摘要:

AbstractWe have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T‐cell specific serine proteinase 1 (TSP‐1) for its effect on the cytolytic potential of cloned cytotoxic T‐cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H‐D‐Pro‐Phe‐Arg‐chloromethyl‐ketone (PFR‐CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule‐associated TSP‐1 activity after extraction with Triton X‐100. Furthermore, PFR‐CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR‐CK inhibited in a dose‐dependent manner both TSP‐1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high‐salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule‐associated proteinase TSP‐1, which probably becomes active upon exocytosis foll

ISSN:0730-2312    

DOI:10.1002/jcb.240400102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe full‐length provirus of human T‐cell leukemia virus type I (HTLV‐I) was isolated from MT‐2, a lymphoid cell line producing HTLV‐I. In transfected cells, structural proteins of HTLV‐I, the gag and env products, were formed and processed in the same manner as observed in MT‐2 cells. The nucleotide sequence was determined for a region between thegagandpolgenes of the proviral DNA clone containing an open‐reading frame. The deduced amino acid sequences show that this open‐reading frame encodes a putative HTLV‐I protease.The protease gene (pro) of HTLV‐I was investigated using a vaccinia virus expression vector. Processing of 53kgagprecursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entiregag‐ and protease‐coding sequence. We demonstrated that the protease processed thegagprecursor polyprotein in atrans‐action. A change in the sequence Asp(64)‐Thr‐Gly, the catalytic core sequence among aspartyl proteases, to Gly‐Thr‐Gly was shown to abolish correct processing, suggesting that HTLV‐I protease may belong to the aspartyl protease group. The 76kgag‐proprecursor polyprotein was identified, implying that acis‐acting function of HTLV‐I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53gagprecursor protein. The p53gagprecursor protein is then processed by thetrans‐action of th

ISSN:0730-2312    

DOI:10.1002/jcb.240400103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractDespite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse‐chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino‐terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high‐mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino‐terminal activation peptides. Removal of the propeptide generates an active single‐chain enzyme. Whether the single‐chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to t

ISSN:0730-2312    

DOI:10.1002/jcb.240400104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe investigated the specificity of circulating autoantibodies to a heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), obtained by recombinant DNA technique, in different rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, primary Sjogren's syndrome (SS), idiopathic Raynaud (IR), mixed connective tissue disease (MCTD), and healthy donors. All sera were tested by ELISA on hnRNP A1 protein. Positive values were obtained in 22% SLE, 19% scleroderma, 10% IR, 40% (2/5) MCTD, 5% SS, and 50% RA patients. The majority of patients reacted with the aminoterminal part (UP1) of hnRNP A1; however, some RA patients reacted also with the carboxy‐terminal part that shows partial homology with keratin. Therefore, hnRNP A1 (UP1) can be considered a target of antinuclear autoimmunity in various rheumatic disorder

ISSN:0730-2312    

DOI:10.1002/jcb.240400105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractResistance (low dose tolerance) to adjuvant arthritis was induced by intradermal immunization with 10 μgMycobacterium tuberculosisadministered 5 and 3 weeks before induction of arthritis. With the purpose of determining phenotypes of cells which participate in the maintenance of the induced resistance to adjuvant arthritis, tolerized rats were treated with two different anti‐T‐cell monoclonal antibodies. In tolerized rats, it was shown that anti‐CD8 (OX8) antibodies, which caused an elimination of CD8+lymphoid cells as determined by immunofluoresnce analysis, made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 19 (47.4%) rats developed the disease as compared with 2 of 18 (11.1%) (P<0.05) in the control antibody‐treated group. Also, in vivo treatment with anti‐CD5 (OX19) monoclonal antibodies made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 15 (60%) anti‐CD5‐treated rats developed the disease as compared with 2 of 18 (11.1%) (P<0.01) rats in the control group. Immunofluorescence analysis performed after anti‐CD5 treatment showed a reduction of staining of CD5+cells as well as a down‐regulation of the staining intensity of CD5 cell surface receptors on the remaining CD5+cells. These data indicate that CD8+‐ as well as CD5+cells participate in the maintenance of low dose tolerance t

ISSN:0730-2312    

DOI:10.1002/jcb.240400106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractInsulin‐dependent (type 1) diabetes mellitus (IDDM) is due to the selective autoimmune‐mediated destruction of pancreatic beta cells possibly initiated by viruses. To elucidate the possible role of viruses and cytokines in the pathogenesis of IDDM, we have examined the effect of reovirus infection on beta cell major histocompatibility complex (MHC) expression and the effect of interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) on beta cell function in vitro. Infection of RIN‐m5F (rat insulinoma) cells with reovirus‐1 or reovirus‐3 was associated with a tenfold increase in class 1 MHC protein and mRNA expression. Reovirus infection did not induce the expression of class 11 MHC by RIN‐m5F cells. Exposure of reovirus to ultraviolet light almost completely abolished its ability to induce class 1 MHC protein expression on infected cells.Murine islets cultured for 3 days with IFN‐γ and/or TNF‐α had a significantly reduced insulin response to glucose, which was more marked with a combination of the cytokines. During 6 days of culture in IFN‐γ plus TNF‐α islets underwent noticeable degeneration associated with an 80% reduction in insulin content. These findings together with previous data suggest viruses and cytokines may have multiple roles in beta cell destruction, indirectly through enhanced MHC protein expression and directly through function

ISSN:0730-2312    

DOI:10.1002/jcb.240400107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractASGP‐1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O‐linked oligosaccharide chains. We have used this system to investigate the O‐glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S‐1 and S‐2) were isolated as their oligosaccharitols by alkaline boro‐hydride elimination, anion exchange HPLC, and ion‐suppression HPLC. From structural analyses S‐1 is proposed to be a branched, sulfated trisaccharide−O4S‐GlcNAcβ1,6‐(Galβ1,3)‐GalNAc and S‐2 its sialylated derivative−O4S‐GlcNAcβ1,6‐(NeuAcα2,3‐Galβ1,3)‐GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP‐1 intermediate in size between immature and mature sialomucin. Pulse‐chase analyses showed that the intermediate could be chased into mature ASGP‐1. The concomitant conversion of S‐1 into S‐2 had a half‐time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAcβ1,6‐(Galβ1,3)‐GalNAc and sialylated derivative GlcNAcβ1,6‐(NeuAcα2,3‐Galβ1,3)‐GalNac. These data suggest that sulfation of ASGP‐1 is an intermediate synthetic step, which competes with β‐1,4‐galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as β‐1,4‐galactosylation. Moreover, sulfation precedes sialylation, but the two

ISSN:0730-2312    

DOI:10.1002/jcb.240400108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractNa+/D‐glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75‐kD protein in apical membranes isolated from highly differentiated LLC‐PK1cultures, an epithelial cell line of pig renal proximal tubule origin. The 75‐kD antigen was enriched from solubilized LLC‐PK1apical membranes by means of high‐pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long‐term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter‐positive cells was dramatically increased after inducer treatment as predicted for differentiation‐regulated expression. These results identify a 75‐kD protein as a component of a developmentally regulated renal Na+/glucose symporter expres

ISSN:0730-2312    

DOI:10.1002/jcb.240400109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe single gene for human macrophage colony‐stimulating factor (M‐CSF, or CSF‐1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M‐CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV‐1 cells expressing a short M‐CSF clone secrete only 24 kd recombinant M‐CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M‐CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M‐CSF protein heterogeneity. Most secreted MIA PaCa‐2 M‐CSF reacts with long‐clone‐specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa‐2 M‐CSF size heterogeneity. In addition, cell lysates also contain larger M‐CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M‐CSF protein heterogeneity results from both pre

ISSN:0730-2312    

DOI:10.1002/jcb.240400110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractExperiments were undertaken to determine whether the method of iodination of epidermal growth factor (EGF) affects its binding to rat liver plasma membranes and its uptake, processing, and secretion into bile by intact rat hepatocytes. EGF was iodinated using one of three oxidative reagents: chloramine T (CT), lactoperoxidase (LP), or monochloride (MC). Quantitative receptor binding studies on plasma membranes isolated from male rat livers with either CT‐, LP‐or MC‐125I‐EGF indicated no significant difference in the apparent binding constants of the three preparations. To determine whether these three preparations were capable of forming a covalent‐like complex with the EGF receptor, they were individually incubated with isolated plasma membranes and subjected to polyacrylamide gel electrophoresis under reducing conditions, followed by autoradiography. Each preparation formed a major radioactive protein band of ∼180 kD, identified as the EFG receptor by immunoprecipitation with monoclonal anti‐EGF receptor antibodies. Furthermore, even unlabeled EGF incubated with plasma membranes formed this same 180 kD band, as revealed on Western blots using anti‐EGF antibody. The biliary secretion of CT‐, LP‐, and MC‐125I‐EGF was compared by injecting each one into rat portal veins and measuring the total and immunoprecipitable radioactivity in bile. The amount of immunologically intact CT‐125I‐EGF in bile was significantly greater than the others, whereas MC‐125I‐EGF transport was significantly reduced. We conclude that the method of iodination does not affect the covalent‐like binding properties of EGF. Furthermore, since unlabeled EGF displayed these same binding properties, oxidative iodination procedures per se do not account for the covalent‐like association between EGF and its receptor. However, the method of iodination used did affect the intracellular transport and processing of EGF by hepatocytes. The structural modification responsible for this alteration in transport

ISSN:0730-2312    

DOI:10.1002/jcb.240400111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY