ABSTRACT
The chemical composition of the cervical mucus (CM), its physical characteristics and the volume of secretion change cyclically throughout the menstrual cycle. The aim of this study was to identify the constitutive protein composition of CM of fertile women and the changes in the CM proteome throughout the menstrual cycle. Five fertile women who had a term delivery within 1 year before the study were enrolled. Proteomic analysis was performed using an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; bioinformatic software was used for functional and quantitative analysis. 59, 81 and 43 proteins (mean) were respectively identified in the pre-ovulatory, ovulatory and post-ovulatory samples. 38 common proteins were identified. 42, 38 and 17 exclusive proteins were respectively identified in pre-ovulatory, ovulatory and post-ovulatory CM. The main part of CM constituents has a catalytic activity, which is mainly related to hydrolase activity. The label-free quantitative analysis of the common proteins revealed a significant reduction in the protein abundance index for antileukoproteinase, after the ovulation, and a peak of haptoglobin at ovulation. This is the first application of high-resolution MS-based proteomics for the identification of protein constituents of CM. This approach may contribute to the identification of putative biomarkers of the female reproductive tract.
Subject(s)
Cervix Mucus/chemistry , Menstrual Cycle/metabolism , Proteins/analysis , Proteome/analysis , Adult , Cervix Mucus/metabolism , Female , Humans , Proteins/chemistry , Proteins/metabolism , Proteome/chemistry , Proteome/metabolism , ProteomicsABSTRACT
Bezafibrate is a hypolipidemic drug that belongs to the group of peroxisome proliferators because it binds to peroxisome proliferator-activated receptors type alpha (PPARs). Peroxisome proliferators produce a myriad of extraperoxisomal effects, which are not necessarily dependent on their interaction with PPARs. An investigation on the peculiar activities of bezafibrate could clarify some of the molecular events and the relationship with the biochemical and pharmacological properties of this class of compounds. In this view, the human acute promyelocytic leukemia HL-60 cell line and human rabdomiosarcoma TE-671 cell line were cultured in media containing bezafibrate and a number of observations such as spectrophotometric analysis of mitochondrial respiratory chain enzymes, NMR metabolite determinations, phosphofructokinase enzymatic analysis, and differentiation assays were carried on. Bezafibrate induced a derangement of NADH cytochrome c reductase activity accompanied by metabolic alterations, mainly a shift to anaerobic glycolysis and an increase of fatty acid oxidation, as shown by NMR analysis of culture supernatants where acetate, lactate, and alanine levels increased. On the whole, the present results suggest a biochemical profile and a therapeutic role of this class of PPARs ligands more complex than those previously proposed.
Subject(s)
Bezafibrate/adverse effects , Mitochondrial Diseases/chemically induced , Peroxisome Proliferators/adverse effects , Tumor Cells, Cultured , Acetates/chemistry , Acetates/metabolism , Alanine/chemistry , Alanine/metabolism , Animals , Bezafibrate/metabolism , Bezafibrate/pharmacology , Dose-Response Relationship, Drug , Humans , Hypolipidemic Agents/adverse effects , Hypolipidemic Agents/metabolism , Hypolipidemic Agents/pharmacology , Italy , Lactic Acid/chemistry , Lactic Acid/metabolism , Magnetic Resonance Spectroscopy , Microscopy, Electron , Mitochondria/drug effects , Mitochondria/physiology , Peroxisome Proliferators/metabolism , Peroxisome Proliferators/pharmacology , Rats , Time FactorsABSTRACT
A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.
