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Notes: Abstract: Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-l, β2-microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (Mr 28,000) and cerebrin 30 (Mr 30,000) that have an N-terminal amino acid sequence of NH2-APPAQVSVQPNF and NH2-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis Chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of 〉90%. The potential use of this technology for micropurification of proteins was discussed.

Notes: [Auszug] Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is ...

Notes: Abstract Recent studies from this laboratory have shown that a monoclonal antibody prepared against a specific epitope on α1-antitrypsin is a valuable diagnostic marker for autoimmune conditions. In the present study we have further characterized this monoclonal antibody and reassessed its diagnostic value in screening samples from patients with various autoimmune conditions. α1-Antitrypsin was micropurified from patients with selected autoimmune conditions and from normal donors. The purified α1-antitrypsin isolated. from patients with autoimmune conditions and normal donors was deglycosylated losing both a mixture of exoglycosidases and endoglycosidase F. The immunoreactivity of the native and deglycosylated α1-antitrypsin was examined using both a monoclonal antibody and a polyclonal antibody in enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), respectively. It was noted that α1-antitrypsin isolated from patients with autoimmune diseases generated a displacement curve dissimilar to α1-antitrypsin purified from normal donors or α1 antitrypsin from patients with autoimmune diseases subjected to deglycosylation when these samples were examined by ELISA using the monoclonal antibody. However, when the polyclonal antibody was used for these studies, no difference was found between the native and deglycosylated ga1,-antitrypsin suggesting that the monoclonal antibody recognized an epitope not detectable by the polyclonal antibody. We have also assessed the diagnostic usefulness of this monoclonal antibody using a battery of 530 serum samples obtained from patients with different autoimmune diseases and compared to normal human serum (NHS,N−66); these include: systemic lupus erythematosus (SLE,N=149), rheumatoid arthritis (RA,N=64), renal diseases (NP,N=33), liver diseases (HP,N=33), mixed connective tissue disease (MCTD,N = 12), diabetes (DB,N=40), SjÖgren's syndrome (SS,N = 41), polymyositis (PM,N=20), scleroderma (SCL,N=20), Alzheimer's disease (AZ,N=11), and patients with elevated levels of carcinoembryonic antigen (CEA,N=41). The results of this study demonstrated that this monoclonal antibody is positively correlated with SLE and SS. The significance of the monoclonal antibody in connection with the pathogenesis of autoimmune diseases was discussed.

Notes: Abstract Previous studies from this and other laboratories have shown that abnormal glycosylation of several acute-phase proteins can be detected in various pathological conditions including autoimmune diseases. In the present study, we have investigated if abnormal glycosylation is limited to acute-phase proteins. We used the concanavalin A (Con A) blots in conjunction with the peptide mapping techniques to analyze serum samples and cerebrospinal fluids (CSF) obtained from patients with autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), scleroderma (SCL), Sjögren's syndrome (SS), and polymyositis (PM); diseases of probable autoimmune origin: hepatopathies (HP); diseases of suspected autoimmune origin: schizophrenia and Alzheimer's disease (AZ); and conditions not related to autoimmunity: pregnancy (PG) and elevation of the carcinoembryonic antigen (CEA), in comparison to normal donors (NHS). We have micropurified two human proteins;α 2-macroglobulin, a non-acute-phase protein, andΒ-chain of haptoglobin, a known acute-phase protein, from serum samples of individual patients with SLE, RA, MCTD, SCL and SS, and from PG and NHS for analysis. The identity of the purified proteins was confirmed by immunoblots using either monospecific polyclonal or monoclonal antibodies, and by directN-terminal amino acid sequencing. Peptide maps for each of these proteins were generated usingStaphylococcus aureus protease V8, a Glu-C endopeptidase. When the peptide fragments ofα 2-macroglobulin were resolved by SDS-PAGE and visualized using silver staining, no differences were noted between patient samples and controls. However, when they were examined by lectin blots using Con A, the Con A-reactive fragments increased specifically and significantly in samples derived from patients of SLE, SCL, MCTD, and RA. Similarly when the peptide fragments of theΒ-chain of haptoglobin were visualized by silver staining, no differences were noted; however, the Con A reactivity of specific fragments increased in SLE, RA, SCL, and SS patients. Analysis of these results indicated that there has been a selective increase in Con A-reactive fragments in both acute-phase and non-acute-phase proteins in autoimmune conditions. Thus, the study of changes in glycosylation patterns in selected serum proteins may be a valuable diagnostic approach to define the pathophysiology of inflammatory and autoimmune disorders.