转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

Interaction of Keratin K1 with Nucleic Acids on the Cell Surface

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.     

The effect of the iron content in the culture medium on the nature of the protein spectrum in Neisseria meningitidis

The protein spectra of Neisseria meningitidis, strain A 208, in the process of its cultivation on solid culture medium based on peptone agar under the conditions of the surplus or deficiency of ions of trivalent iron in the medium. The creation of iron deficiency by the introduction of two iron-binding admixtures, desferol and diethylenetriaminepentaacetic acid (DTPA), was found to lead to the production of two additional proteins with molecular weights of 72 kD and 36-37 kD by meningococcal cells. Of these two admixtures, DTPA more readily stimulated the production of low-molecular protein with a molecular weight of 36-37 kD. This protein was found to be noticeably labile, while protein with a molecular weight of 72-73 kD showed no such lability. As the result of the cultivation of meningococci in iron-deficient medium, the content of protein in microbial residue was 2- to 3-fold greater than that obtained by the cultivation of meningococci in culture medium with the surplus of iron in the form of ferric nitrate.     

Existance of a Bovine Sperm Antibody Recognizable Antigen-Epitope on the Plasma Surface of Lily Sperm Cells

By using Western blot and rabbit-anti-bovine sperm IgG as the first antibody, a 64 kD protein from sperm cells of Lilium davidii Duch. and the same molecular weight protein from generative cells of L. davidii as well as a 22 kD and a 65 kD protein from sperm cells of Zea mays could be detected. Nevertheless, proteins from filament and anther wall of L. daviclii or etiolated shoot of corn manifested negative reaction. By indirect immunofluorescence assay using the same antibody, the surface of sperm cells from L. daviclii showed positive reaction. Based on the results, the authors believed that a plant sperm cell might have the same epitope (s) as that of an animal sperm cell; the epitope (s) might locate on the surface of a sperm cell and be specific to sperm cells.     

cAMP-binding protein--a protein kinase of the plague pathogen]

In Y. pestis a cyclic AMP-binding protein was detected, isolated to a homogeneous state, and its physico-chemical properties were studied. The protein is a highly molecular compound with a molecular weight of 180 kD, capable of being released into the environment in the process of cell growth and having protein kinase activity, not depending on the presence of cyclic AMP. Y. pestis neuraminidase is one of the substrates appearing due to the action of protein kinase detected in this study. Y. pestis protein kinase may alter the spectrum of protein phosphorylation in the leukocytes of white mice. The direct participation of this protein in the development of infection is supposed.     

肾综合征出血热纯化疫苗的SDS-PAGE分析

为了证明蛑综合征出血热纯化疫苗的主要成分坦病毒蛋白,采用出血热纯化疫苗经浓缩后进行SDS－PAGE和Western-blotting分析。结果 经SDS－PAGE显示,肾综合征出血热纯化疫苗有三条蛋白带,分子量分别约为70kD、55kD和50kD,与汉坦病毒三种结构蛋白（糖蛋白G1、G2和核蛋白NP）的分子量相符;经Western-blotting显示,分子量50kD的蛋白带反应阳性,分子量70kD和55kD的蛋白带无反应,认定出血热纯化疫苗的主要成分为汉坦病毒蛋白,主要由G1、G2和NP三种结构蛋白构成。     

Antibodies to meningococcal iron regulated protein FbpA and minor proteins in the sera of patients with meningococcal meningitis

Altogether 7 blood serum specimens taken from 3 patients with meningococcal meningitis were studied by the method of immunoblotting. The study revealed that on day 7 and especially on day 10 from the onset of the disease antibodies to periplasmatic iron-regulated protein FbpA with a molecular weight of 34 kD appeared in the serum of one patient. In the sera of two other patients the appearance of antibodies to minor iron-regulated proteins with molecular weights of 43 kD and 46 kD, absent at the acute stage of the disease, were detected on day 7. As the process of convalescence was progressing, in all patients under observation an increase in the specific immune response to proteins of class 5 with molecular weights of 20-14 kD was observed.     

Chemical Cross-linking of Neighboring Thylakoid Membrane Polypeptides

Cross-linking between protein components of whole spinach (Spinacia oleracea var. Nobel) thylakoids and of photosystem I- and II-enriched thylakoid fractions has been produced by reaction with the bifunctional imidoester dimethyl-3,3′-dithiobispropionimidate dihydrochloride as well as by the oxidation of intrinsic sulfydryl groups with an orthophenanthrolinecupric ion complex. The mixture of membrane proteins and their cross-linked products has been analyzed by two-dimensional sodium dodecyl sulfate electrophoresis, with a reductive cleavage step of the cross-linkages before the second dimension. Cross-linked aggregates up to a molecular weight of about 130 kilodaltons (kD) were analyzed, and it was inferred that the polypeptides appearing together in the same aggregates were neighbors within the membrane.

In thylakoids as well as in isolated photosystem fractions, oligomers were formed by cross-linking polypeptides of the 60 to 90 kD range, among them the polypeptides of the chlorophyll-protein complex I. Polypeptides of 46, 19, and 12 kD were cross-linked to these complexes. Polypeptides of 25 and 22 kD, which are related to the chlorophyll-protein complex II, were cross-linked in thylakoids as well as in photosystem II fractions, suggesting that in the membrane these molecules are close together. In photosystem II fractions an oligomer having a molecular weight of about 60 kD was formed by cross-linking several polypeptides of different molecular weights: 40, 25, and 22 kD.

Our cross-linking experiments show that protein interactions in the thylakoid membrane occurred mainly among the polypeptides of the two chlorophyll-protein complexes, thus suggesting an oligomeric nature of these apoproteins.

     

Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture

Cocultures of rat Sertoli-spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6-12 h and then maintained in serum free, hormone/growth factor-supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2-3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin-68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin 1) behaves as a carrier protein for growth factors, 2) has protease inhibitory activity, 3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and 4) fetal bovine serum-supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long-term viability and differentiation of spermatogenic cells in vitro.     

嗜肺军团菌效应蛋白质介导的新型泛素化过程

泛素化是真核细胞特有的蛋白质翻译后修饰方式,调节真核细胞内多种重要生理过程,例如蛋白质稳态、细胞周期、免疫反应、DNA修复以及囊泡转运等。鉴于泛素化对于生命活动的重要性,病原菌在与宿主细胞的长期进化过程中衍生出一系列针对宿主泛素化过程的效应蛋白质,调控宿主体内泛素化过程,从而构建有利于病原菌自身生长繁殖的内环境。嗜肺军团菌是一种革兰氏阴性菌,是军团菌肺炎的致病菌,能够引起发热和肺部感染,重型病死率高达15%~30%。Dot/Icm Ⅳ型分泌系统是嗜肺军团菌侵染过程中最主要的毒力系统。在侵染宿主细胞的过程中,嗜肺军团菌利用该分泌系统,分泌超过330种效应蛋白质,协助细菌在宿主胞内生存、增殖和逃逸。多种嗜肺军团菌效应蛋白质通过直接或者间接的方式对宿主泛素化过程进行调控。近年的研究发现,多种效应蛋白质可以介导不同于真核生物经典泛素化的新型泛素化过程。本文介绍了嗜肺军团菌效应蛋白质介导的新型泛素化过程的最新研究进展,为理解泛素化过程在嗜肺军团菌致病过程中的重要作用提供参考依据。     

Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

兰州百合精细胞表膜上存在被牛精子抗体识别的抗原决定簇

以兔抗牛精子 Ig G为一抗 ,对植物精细胞蛋白进行 Western blot分析 ,发现兰州百合 (Liliumdavidii Duch.)精细胞和生殖细胞中各有一分子量为 64k D的蛋白显示阳性反应 ;在玉米 (Zea mays)精细胞蛋白中也发现了阳性反应条带 ,其分子量为 65 k D和 2 2 k D;而兰州百合花丝、花药壁和玉米黄化苗的蛋白中均没有阳性条带。用兔抗牛精子 Ig G对兰州百合精细胞进行间接免疫荧光标记 ,结果表明在兰州百合精细胞表面 ,有兔抗牛精子 Ig G的识别位点。根据以上结果 ,作者认为植物精细胞中有与动物精子相同或相似的抗原决定簇 ,它 (们 )主要分布于精细胞表膜上 ,并为精细胞所特有     

Antibodies to meningococcal antibodies in the sera of patients and donors

The comparative study of sera taken from healthy persons (pooled sera of 100 donors, 6 individual serum specimens) and sera taken from patients with meningococcal meningitis (pooled sera of 10 patients with meningococcal infection, group A, and 6 individual serum specimens from patients with meningococcal infection, groups A, B, C) was carried out by the method of immunoblotting. All proteins from healthy donors were found to contain antibodies to meningococcal iron-regulated protein (IRP) of 85 kD, designated as TbpB. In 30% of donor sera the presence of antibodies to meningococcal IRP of 34 kD (FbpA) was registered. Moreover, donor sera were found to contain antibodies to meningococcal IRP of 45 kD. The sera taken from convalescents were found to have the increased content of antibodies to IRP of 70 and 85 kD and somewhat lesser content of antibodies to proteins of 98, 44 and 34 kD. As regards other (non iron-regulated) proteins, in the process of convalescence the most intensive antibody production was observed with respect to minor protein with a molecular weight of 50 kD, as well as proteins of class 5, characterized by molecular weights of 30 kD and less.     

In vitro expression of a 38,000 dalton heparin-binding glycoprotein by morphologically differentiated smooth muscle cells

In vitro, high density monolayer cultures of vascular smooth muscle cells can be induced to form multicellular nodules. The nodular cells appear to be morphologically differentiated smooth muscle cells. Sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis was used to compare the proteins synthesized and secreted by monolayer and nodular cultures of smooth muscle cells. Although most proteins appeared to be similar, the nodular cultures contained a unique heparin binding protein of Mr = 38,000 (38kD protein) (Millis, A.J.T., Hoyle, M., Reich, E., and Mann, D.M., 1985, J. Biol. Chem., 260:3754-3761). The 38kD protein was glycosylated and its apparent molecular weight was shifted to Mr = 32,500 after synthesis in the presence of tunicamycin or digestion with endoglycosidase F. The production of 38kD protein by nodular cell cultures did not appear to result from the degradation of a high molecular weight precursor in nodular conditioned medium. Further, it was not detected in monolayer cell conditioned medium that had been incubated with nodular cells. Finally, its synthesis was not induced in monolayer cell cultures that had been labeled in nodular cell conditioned medium. The 38kD protein appears to be uniquely associated with nodular cultures of smooth muscle cells.     

兰州百合精细胞特异蛋白的研究

通过低渗冲击及Percoll密度梯度离心的方法,成功地分离并纯化了兰州百合(Lilium davidiiDuch.)生活的生殖细胞及精细胞。从精细胞、生殖细胞及叶片中提取了全蛋白,并通过双向电泳技术对它们进行了比较。在双向电泳图谱上精细胞比生殖细胞显示更多的蛋白斑点,特别是在碱性端。通过混合酶解及离心,分离了生活的叶肉原生质体。用生物素的琥珀酰胺酯衍生物(NHS-biotin)对精细胞、生殖细胞及完整的叶肉原生质体质膜蛋白进行标记,然后进行Western blot分析,用辣根过氧化物酶酶标链霉抗生物素蛋白及其底物4-氯-1-萘酚反应显色,比较了3种质膜蛋白。发现分子量为46kD及50kD的两种蛋白是精细胞质膜特异的。在双向电泳图谱上也可找到与这两种蛋白相对应的斑点,它们很可能与受精过程中精卵的识别有关。     

Light-Induced Damage of Amino Acid Residues and Degradation of Polypeptides in D1/D2/Cytochrome b559 Complex

Photosystem Ⅱ reaction center D1/Dg/Cyt b559 complex is very sensitive to light. Besides pigments, some amino acids, like histidine and methionine residues on the polypeptide chain, were damaged and D1 and D2 proteins were degraded by illumination. SDS-PAGE analysis demonstrated an increased content of the D1 and D2 protein dimers and a new band with molecular weight of 41 kD after light treatment. Meanwhile, the D1 and D2 bands were shifted to apparent positions of higher molecular weight. During the consequent incubation in the dark following illumination, although there was no change in the composition of amino acids, the degradation process of D1 and D2 proteins and the production of 41 kD fragment continued. It was proposed that degradation of D1 and D2 proteins was probably due to the photodamage of some amino acids via chemical splitting and co-valent cross-linkage in this process.     

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.     

Characterization of a high-molecular-weight protein immunoprecipitated by platelet-derived growth factor antisera

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.