骨骼肌M蛋抗血清的制备及正常人骨骼肌M蛋白的免疫电镜定位

分别制备了兔抗人Ｍ蛋白（Ｂ成分）抗血清和兔抗人Ｃ成分＾［１］抗血清。用蛋白Ａ－胶体金作标记物，对经Ｌｏｗｉｃｒｙ１Ｋ４Ｍ低温包埋的人骨骼肌超薄切片中Ｍ蛋白和分子量１４００００的Ｃ成分进行免疫电镜定位。发现Ｍ蛋白分布于整个Ｍ线，而Ｃ成分虽然也集中于Ｍ线以内，但主要分布于Ｍ线内的边缘区域。     

人骨骼肌M蛋白的提取与纯化

依据Ｔｒｉｎｉｃｋ－Ｅｐｐｅｎｂｅｒｇｅｒ对鸡骨骼肌Ｍ蛋白的提取方法,由人骨骼肌中得到的Ｍ蛋白粗提物除含分子量为１６５０００的Ｍ蛋白外,还含有分子量为１８５０００和１４００００（Ｃ成分）的两组分。由于在粗提物中未发现分子量为９００００的磷酸化酶,我们将最终纯化步骤中的亲和层析改为制备电泳,同样获得了纯化的Ｍ蛋白。     

人骨骼肌M蛋白的提取与纯化

依据Ｔｒｉｎｉｃｋ－Ｅｐｐｅｎｂｅｒｇｅｒ对鸡骨骼肌Ｍ蛋白的提取方法,由人骨骼肌中得到的Ｍ蛋白粗提物除含分子量为１６５０００的Ｍ蛋白外,还含有分子量为１８５０００和１４００００（Ｃ成分）的两组分。由于在粗提物中未发现分子量为９００００的磷酸化酶,我们将最终纯化步骤中的亲和层析改为制备电泳,同样获得了纯化的Ｍ蛋白。     

重组人GM－CSF／MCAF融合蛋白抗血清的制备与鉴定

重组人粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人单核细胞趋化激活因子（ＭＣＡＦ）融合蛋白经ＳｅｐｈａｄｅｘＧ－７５和ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ两步柱层析,获得了电泳纯的ＧＭ－ＣＳＦ／ＭＣＡＦ融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ试验表明,该抗血清效价高、特异性好,可分别与ＧＭ－ＣＳＦ／ＭＣＡＦ、ＧＭ－ＣＳＦ和ＭＣＡＦ发生反应。     

重组人GM－CSF／MCAF融合蛋白抗血清的制备与鉴定

重组人粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人单核细胞趋化激活因子（ＭＣＡＦ）融合蛋白经ＳｅｐｈａｄｅｘＧ－７５和ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ两步柱层析,获得了电泳纯的ＧＭ－ＣＳＦ／ＭＣＡＦ融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ试验表明,该抗血清效价高、特异性好,可分别与ＧＭ－ＣＳＦ／ＭＣＡＦ、ＧＭ－ＣＳＦ和ＭＣＡＦ发生反应。     

基因免疫和蛋白免疫制备抗P16抗血清

蛋白免疫,是传统的抗体制备法,基因免疫是新型的抗体制备法。为了制备抗这Ｐ１６抗血甭以及比较基因免疫和蛋白免疫制备抗体,分别以融合谷胱甘肽２－转移酶－Ｐ１６５和克隆有ｐ１６肿瘤抑制基因相关ｃＤＮＡ的裸露真核表达载体ｐＣＭＶ－ｐ１６经皮内多点注射接种家兔,制备抗Ｐ１６抗血清,Ｗｅｓｔｅｒｎ印迹法检测到蛋白免疫的抗Ｐ１６抗血清滴度为１：６２５,明显高于基因免疫制备的Ｐ１６抗血清滴度。     

层粘连蛋白、纤粘连蛋白及Ⅳ型胶原抗血清对小鼠胚泡着床的影响

文中报道了子宫角单次注射层粘连蛋白（ＬＮ）抗血清、纤粘连蛋白（ＦＮ）抗血清以及Ⅳ型胶原（ＣｏｌⅣ）抗血清对小鼠胚胎植入的作用。选用６０只３０ｇ左右的昆明品系雌性成年小鼠,与有生殖能力的雄性小鼠按１：１合笼,翌晨检查外阴,以出现阴栓为妊娠第１天。于妊娠第３天下午,给每个雌性小鼠 侧子宫角单次注射５μｌ ＬＮ抗血清ＮＦ抗血清或Ｃｏｌ Ⅳ抗血清作为实验组;给同一小鼠右侧子宫角注射５μｌ正常兔血清（Ｎｏｒ     

一种与受精有关的人精子膜蛋白（BS－17）的分离纯化

本文用盐分级分离,ＭｏｎｏＱＦＰＬＣ及ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法从人精子ＣＨＡＰＳ抽提液中分离纯化出一种与不育病人血清中抗精子抗体发生特异反应的ＢＳ－１７人精子膜蛋白。该蛋白为一糖蛋白,分子量为１７．５５±２．１５ｋＤ,等电点为５．６５,中性己糖含量为１６．６７％。在人精子上主要分布于顶体区域,不同于已有报导的人精子膜蛋白。在体外实验中抗ＢＳ－１７多克隆血清可以显著影响人精子的获能（ｐ＜０．０２５）和对去透明带仓鼠卵的穿透（ｐ＜０．００５）,但不影响人精子运动性及与去透明酯酸带仓鼠卵的结合。小鼠体内被动免疫实验结果证明抗ＢＳ－１７多抗血清具有明显地抑制受精的功能（ｐ＜０．００１）。     

人心肌肌凝蛋白轻链(CMLC)的分离及临床应用

从人心肌提取了肌凝蛋白,并检测了其ATP酶活力的稳定性。用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)法纯化并鉴定了人心肌肌凝蛋白轻链(CMLC),结果表明CMLCI的分子量为27000;CMLCⅡ的分子量为20 000。它们的紫外吸收光谱显示A260>A280,表明其高含苯丙氨酸。制备了兔抗人CMLC抗血清,免疫双扩散的结果表明,纯化后的CMLC与肌凝蛋白有相同的抗原性。用阳性的兔抗人CMLC抗血清为对照作ELISA实验,不仅从免疫学角度进一步证实了人心肌CMLC的特性且为其在临床诊断应用提供了可靠的方法。     

利用大肠杆菌在菌体外分泌生产人C反应性蛋白(CRP)

酵母工业长浜生物科学研究所利用大肠杆菌确立了在菌体外分泌生产在体内发生急性炎症和组织损伤时能在血清中迅速增加的一种蛋白——人C反应性蛋白(CRP)技术。从94年开始商业生产,抗CRP抗血清可用于急性感染疾病的诊断。目前,该公司一年可供应1000升左右抗人CRP的兔抗血清(及抗体)。作为诊断用容积需要系统化。该公司目前引进含CRP的血清和腹水,高纯度地纯化CRP,将其给兔免疫,制造抗血清。由于生产重组人CRP,省去了引进原料需花时间高纯度地纯化CRP的作业。不仅获得了抗体,还能确保作为CRP测定药盒的标准物质所必需的高品质CRP。     

Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle

To better understand the relationship between the Mr 165,000 M-line protein (M-protein) and H-zone structure in skeletal and in cardiac muscle, as well as the possible interaction of M-protein with another skeletal muscle M-line component, the homodimeric creatine kinase isoenzyme composed of two M subunits (MM-CK), we performed biochemical, immunological, and ultrastructural studies on myofibrils extracted by different procedures. In contrast to MM-CK, M-protein could not be completely removed from myofibrils by low ionic strength extraction. Fab-fragments of antibodies against M-protein could not release M- protein quantitatively from either breast or heart myofibrils but remained bound to the myofibrillar structure, whereas monovalent antibodies against MM-CK cause the specific release of MM-CK and the concomitant disappearance of the M-line from chicken skeletal muscle myofibrils. When MM-CK was removed from skeletal myofibrils by low ionic strength extraction or, more specifically, by incubation with anti-MM-CK Fab, M-protein was still not released quantitatively upon treatment with anti-M-protein Fab as judged from immunofluorescence data. In the ultrastructural investigation of low ionic strength extracted muscle fibers, M protein could be localized in two stripes on both sides of the former M-line, suggesting a reduced attachment to the residual H-zone structure, whereas the specific removal of MM-CK resulted in the same dense staining pattern for M-protein within the M- line as observed in untreated fibers. However, the binding of M-protein to the residual M-line structure seemed to be reduced, as a considerable amount of this protein could be identified in the supernate of sequentially incubated myofibrils. The results indicate a strong binding of M-protein within the H-zone structure of skeletal as well as heart myofibrils.     

M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

CPNA-1, a copine domain protein,is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1–null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.     

Effects of the components of Newcastle disease virus on the structural order of lipid assemblies

The membrane M-protein of Newcastle disease virus is localized directly beneath the lipid bilayer. Although this protein is the major constituent of the virus, its structural relationship to the lipid or to the other viral component hemagglutininneuraminidase, the so called HN-glycoprotein, is still unknown. The effects of either M-protein alone or both M-protein and HN-glycoprotein on the lipid assemblies in reconstituted liposomes were determined by differential polarized phase fluorometry, steady-state fluorescence anisotropy and emission lifetime measurements.It is demonstrated that the degree of rotation of fluorophores in reconstituted liposomes is restricted by the molecular packing of lipids in the bilayer and this in turn can be correlated with the structural order of the lipids in the membrane. The experimental results show that the structural order parameters calculated from the fluorescence measurements are strongly influenced by the presence of both M-protein and HN-glycoprotein in the lipid assemblies.     

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.     

Interaction of studies of a 100,000 dalton chymotryptic fragment of rabbit skeletal M-line protein with the S2 subfragment of myosin

Controlled chymotryptic digestion of the 165,000 dalton component of the M-line of rabbit skeletal muscle, followed by Biogel P-150 chromatography of the digest, has led to the isolation of a homogeneous 100,000 dalton species. This fragment was found, by both sedimentation equilibrium and gel filtration chromatography, to interact with heavy meromyosin subfragment 2 (HMM-S2). The persistence of this fragment, after chymotryptic digestion, to bind HMM-S2, along with the known insensitivity of the M-line to proteolysis, suggests a structural role for the parent 165,000 dalton component along the lines of the M-filament, as suggested by the Knappeis and Carlsen model for M-line structure.     

Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.     

Size and shape of skeletal muscle M-protein

M-protein was isolated from chicken pectoralis muscle and shown to be relatively homogeneous by the criterion of high-speed equilibrium ultracentrifugation. A sample of the protein was rotary shadowed with platinum and examined by electron microscopy. M-protein appears to be elongated with a length of 360 Å and a width of 41 Å to give an axial ratio of 9:1. These results are compatible with the suggestion that M-protein is a component of the M-filaments in the M-band of skeletal muscle myofibrils.     

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross- striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M- line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'- bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.