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MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the “Rovabio™ Excel”, an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed ‘peptidic shotgun’, which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum. [ABSTRACT FROM AUTHOR]

Background: Many antiangiogenic molecules are proteolytically cleaved from larger plasma proteins. For example, plasminogen activators cleave plasminogen into plasmin, and plasmin is converted into angiostatin in the presence of sulfhydryl donors. We thus investigated whether the antiangiogenic activity in plasma could be increased by treatment with recombinant tissue plasminogen activator (rt-PA) and the sulfhydryl donor captopril.Methods: Human plasma was treated with rt-PA (10 micro g/mL) and/or captopril (1 micro M). Angiogenesis was measured in vitro by human endothelial cell tube formation and endothelial cell proliferation and in vivo in mice with the Matrigel plug assay. Angiostatin was removed from treated plasma by affinity chromatography, immunoprecipitation, or ion-exchange chromatography, and the antiangiogenic activity of the depleted plasma was assessed by tube formation. Three cancer patients were treated with rt-PA and captopril, and their pretreatment and post-treatment plasmas were tested for antiangiogenic activity in vitro.Results: Angiogenesis in vitro was stimulated by untreated plasma and inhibited by plasma that had been treated with rt-PA and captopril but was not affected by treatment with rt-PA and/or captopril alone. In vivo angiogenesis in Matrigel plugs was substantially lower in mice treated with rt-PA and captopril than in untreated control mice. Antiangiogenic activity in treated plasma was largely retained after angiostatin was removed: treated plasma inhibited angiogenesis by 64.3% (95% confidence interval [CI] = 46.4% to 82.2%), relative to untreated plasma, and treated plasma depleted of angiostatin by affinity chromatography or immunoprecipitation inhibited angiogenesis by 65.1% (95% CI = 53.8% to 76.4%) or 63.7% (95% CI = 50.9% to 76.5%), respectively. Antiangiogenic activity of plasma from three cancer patients was higher after treatment with rt-PA and captopril than before such treatment.Conclusion: Treatment with rt-PA and captopril induced antiangiogenic activity in vitro and in vivo that appears to be independent of angiostatin. [ABSTRACT FROM AUTHOR]

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither lo the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation. [ABSTRACT FROM AUTHOR]

The Spectrotypic patterns of anti-Echinococcus granulosus and anti-F(ab')2 antibodies were analysed in 13 patients with different forms of hydatidosis by means of isoelectric focusing (IEF), Specific anti echinococcus antibodies and specific echinococcus antigens were obtained by crossed affinity chromatography between patient serum and cyst fluid, as previously described (D'Amelio et al., 1985). A wide spectrotype in the pattern of anti-Echinococcus antibodies was observed, with strong homologies among the different sera mainly at acid and neutral pH. One serum displayed faint, nearly in apparent bands. Conversely, the pattern of anti-F(ab')2 antibodies in the six patients in whom they were positive was limited but heterogeneous, ranging from monoclonal through oligoclonal to polyclonal. A predominant clone was identified in the patient with a polyclonal pattern. A correlation between qualitative IEF spectrotypic patterns and the quantitative results of immunoenzymatic assays was observed for both types of antibodies. [ABSTRACT FROM AUTHOR]

A component of Aspergillus fumigatus, Ag 7, previously identified as a major antigen for patients with allergic bronchopulmonary aspergillosis, has been isolated by gel filtration and further purified by affinity chromatography using monospecific antiserum. The antigen, which binds both specific IgG and IgE antibodies, was shown to be a high molecular weight, 150-200 kD, heat-stable glycoprotein, which binds to concanavalin A. suggesting the presence of α-D mannopyrannoside, or α-D glucopyrannoside and residues. On sodium dodecyl sulfate polyaerylamide gel electrophoresis (SDS-PAGE) it had a subunit of molecular weight 36 kD (with or without prior reduction), which retained antigenicity and allergenicity when tested with patients" sera. [ABSTRACT FROM AUTHOR]

Thyroglobulin antibodies in the sera from 31 patients with a variety of disorders were studied by isoelectric focusing. Only one gave a spectrotype indicative of a monoclonal response. the other 30 giving spectrotypes characteristic of polyclonal responses. There was evidence of clonal dominance in some of the sera and each gave a different spectrotype. Light chains were prepared from five thyroglobulin antibodies purified by affinity chromatography. There was no restriction in the speclrotypes when compared with light chains prepared from normal immunoglohulin. [ABSTRACT FROM AUTHOR]

Oral contraceptives (OC) can induce in some women, scrum immune complexes precipitated in 25% saturated (NH4)2SO4, especially in the case of a thrombotic complication. In this work, the presence of antiethinyloestradiol (anti-EE) antibodies in the complexes was investigated. Binding of ethinyloestradiol-³H (EE-³H), either by (NH4)2SO4, precipitate directly, or in equilibrium dialysis experiments with the anti-EE gamma-globulin isolated through a purification-activation method combining dilution chromatography and affinity chromatography, was measured. The purified proteins were identified by immunoprecipitation and isoelectrophoresis. Twelve sera from OC users and three from women who had never used them were tested. Eleven of the twelve users had serum immune complexes. Normal human gamma-globulin pools and gamma-globulins prepared from a rabbit immunized with EE, were used as negative and positive controls. Results demonstrated that serum complexes precipitated in 25% saturated ammonium sulphate contained EE-³H binding gamma-globulins. For seven out of eleven cases, the association constant Ka was calculated and varied from 4 × 105 M-1 to 2.6 × 107 M-1, and the valence from 1.4 to 2, a result consistent with an IgG antibody activity. These anti-EE antibodies were found in the serum complexes from women on OC with or without thrombosis. However their amount seemed to be greater in the cases with thrombosis. In the serum of women who had never used the pill, in the only OC user who had no serum complexes in 25% saturated ammonium sulphate, and in the pool of normal human gammaglobulins, no anti-EE activity was detected. [ABSTRACT FROM AUTHOR]

Summary: Chromaligner is a tool for chromatogram alignment to align retention time for chromatographic methods coupled to spectrophotometers such as high performance liquid chromatography and capillary electrophoresis for metabolomics works. Chromaligner resolves peak shifts by a constrained chromatogram alignment. For a collection of chromatograms and a set of defined peaks, Chromaligner aligns the chromatograms on defined peaks using correlation warping (COW). Chromaligner is faster than the original COW algorithm by k2 times, where k is the number of defined peaks in a chromatogram. It also provides alignments based on known component peaks to reach the best results for further chemometric analysis. [ABSTRACT FROM PUBLISHER]