in clathrin‐mediated endocytosis. FCHO2 knockdown reduced transferrin endocytosis. These results suggest that FCHO2 regulates clathrin‐mediated endocytosis through its interactions with membranes and Eps15. These properties of FCHO2 are similar to those of SGIP1α. FCHO2 is likely to be a ubiquitous homologue of SGIP1α. We furthermore found that FCHO2 was subjected to monoubiquitination, and gel filtration analysis showed that FCHO2 formed an oligomer. These new properties might also contribute to the role of FCHO2 in clathrin‐mediated endocytosis.  相似文献   

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Following antigen/IgE‐mediated aggregation of high affinity IgE‐receptors (FcεRI), mast cells (MCs) degranulate and release inflammatory mediators leading to the induction of allergic reactions including anaphylaxis. Migration of MCs to resident tissues and sites of inflammation is regulated by tissue chemotactic factors such as stem cell factor (SCF (KIT ligand)). Despite inducing similar early signaling events to antigen, chemotactic factors, including SCF, produce minimal degranulation in the absence of other stimuli. We therefore investigated whether processes regulating MC chemotaxis are rate limiting for MC mediator release. To investigate this issue, we disrupted actin polymerization, a requirement for MC chemotaxis, with latrunculin B and cytochalasin B, then examined chemotaxis and mediator release in human (hu)MCs induced by antigen or SCF. As expected, such disruption minimally affected early signaling pathways, but attenuated SCF‐induced human mast cell chemotaxis. In contrast, SCF, in the absence of other stimuli, induced substantial degranulation in a concentration‐dependent manner following actin disassembly. It also moderately enhanced antigen‐mediated human mast cell degranulation which was further enhanced in the presence of SCF. These observations suggest that processes regulating cell migration limit MC degranulation as a consequence of cytoskeletal reorganization.  相似文献   

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Mouse collagen‐induced arthritis (CIA) is the most commonly used animal model to investigate underlying pathogenesis of autoimmune arthritis and to demonstrate the therapeutic efficacy of novel drugs in autoimmune arthritis. The conventional read‐outs of CIA are clinical score and histopathology, which have several limitations, including (i) subjected to observer bias; and (ii) longitudinal therapeutic efficacy of a new drug cannot be determined. Thus, a robust, non‐invasive, in‐vivo drug screening tool is currently an unmet need. Here we have assessed the utility of 18F‐fluorodeoxyglucose positron emission tomography (18F‐FDG) as an in‐vivo screening tool for anti‐inflammatory drugs using the mouse CIA model. The radiotracer 18F‐FDG and a PET scanner were employed to monitor CIA disease activity before and after murine anti‐tumour necrosis factor (TNF)‐α antibody (CNTO5048) therapy in the mouse CIA model. Radiotracer concentration was derived from PET images for individual limb joints and on a per‐limb basis, and Spearman's correlation coefficient (ρ) was determined with clinical score and histology of the affected limbs. CNTO5048 improved arthritis efficiently, as evidenced by clinical score and histopathology. PET showed an increased uptake of 18F‐FDG with the progression of the disease and a significant decrease in the post‐treatment group. 18F‐FDG uptake patterns showed a strong correlation with clinical score (ρ = 0·71, P < 0·05) and histopathology (ρ = 0·76, P < 0·05). This study demonstrates the potential of 18F‐FDG PET as a tool for in‐vivo drug screening for inflammatory arthritis and to monitor the therapeutic effects in a longitudinal setting.  相似文献   

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We used phalloidin staining and immunocytochemistry at the light and electron microscope level to determine the localization of actin in the cell bodies of rat spinal ganglion neurons. The results show that actin is mostly concentrated along the periphery of the neuronal perikaryon, including the perikaryal projections. This localization places actin in a strategic position to be influenced by incoming signals and to produce mechanical tensions able to shape the perikaryal surface.  相似文献   

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Mammalian retinal projections are divided into two anatomically and functionally distinct systems: the primary visual system, which mediates conscious visual processing, and the subcortical visual system, which mediates nonconscious responses to light. Light deprivation during a critical period in development alters the anatomy, physiology, and function of the primary visual system in many mammalian species. However, little is known about the influence of dark‐rearing on the development of the subcortical visual system. To evaluate whether the early lighting environment alters the anatomy of the subcortical visual system, we examined the retinas and retinofugal projections of rats reared in a 12:12 light/dark cycle or in constant dark from birth to 4 months of age. We found that dark‐rearing was associated with a reduction in the distribution of retinal fibers in the stratum opticum of the contralateral superior colliculus. In contrast to the plasticity of the retinocollicular projection, retinal input to sleep, circadian, and pupillary control centers in the hypothalamus, pretectum, and lateral geniculate complex was unaffected by dark‐rearing. A decrease in retinal innervation of the stratum opticum and intermediate layers of the superior colliculus may account for some of the deficits in multisensory integration that have been observed in dark‐reared animals of several species. Anat Rec 2007. © 2007 Wiley‐Liss, Inc.  相似文献   

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Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75‐fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15–30 min. Focal adhesion kinase, p190 Rho‐GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk‐1, erk‐2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation. Anat Rec 254:348–359, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

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Recently, important differences between myofibrillogenesis in cultured cardiomyocytes vs. the three-dimensional setting in situ could be determined. We investigated thin filament assembly in situ by confocal microscopy of whole-mount preparations of immunostained embryonic chicken hearts. Of interest, a distinct localisation of different actin isoforms was observed in immature thin filaments. Cardiac alpha-actin is restricted to filaments with a length comparable to mature thin filaments as soon as the first contractions occur, while vascular alpha-actin makes up filaments that extend toward the M-band. The pointed-end actin filament capping protein tropomodulin can be found initially in close association with the plasma membrane, but attains its mature localisation pattern at the ends of the thin filaments only comparatively late during myofibrillogenesis. Thus tropomodulin acts as a length stabilising element of actin filaments also in developing cardiomyocytes in situ, but plays an additional role together with membrane-associated actin filaments in the earliest steps of myofibril assembly.  相似文献   

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Turtle horny shell has a scute pattern, which is conservative through evolution and across species. The discovery of epidermal placodes as the scute primordia and their strict topographical association to the somites of the turtle embryo suggested a new interpretation of the developmental mechanism of the scute pattern. Here, we tested the hypothesis that horny scutes develop from a mosaic of placodes corresponding exactly to the paths of myoseptae, with vertebral and pleural scutes developing staggered in adjacent segments, and marginal scutes developing in every segment. This scheme predicts little variation in marginals and suggests intercalary supernumerary scutes as potential variations for the vertebral and pleural rows. We examined spatial and numerical variations of the horny shell in 655 newly hatched olive ridley sea turtle, Lepidochelys olivacea, which is known to have a highly variable horny shell. In total, 120 patterns of carapacial scutes and 10 patterns of scutes on plastron, differing in the number and position of scutes were found. The number of vertebral scutes varied from 4 to 10. Variations with five, six and seven vertebrals occurred with the greatest and nearly equally frequency (31.5% on average). Pleural scutes were from 5 to 10 at one or both sides, and the typical symmetric pattern for sea turtles with five pairs of pleurals was only seen in ca. 12% of specimens. In contrast, the majority of the specimens (92.7%) had just 13 pairs of marginals, showing a stable normal pattern. Similarly, on plastron the horny scutes were conservative, too; about 85% of specimens standardly had six pairs of plastral scutes and all specimens had four pairs of inframarginals. Despite a high level of variation of vertebral and pleural scutes in olive ridley turtle, all patterns fall into the theoretical spectrum of possible variants predicted by the segment‐dependent model of development of the turtle horny shell. Therefore, the results of our analysis support the existence of direct morphogenetic correlation between the number and distribution of normal and supernumerary scutes and metamere organization of the turtle embryo.  相似文献   

17.
Zhang X  Jin G  Li W  Zou L  Shi J  Qin J  Tian M  Li H 《Stem cells and development》2011,20(9):1627-1638
Lesions to the fimbria fornix (FiFx) plus cingulate bundle (CB), the principal routes of communication of forebrain cholinergic regions, produce lasting impairment of spatial learning and memory in mice. We report that extensive neurogenesis takes place in the FiFx, CB, and basalis magnocellularis following FiFx plus CB transection. Immunofluorescence revealed that nestin-expressing cells were present in all 3 areas following lesion; the majority of nestin-positive cells were also positive for 5-bromo-2-deoxy-uridine, a marker of DNA synthesis. Nestin-positive proliferative cells were almost entirely absent from unlesioned tissue. Neurospheres cultured in vitro from lesioned FiFx displayed the characteristics of neural stem cells--proliferation, expression of embryonic markers, and multipotential differentiation into neurons, astrocytes, and oligodendrocytes. At early stages after transection, a small number of immature and migrating doublecortin-immunopositive neurons were detected in lesioned FiFx, where neuronal cell bodies are normally absent. At later stages, postlesion immature neurons developed into β-tubulin III-positive mature neurons. Lentivirus labeling assay implied that the injury-induced neurogenesis in FiFx may be from local neurogenic astrocytes but not from dentate gyrus. These results demonstrate that insult to cholinergic tracts can stimulate neural stem cell proliferation and neuronal regeneration not only in innervated regions but also in the projection pathways themselves. Ectopic neurogenesis in cholinergic system-related areas provides an additional mechanism for repair of cholinergic innervation following damage.  相似文献   

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Prostaglandin E2 (PGE2) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE2 production is regulated by arachidonic acid release and cyclooxygenase‐2 (COX‐2) expression. In the present study, we found that COX‐2 expression can be achieved by activating Ca2+/Calmodulin (CaM)‐dependent protein kinase II (CaMKII) and cAMP‐response element‐binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca2+]i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX‐2 expression and PGE2 production in a chronological order. These results suggested that Ca2+/CaM‐dependent CaMKII plays an important role in mediating COX‐2 expression and PGE2 production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE2 production, which plays an essential role in inflammation and cancers.  相似文献   

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House dust mites (HDM s) are a common source of allergens that trigger both allergen‐specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll‐like receptor 4 (TLR 4) in the process of sensitization to house dust mite allergens in an HDM extract‐induced asthma mouse model. Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose‐dependent manner from 2.5 to 30 μg/dose. In TLR 4‐knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild‐type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR 4‐knockout mice than in wild‐type mice. This suggests that the roles of TLR 4 signaling are different between the early phase and the later phase of HDM allergen‐induced inflammation. Thus, innate immune response through TLR 4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.  相似文献   

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