运用酵母双杂交系统筛选肌营养不良相关蛋白2(DRP2)相互作用蛋白

目的应用酵母双杂交系统筛选小鼠、大鼠、人类大脑文库中与DRP2相互作用的蛋白,分析中枢神经系统中DRP2分子复合体的组成成分。方法设计3个含有DRP2不同蛋白结构域的诱饵质粒,在完成诱饵质粒的自激活性的鉴定后,对小鼠、大鼠、人类脑MATCHMAKER cDNA文库进行筛选。结果运用DRP2蛋白N-Bait筛选大鼠脑MATCHMAKER cDNA文库,笔者获得了一个阳性文库质粒。该质粒的插入子编码一种SNAP25相互作用的蛋白质Scoilin。Scoilin又被命名为Zwint-1蛋白。同时笔者还确定了这两种相互作用蛋白质的精确结构域:DRP2蛋白以其含有两个Spectrin重复和一个WW蛋白结构域的氨基端与Scoilin(Zwint-1)全蛋白相互作用。结论应用酵母双杂交,笔者于大鼠大脑中找到了DRP2的相互作用的蛋白质Scoilin(Zwint-1),并且确定了这两种相互作用蛋白质的精确结构域。     

The genomic organization of human dystrobrevin

Dystrophin-related and dystrophin-associated proteins (DAPs) are thought to play an important role in the stability and maintenance of the plasma membrane during muscle contraction and relaxation. Studies conducted on the electric organg of Torpedo californica have shown that some of the DAPs are also involved in the formation and maintenance of neuromuscular junctions (NMJs). In addition, dystrophin and several DAPs have been shown to be the primary genetic defect in a number of phenotypically similar muscular dystrophies. We previously reported the identification and characterization of human dystrobrevin, a protein which is unique in being both homologous to dystrophin and a dystrophin-associated protein. Here we describe the genomic organization of the human dystrobrevin gene. It is encoded by 23 exons spanning at least 180 kb of chromosome 18q12. Three different C-termini of dystrobrevin are generated by the mutually exclusive mRNA splicing of three exons. Two alternatively spliced exons (exons 11A and 12) are utilized exclusively in striated muscles. A comparison between the genomic organization of dystrophin and human dystrobrevin shows that the two genes have significant similarities in their genomic structure, implying an ancestral or evolutionary relationship. Based on intronic sequence, a primer set was designed to specifically amplify each exon of dystrobrevin to screen for mutations by SSCP in patients with neuromuscular diseases for which dystrobrevin could be a candidate. Received November 7, 1996; Revised and Accepted January 8, 1997     

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. NeuroReport 1992; 3; 857–860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by 0.3 μm, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.     

Expression of utrophin (dystrophin-related protein) and dystrophin-associated glycoproteins in muscles from patients with Duchenne muscular dystrophy

We examined whether the dystrophin-associated glycoprotein complex (GPC), which serves to fix dystrophin to cell membranes, is present at the sarcolemma in Duchenne muscular dystrophy (DMD) muscles using an immunohistochemical method. Antibodies against 50DAG (A2) and 43DAG (A3a). the components of GPC, were used for the detection of GPC. We found that, although the amount of GPC was reduced in DMD muscles where ulrophin but not dystrophin was distinctly present, 43DAG (A3a) was fairly heavily and 50DAG (A2) was lightly but distinctly stained on the cell surfaces. It is likely that the capability of utrophin to preserve 50DAG (A2) is less than that of dystrophin, although utrophin has been reported to bind to GPC. We also found that 43DAG (A3a) but not 50DAG (A2) was detected in the peripheral nerves where utrophin was detected. Therefore, it is likely that 43DAG (A3a) is essential for the fixation of utrophin to cell membranes, as in the case of dystrophin. 50DAG (A2) may play other important roles in the pathogenesis of DMD. © 1994 John Wiley & Sons, Inc.     

Dystrophin and dystrophin-related protein (utrophin) distribution in normal and dystrophin-deficient skeletal muscles

The respective localizations of dystrophin and dystrophin-related protein (DRP or utrophin) along the sarcolemmal membrane and at the neuromuscular junctions (NMJs) in normal and dystrophin-deficient skeletal muscles, were determined using confocal laser microscopy. The analysis was prompted by the recent availability of a new anti-utrophin mAb [Bewick et al. Neuro Report 1992; 3: 857–860] and different mAbs that react with dystrophin or both dystrophin and utrophin. In dystrophin-deficient muscles, utrophin was expressed and detectable over large subcellular areas normally occupied by dystrophin along the sarcolemmal membranes and at the NMJs. Utrophin was expressed in a non-uniform, discontinuous way on the sarcolemmal membrane in dystrophin-deficient skeletal muscles, similar to dystrophin in normal muscle fibres. The respective distributions of both related muscle proteins and their positions relative to the -bungarotoxin acetylcholine (ACh) receptor marker were determined. Double-staining experiments and superimposition of the confocal images showed that utrophin was more closely associated with ACh receptors than dystrophin at the NMJs in normal muscles. Utrophin distribution consequently differed from that of dystrophin.     

Expression of dystrophin-associated glycoproteins and utrophin in carriers of Duchenne muscular dystrophy

The expression of dystrophin, the dystrophin-associated proteins and utrophin has been studied immunocytochemically in three young, manifesting carriers of Duchenne muscular dystrophy, aged 3, 5 and 12 yrs, one adult manifesting carrier, aged 60 yrs, and one presumptive carrier with a raised serum creatine kinase, aged 24 yrs, the mother of the 5-yr-old manifesting carrier. The manifesting carriers had variable degrees of weakness; the presumptive carrier had no weakness. Morphological abnormalities were also variable and were most marked in the young manifesting carriers. The three young manifesting carriers and the presumptive carrier had a mosaic pattern of dystrophin-positive and dystrophin-negative fibres. All the dystrophin-associated proteins were reduced in the dystrophin-deficient fibres, giving a similar mosaic pattern to dystrophin. Expression of dystrophin and the dystrophin-associated proteins was normal in the adult manifesting carrier. Utrophin was detected on the sarcolemma of fibres both with and without dystrophin and the dystrophin-associated proteins. Thus, dystrophin and utrophin are co-expressed in several fibres in carriers. The results emphasize the close association between dystrophin and the glycoprotein complex and their role in the pathogenesis of muscle damage. In addition, the presence of utrophin in fibres with greatly reduced glycoproteins suggests that very little of the glycoprotein complex may be required to anchor the amount of utrophin expressed at the sarcolemma in these particular cases.     

Dystrophin and dystrophin-related protein in the brains of normal and mdx mice

To clarify the localization and characterization of dystrophin and dystrophin-related protein (DRP) in the brains of normal and mdx mice, we carried out immunostaining and immunoblotting studies using four region-specific antidystrophin and anti-DRP antibodies. With immunostaining, punctate immunoreactivity of dystrophin was seen along the cell bodies and dendrites of the cerebral cortical neurons in the normal mice. By contrast, dystrophin was not detected at all in the brains of mdx mice. Immunoreactivity of DRP was observed in the vascular walls, pia mater, and choroid plexus of both normal and mdx brains, but not in the neuronal cells. The possible compensatory increase of DRP as seen in the skeletal muscles of mdx mice was not noted in the brains. The immunoblot findings were very consistent with those of immunostain. Although further studies of brain-type dystrophin are necessary, it seems unlikely that DRP participates in any physiological function of the neuronal cells. © 1994 John & Sons, Inc.     

Dystrophin-related protein is found in the central nervous system of mice at various developmental stages,especially at the postsynaptic membrane

Dystrophin deficiency is known to be the cause of X-linked Duchenne muscular dystrophy (DMD). A recently cloned B3-cDNA shares 80% homology with the C-terminus and actin-binding portion of dystrophin. This autosome-derived gene product is called dystrophin-related protein (DRP). DRP is known to exist in fetal muscles even in mdx mice, an animal model for X-linked DMD, but not in mature mouse muscles. We raised a polyclonal antibody against a B3-unique amino acid sequence (Ab-LDP) and investigated the existence and distribution of DRP in the central nervous system (CNS) tissues of (mdx) and normal control B10 mice at various stages of development using immunoblotting and immunohistochemical methods. The former shows that DRP exists in the CNS of both B10 and mdx mice, regardless of the developmental stage, with the exception that the 420 kDa DRP band of the 15-day fetus is faint. In immunohistochemical studies, the choroid plexus, some neurons, glial cells, pia mater, and blood vessels were stained with Ab-LDP. Staining intensity did not differ between B10 and mdx mice of between developmental stages except that the 15-day fetus stained only faintly. This is in contrast of the results obtained for muscles in which DRP localized to muscle membrane in embryo decreases and is assembled at the neuromuscular junction in adults. In addition, an electron microscopic study on the cerebral cortex from adult B10 mice was also performed and revealed Ab-LDP staining of the postsynaptic membrane of dendrite and the rough endoplasmic reticulum of neurons. This is a new finding and is of interest because DRP is expressed at the neuromuscular junction and its localization is similar to that of the acetylcholine receptor. Wiley-Liss, Inc.     

Absence of correlation between utrophin localization and quantity and the clinical severity in Duchenne/Becker dystrophies

While present in the surface membrane of embryonic muscle fibers, in adult normal muscle fibers, utrophin is restricted to the motor endplate and cell of blood vessel walls. However, the observation that utrophin is maintained in the extrajunctional plasma membrane in Duchenne (DMD) and in mdx muscle fibers has led to the suggestion that excess utrophin might compensate for dystrophin deficiency in the Xp21 muscular dystrophies. In order to detect an inverse correlation of utrophin presence and clinical severity, we have assessed utrophin distribution and quantity in DMD and Becker (BMD) patients of different ages and stages of clinical severity. All patients showed a positive discontinuous immunolabeling of utrophin on the sarcolemma, staining equally small and large muscle fibers, indicating that immature characteristics are maintained in such fibers. On Western blot, utrophin bands with concentrations 2- to 10-fold greater than in normal controls were detected in all DMD/BMD patients. However, no negative correlation was found between the amount of utrophin and the severity of clinical course, implying that the detectable utrophin levels in these patients did not compensate for dystrophin deficiency. In a DMD patient with growth hormone (GH) deficiency and a BMD-like clinical course, utrophin levels were comparable to the other typical DMD cases, which reinforces the hypothesis that the observed increase in utrophin is apparently not responsible for a milder clinical course in some patients with Xp21 muscular dystrophies. © 1995 Wiley-Liss, Inc.