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C H Chou

Publications and source records attributed to C H Chou.

At least 55 records · Page 3Linked to original sources
PubMed1994

Peptide bond specificity of calpain: proteolysis of human myelin basic protein.

In order to determine the peptide bond specificity of calpain, human myelin basic protein (HMBP) was treated with purified calpain of bovine brain. Upon incubation, HMBP component I (HMBP-I) was degraded into several peptides as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component I was more susceptible to degradation than components II and III. HMBP degradation products were separated by high performance liquid chromatography (HPLC) and the cleavage sites in HMBP molecules were determined by peptide sequence analysis and by N- and C-terminal analyses. The major cleavage site was found to be 94Val-95Thr with several minor cleavages at 49Arg-50Gly, 18Ala-19Ser, 23His-24Ala, 27Gly-28Phe, 59Asp-60Ser, 70Gly-71Ser, 97Arg-98Thr, 110Ser-111Leu, 145Asp-146Ala, and 156Leu-157Gly. These results indicate that calpain is involved in the limited proteolysis of human myelin basic protein and prolonged incubation causes further digestion of the large peptides.

Amino Acid Sequence↗
PubMed1994

Characterization of the Su antigen, a macromolecular complex of 100/102 and 200-kDa proteins recognized by autoantibodies in systemic rheumatic diseases.

The Su autoantigen was characterized biochemically using human and murine autoimmune sera and the clinical significance of anti-Su antibodies was studied in 236 Japanese and 160 American patients with systemic rheumatic diseases. Anti-Su in immunodiffusion (ID) was strongly associated with immunoprecipitation of one or more 100- to 102-kDa proteins by MRL/lpr mouse sera (27/32 of ID positive vs 4/20 of ID negative, P = 0.000016), and all four human anti-Su reference sera immunoprecipitated the 100/102-kDa protein(s). In addition, all sera immunoprecipitated a less efficiently labeled approximately 200-kDa protein that comigrated on sucrose density gradients with the 100/102-kDa proteins. Based on these data, a complex of the 100/102-kDa and 200-kDa proteins is likely to be the main target of anti-Su antibodies. Three of four anti-Su monospecific sera were negative for immunofluorescent antinuclear antibodies (ANA), suggesting anti-Su antibodies may be associated with a negative ANA in some cases. Autoantibodies to Su were detected frequently by immunoprecipitation in systemic lupus erythematosus (17-21%), scleroderma (13-20%), and overlap syndrome (22-40%) and were associated with autoantibodies to Ku.

Animals↗
PubMed1994

Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers.

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.

Animals↗
PubMed1994

Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones.

Pathogenic autoantibodies to DNA are frequently associated with autoantibodies to chromatin-associated proteins such as histones or the Ku (p70/p80) antigens. In view of the increasing evidence that autoantibody production is antigen-driven, and because DNA is packaged with proteins in the cell, we suggest that anti-DNA antibodies may arise in response to DNA-protein complexes rather than naked DNA. Recent studies of the specificities of autoantibodies directed against the components of nucleosomes and transcriptional complexes are consistent with this hypothesis. The possible clinical significance of immune recognition of various types of chromatin complexes in autoimmune disease is discussed.

Antibodies, Antinuclear↗
PubMed1993

Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.

The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506-541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1992

Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen.

The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.

Amino Acid Sequence↗
PubMed1992

Recognition of multiple epitopes in the coiled-coil domain of lamin B by human autoantibodies.

The nuclear lamina of mammalian cells consists of three major proteins, lamins A, B and C, which form a fibrous meshwork interposed between the inner nuclear membrane and the chromatin. Sera from certain patients with systemic lupus erythematosus (SLE) and autoimmune liver disease contain high titers of autoantibodies against lamin B. We have shown previously that anti-lamin B autoantibodies in SLE recognize epitopes highly specific for lamin B, even though lamin B and lamins A/C are highly homologous proteins. To further characterize the specificities of these autoantibodies, fusion proteins carrying fragments of lamins B and C were tested for reactivity with SLE sera by immunoblotting. Five distinct epitopes of lamin B were identified, at least four of which were located in the highly conserved coiled-coil rod domain. Epitopes located on amino acids (AA) 80-193 and 245-303 were recognized by 4/10 and 8/10 anti-lamin B positive sera, respectively. Affinity purified anti-lamin B autoantibodies reacted preferentially with lamin B, indicating that they recognized mainly portions of lamin B that differ from lamins A and C. On the contrary, most of the affinity-purified anti-lamin C autoantibodies from SLE sera cross-reacted with lamin B, suggesting that the anti-nuclear lamina immune response in these patients is directed primarily against lamin B. The preferential reactivity of these sera with multiple epitopes specific to lamin B, and the finding that the autoantibodies to lamins A and C present in some of these sera cross-react with lamin B suggest that autoantibodies to lamin B are generated in response to the authentic lamin B protein rather than a cross-reactive foreign protein.

Adult↗
PubMed1991

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.

Antibody Specificity↗
PubMed1991

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.

Animals↗
PubMed1991

Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein.

Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)-specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu-BP)-reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu-BP. The MS T cell lines responded to more Hu-BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8+ T cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA-DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149-170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA-DR2 homozygous donor, four of eight clones utilized V beta 5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response to BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP-reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP-reactive T cells play a critical role in the pathogenesis of MS.

Adult↗
PubMed1991

Antibodies to myelin basic protein and measles virus in multiple sclerosis: precursor frequency analysis of the antibody producing B cells.

Antibody-producing B lymphocytes were polyclonally activated and transformed, by Epstein-Barr virus (EBV), into multiple B lymphoblastoid cell lines in a microculture system. The frequencies of B precursor cells producing antibodies to myelin basic protein (MBP) and measles virus were analyzed in peripheral blood of patients with multiple sclerosis (MS) and control subjects. Measles virus-specific B cells were detected at a significantly higher frequency in MS patients (n = 10, P less than 0.005) than patients with other neurological diseases (n = 10) and normal subjects (n = 10). In contrast, the frequencies of B cells producing anti-MBP antibodies and natural antibodies did not differ statistically among the three groups tested (P greater than 0.05). In addition, the anti-MBP antibodies produced by a panel of stable B cell lines obtained were found to react selectively with an epitope(s) within the C-terminal half fragment 90-171 of the human MBP molecule. In our experiments, no antibody cross-reactivity between MBP and measles virus could be detected in a total of 2760 B cell cultures.

B-Lymphocytes↗
PubMed1991

Onset and regulation of anti-lamin B autoantibody production is independent of the level of polyclonal activation.

Anti-lamin B autoantibodies are associated with both systemic lupus erythematosus (SLE) and autoimmune liver disease. We examined the possibility that the underlying clinical feature in patients with anti-lamin B autoantibodies might be chronic autoimmune liver disease, and whether the hypergammaglobulinemia present in both disorders is involved in generating anti-lamin B autoantibodies. A lamin B fusion protein (MLB1), consisting of amino acids 77-533 of lamin B fused to TrpE, was used to screen sera from 84 patients with SLE for anti-lamin B autoantibodies. 3/4 prototype human lamin B antisera, 5/84 SLE sera (6%), and 0/30 sera from healthy individuals reacted with MLB1 on immunoblots at a 1:500 dilution. Of the 9 anti-lamin B autoantibody positive patients studied, all but 1 fulfilled at least four ARA criteria for SLE. None of the patients displayed evidence of chronic autoimmune liver disease, suggesting that autoimmune liver disease is not strongly associated with anti-lamin B antibodies in SLE. In SLE, as in "lupoid hepatitis", anti-lamin B autoantibodies are often produced transiently during periods of increased disease activity. Although polyclonal hypergammaglobulinemia is also associated with increased activity of both diseases, anti-lamin B autoantibody production in 2 patients was independent of total immunoglobulin levels, antibodies to irrelevant proteins, and production of some other autoantibodies. Thus, polyclonal activation is insufficient to explain either the initiation or regulation of anti-lamin B autoantibody production, supporting the hypothesis that antinuclear antibodies are antigen-selective.

Adult↗
PubMed1991

Determination of vecuronium in blood by HPLC with UV and electrochemical detection: a pilot study in man.

Vecuronium bromide is a non-depolarizing neuromuscular blocking agent with a rather low therapeutic level. Rapid, sensitive, and selective determination of vecuronium bromide in human blood or plasma was essential for pharmacokinetic study. We developed such a method using HPLC with electrochemical detection. Samples were first acidified, followed by a one-step liquid-liquid extraction. Tubocurarine, which has a structure similar to that of vecuronium, was used as the internal standard. The electrochemical detector, Ag/AgCl electrodes, was operated by setting the working electrodes, W1 and W2, at +0.65 V and +1.05 V, respectively. When vecuronium blood concentration was plotted versus peak area ratios (PAR) of vecuronium over tubocurarine, a linear relationship was observed over the range of 25 ng/mL to 500 ng/mL with a correlation coefficient greater than 0.997. Clinically possible sources of interference, such as atropine, apresoline, droperidol, fentanyl, labetalol, thiopentone, atracurium, and valium, were examined and none showed interference in the assay of vecuronium and tubocurarine. This method has been successfully applied in a preliminary study of the pharmacokinetics of vecuronium in a patient undergoing surgery. The low detection limit of this method in the patient was 3 ng/mL.

Chromatography, High Pressure Liquid↗
PubMed1990

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients.

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.

Amino Acid Sequence↗
PubMed1990

Localization of an encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein.

T cells from SJL mice reactive with myelin basic protein peptide 1-38 have been reported to be encephalitogenic when adoptively transferred into naive syngeneic recipients. To determine whether the encephalitogenic epitope recognized by peptide 1-38-specific SJL T cells was different from those recognized by H-2u-restricted MBP peptide 1-38-specific T cells, peptide 1-38-specific SJL T cell lines were developed following immunization with guinea pig MBP peptide 1-38. Following a period of in vitro selection in the presence of peptide 1-38 and syngeneic antigen-presenting cells, one of two T cell lines transferred severe clinical disease adoptively. The second line was not encephalitogenic. When the fine specificity for antigen of the two T cell lines was determined by the use of overlapping synthetic peptides, the encephalitogenic epitope recognized by the encephalitogenic line was localized to residues 17-27. This epitope is clearly distinct from that recognized by H-2u mice. The non-encephalitogenic line was found to react only with peptide 1-38, and did not react with mouse MBP.

Animals↗
PubMed1989

Human T lymphocyte response to myelin basic protein: selection of T lymphocyte lines from MBP-responsive donors.

The goal of this study was to delineate the importance of blood T lymphocyte responses to several myelin basic protein (MBP) preparations in the ultimate selection of MBP-specific T lymphocyte lines. Proliferation responses to human myelin basic protein (MBP) were assessed in blood samples from 27 multiple sclerosis (MS) patients, 20 patients with other neurologic diseases (OND), and 26 normal subjects, using five MBP preparations with different histories and electrophoretic characteristics to enhance the spectrum of epitopes represented. Substantial variations were observed in the ability of different MBP preparations to induce blood T cell proliferation in a given donor. However, four out of five of the MBPs induced modest but significant proliferation in the MS study population relative to normal individuals, with intermediate responses occurring in OND patients. Positive responses occurred more frequently in MS patients (78%) than in normal donors (31%), and were an important prerequisite for the successful selection of MBP-specific T cell lines.

Adult↗