Caveolae、Caveolin-1与肿瘤的研究进展

Caveolae是细胞膜内陷形成的烧瓶状的特殊膜结构,参与包括细胞的分子运输、细胞粘附和信号转导在内的多种细胞活动。Caveolin-1是Caveolae中重要的结构蛋白,抑制细胞生长,与多种人类肿瘤发生发展相关的信号分子相互作用,如G蛋白、Src家族激酶、Ras、蛋白激酶C、内皮生长因子(EGF)受体、内皮型一氧化氮合成酶(eNOS)及整合素等。因此,Caveolin-1是细胞生长相关信号途径及肿瘤发生发展过程中重要的抑制因子。     

Endothelial Caveolae and Caveolin-1 as Key Regulators of Atherosclerosis

This commentary discusses the role of caveolin-1 in atherosclerosis.     

Myoglobin in Primary Muscular Disease: I. Duchenne Muscular Dystrophy: and: II. Muscular Dystrophy of Distal Type

Skeletal myoglobin from two cases of muscular dystrophy, one of Duchenne muscular dystrophy, and one of muscular dystrophy of distal type, have been examined and no differences from normal human myoglobin were found. The opportunity has been taken to discuss the nature of minor fractions of myoglobin-like material which are found when human skeletal myoglobin is isolated. Those which have been observed in the present study have been artefacts and it was possible to demonstrate that they were due to deamidation of certain glutamine and asparagine residues.     

Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages.Plasma membrane damage in mechanically active cells such as the myocyte is inevitable even under normal physiological conditions.^1,2 Since membranes are not self-sealing, effective and efficient repair mechanisms are necessary to maintain cell viability. Dysferlin plays a central role in this active repair mechanism in skeletal muscle. In the absence of dysferlin disruptions of the skeletal muscle plasma membrane are not repaired leading to cell death.³ Skeletal muscle can regenerate new cells from satellite cells but eventually even this response is exhausted, and lost myocytes are replaced by fat and fibrosis resulting in debilitating muscular dystrophy.Limb-girdle muscular dystrophy type 2 B (LGMD2B), Miyoshi myopathy, and distal myopathy of anterior tibialis are three clinically distinct forms of muscular dystrophy that are caused by mutations within the dysferlin (DYSF) gene resulting in severe to complete deficiency of dysferlin expression.^4,5 Clinically, these dysferlinopathies start in young adulthood with progressive muscle weakness and atrophy that advances to severe disability in older adulthood.Dysferlin is a 273 kDa membrane-spanning protein with multiple C2 domains that bind calcium, phospholipids, and proteins to then trigger signaling events, vesicle trafficking, and membrane fusion.^6,7 The name “dysferlin” reflects the homology with FER-1, the Caenorhabditis elegans spermatogenesis factor involved in the fusion of vesicles with the plasma membrane, as well as the dystrophic phenotype associated with its deficiency.⁵ Dysferlin is crucial to calcium dependent membrane repair in muscle cells.^3,8 In normal skeletal muscle, sarcolemma injuries lead to the accumulation of dysferlin-enriched membrane patches and resealing of the membrane in the presence of Ca²⁺.^3,9While the profound effect of dysferlin deficiency in skeletal muscle has been the subject of much investigation, the effect of dysferlin deficiency in cardiac muscle has largely been ignored. However, in 2004, Kuru et al¹⁰ reported on a 57-year-old Japanese woman with LGMD2B and dilated cardiomyopathy; more recently, Wenzel et al¹¹ described dilated cardiomyopathy in two out of seven patients with LGMD2B and other cardiac abnormalities in three of the others. These observations suggest that dysferlin deficiency can lead to cardiomyopathy as well as to muscular dystrophy. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known.Spontaneous mutations in the mouse are valuable resources in understanding human disease processes. Genetically defined mice develop dysferlinopathies closely resembling LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² In 2004, Ho et al¹² identified A/J mice as dysferlin deficient. A retrotransposon insertion in the dysferlin gene was found to result in a null allele, resulting in skeletal muscle dystrophy that shows histopathological and ultrastructural features that closely resemble the human dysferlinopathies of LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² The onset of dystrophic features in A/J mice begins in proximal limb muscles at 4 to 5 months of age and progresses to severe debilitating muscular dystrophy over several months. Ho et al¹² also found that human and murine dysferlin share very similar (approximately 90% identity) amino acid sequences. Cardiac muscle was not included in their study.Recently, Han et al,⁸ using sucrose gradient membrane fractionation on homogenates of wild-type C57BL/6J mouse heart muscle, showed that dysferlin is present in the cardiomyocyte plasma membrane and intracellular vesicle fractions. It was proposed that dysferlin is localized to the cardiomyocyte sarcolemma and some unidentified type of vesicles.⁸ Han et al⁸ in one study and Wenzel et al¹¹ in another study showed that the induction of significant cardiac stress lead to cardiac dysfunction in dysferlin-deficient mice, but to what extent dysferlin deficiency causes cardiomyopathy by aging alone in patients clinically affected with the debilitating effects of LGMD2B, Miyoshi myopathy, or distal myopathy of anterior tibialis is unknown.In this study, we used the A/J mouse model to study the effects of aging in mice affected by genetically determined dysferlin deficiency by using echocardiography and conscious electrocardiography to determine functional changes in vivo, followed postmortem by light and electron microscopy to determine associated morphological changes. We have determined that the normal primary location for dysferlin in the cardiomyocyte of control A/HeJ mice is the intercalated disk (ID), and to a lesser extent, to a distinctive transverse banding pattern within the sarcoplasm of the cardiomyocyte. We have also determined that in the dysferlin-deficient cardiomyocyte there is evidence of membrane damage at these locations. We also present data that show functional cardiac deficits were present in vivo at around 10 months of age then recovered by 12 months. Histopathology showed that under normal laboratory conditions dysferlin deficiency causes only a mild cardiomyopathy even at advanced ages, suggesting the possibility of dysferlin-independent membrane repair mechanisms in cardiac muscle that do not exist in skeletal muscle.     

Muscular Dystrophy with Reduced β-Sarcoglycan in a Cat

A partial β-sarcoglycan (SG) deficiency with retention of other components of the SG complex (SGC) is described in 6-month-old, intact male domestic shorthaired kitten that was referred for evaluation of weakness, reluctance to move and dyspnoea. Neurological deficits were restricted to the neuromuscular system. Muscle biopsy revealed moderate variability in myofibre size, with numerous atrophic rounded fibres, rare myofibre necrosis, regeneration and moderate perimysial and endomysial fibrosis. Immunohistochemistry revealed decreased expression of β- and γ-SG and western blotting revealed markedly decreased β-SG with normal expression of α-, γ- and δ-SG, caveolin-3 and calpain-3. Sarcoglycanopathy has not previously been described in cats. In human and canine sarcoglycanopathies the deficiency in any one of the SGs leads to secondary deficiency of the entire SGC. Such spontaneously arising muscular disease in animals can provide valuable models for equivalent human disorders.     

Conditional Knockout of Pik3c3 Causes a Murine Muscular Dystrophy

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.Phosphatidylinositols (PIs) comprise a group of low-abundance lipids with hydroxylated inositol head groups that are capable of receiving phosphates at any of the three outer positions. Dynamic phosphorylation of the inositol ring influences many cellular and metabolic processes, including endocytosis, endosomal trafficking, and autophagy.^1–5 The quantity and localization of different PIs are regulated by a group of phosphoinositide kinases and phosphatases that function as key regulatory enzymes and that have been implicated in a number of human diseases, including especially oncologic and neurodegenerative diseases.^6–9Myotubular myopathy (MTM) is a severe childhood-onset disease of skeletal muscle caused by mutations in the phosphoinositide phosphatase myotubularin gene (MTM1).¹⁰ MTM is characterized by profound weakness and hypotonia at birth, persistent life-long disabilities including wheelchair and ventilator dependence, and early mortality.¹¹ As predicted by the fact that MTM1 has been shown in vitro to be responsible for dephosphorylation of PI(3)P, levels of PI(3)P are significantly elevated in animal models of MTM.^12–14 Despite growing knowledge of disease pathogenesis, there are currently no available treatments for MTM. One significant barrier toward therapy development for MTM is the fact that the normal function(s) of PI(3)P in skeletal muscle are unknown.PI(3)P is created through phosphorylation of PI at the D3 position by PI3 kinases,^15,16 or through dephosphorylation of PI(3,5)P₂ by the phosphatase FIG4.¹⁷ There are three classes of PI3 kinases that produce PI(3)P in mammals, with varying tissue expression and substrate specificity.^18,19 In skeletal muscle, the primary sources of PI(3)P are hypothesized (based on gene expression) to be the class III kinase, Pik3c3 (hVPS34), and the class II kinase Pik3c2β, with PIK3C3 considered the major enzymatic regulator of its production.^15,16,20 Previous studies of PIK3C3 have identified it as a regulator of several intracellular processes, including endosome-to-Golgi membrane traffic,⁷ endocytosis,^21,22 mTOR-S6K1 signaling,^23,24 and autophagy. Perhaps its best-studied function is in autophagy, where PIK3C3 and its regulatory subunit PIK3R4 (Vps15) form multiple complexes with other autophagy gene products to regulate several steps of autophagosome formation and maturation.^25–28The goal of the present study was to begin understanding the role of PI(3)P in skeletal muscle by evaluating the function of PIK3C3. As previously reported, whole-animal gene knockout of Pik3c3 in the mouse results in early embryonic lethality.^29,30 Therefore, to study PIK3C3 specifically in muscle, we have used the Cre-lox system. Cre-lox–mediated knockout of Pik3c3 has been performed in kidney,³¹ liver, and heart³²; sensory, cortical, and hippocampal neurons^30,33; and T cells,³⁴ but Pik3c3 has yet to be examined in skeletal muscle. We generated mice with conditional knockout of Pik3c3 in skeletal muscle by combining floxed Pik3c3³⁰ and Cre recombinase under the muscle creatine kinase promoter: Tg(Ckmm-Cre).³⁵ The resulting mice had normal embryonic and early postnatal development, but died by 2 months of age, presumably from severe cardiomyopathy.³² Somewhat surprisingly, examination of skeletal muscle in knockout animals revealed a murine muscular dystrophy. We present the comprehensive characterization of this dystrophic phenotype. In total, we report for the first time a requirement for Pik3c3 in skeletal muscle homeostasis, and further identify loss of Pik3c3 as a cause of muscular dystrophy in the mouse.     

Xenotransplantation of Embryonic Precursors of Human Myogenesis for the Correction of Dystrophinopathy in Mice with Hereditary Muscular Dystrophy

Human embryonic myogenic precursors were transplanted into muscles of mdx mice with hereditary dystrophin-deficient muscular dystrophy. Transplantation induced the synthesis of human dystrophin. The number of dystrophin-positive fibers progressively decreased, however, some of them were preserved even 5 months after transplantation. Our results indicate that xenogeneic transplantation of embryonic myogenic precursors compensates the genetic defect in dystrophin-deficient mice.     

Emery-Dreifuss肌营养不良症的研究进展

Emery-Dreifuss肌营养不良症是一种相对良性的肌营养不良类型。其遗传方式为X-连锁隐性、常染色体显性和隐性遗传。EMD基因和LMNA基因是引起X-连锁EDMD和常染色体遗传EDMD的致病基因，编码产物分别为emerin蛋白和核纤层蛋白（lamin）A/C。该病确切的发病机制目前尚不清楚，临床特点表现为早期出现关节挛缩，受累肌肉呈肱－腓分布并伴有心脏受累。致病基因的研究使基因治疗该病成为可能。     

Clinical and Genetic Analysis of Korean Patients with Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominantly inherited muscular disorder, which is characterized by weakness of facial, shoulder and hip girdle, humeral, and anterior distal leg muscles. The FSHD gene has been mapped to 4q35 and a deletion of integral copies of a 3.3-kb DNA repeat motif named D4Z4 was known to be the genetic background of the disorder. Although FSHD is the second most common muscular dystrophy in adulthood, there were few reports on the genetically confirmed patients in Korea. Recently, we experienced four Korean patients with clinical features resembling FSHD. In order to confirm the diagnosis, conventional Southern blot (SB) analysis by using double digestion with EcoRI and BlnI and hybridization with p13E-11 probe was performed in three patients and newly developed long polymerase chain reaction (PCR) method was used for one patient because genomic DNA was not enough for conventional SB for this patient. All patients were demonstrated to have shortened D4Z4 repeats that were consistent with FSHD. Therefore, we could confirm the diagnosis of FSHD in four Korean patients and appropriate genetic counseling was done for the patients and their families. It is of note that long-PCR method could be a good alternative for conventional SB when D4Z4 repeats were less than 5.     

Duchenne型肌营养不良症肌细胞膜异常的研究

本文应用凝集素免疫电镜和冷冻断裂电镜技术，通过对6例DMD患者组和4例健康对照组骨骼肌细胞膜的观察，发现RCA-I和WGA凝集素受体主要分布在肌质膜上，DMD患者有40％左右的肌质膜存在局限性的受体分布缺损区，并且DMD肌质膜的PF面和EF面上的膜蛋白颗粒还明显减少，从分子病理学水平证实了DMD存在肌细胞膜的异常。     

Cytofluorometric Determination of Nuclear DNA in Heart Muscles of Patients with Muscular Dystrophy

The degree of DNA polyploidy of cardiac muscle cells was investigated by cytofluorometry in 4 cases of progressive muscular dystrophy (DMP) and 2 cases of myotonic dystrophy (DM), and the results were compared with those for non dystrophic hearts of normal or increased weight. The heart muscle cells from all patients with these forms of dystrophy showed marked nuclear DNA hyperploidy reaching 16c, irrespective of cardiac hypertrophy or atrophy. In the non dystrophic group, DNA hypertrophy corresponding to that of dystrophy was only detected in cases of marked cardiac hypertrophy exceeding a weight of 400 g. The wasting of the respiratory musculature, deformity of the thorax and cardiac muscular atrophy appeared to be the principal factors causing DNA polyploidy in patients with muscular dystrophy. Acta Pathol Jpn 39: 566 572,1989.     

Enrichment of Dystrophy-Associated Antigen from Erythrocyte Membranes of Mice with Muscular Dystrophy

Summary

In a previous report, it was shown that erythrocyte membranes from mice with muscular dystrophy of strain 129/ReJ-dy manifest a unique antigen. In the current study, this antigen was enriched from solubilized membranes by a two-step solid phase immunoadsorbent. The enriched fraction retained antigenicity as well as a reduced set of electrophoretic forms of protein over that seen in solubilized membranes. The enriched fraction contained no novel molecular weight species but several were noticeably enriched, particularly at 54,000 daltons.