Importance of associated microorganisms in the development of acute pneumonia

Sputum samples obtained from 106 patients with acute pneumonia have been studied by the quantitative microbiological method. Different microbial associations have been shown to play an important role in the development of acute pneumonia (67.9%). Microbiological studies have revealed the prevalence of pneumococci in the etiological structure of the disease. Staphylococci, hemophilic bacteria and Neisseria have been found to take part in the development of acute pneumonia in a lesser number of cases and more often occur in combination with pneumococci or form different associations themselves. The so-called "etiological" type of microbial associations has been determined, two or three microbial species being isolated at high concentrations (10(6) and over) and the percentage of patients with such combinations being 59.7 +/- 5.8%. The analysis of the clinical course of the disease has revealed that the presence of different microbial species in patients linked, to a great extent, with the clinical picture of the disease. These data may be used for prescribing adequate etiotropic therapy and for prognostication. Quantitative bacteriological studies carried out in the dynamics of the disease permit timely detection of changes in the microflora of the respiratory tracts, the evaluation of the effectiveness of antibacterial therapy in the process of its implementation and the rational correction of therapeutic measures.     

The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.     

Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process

Four mAbs of the IgG(1) class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bbeta15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bbeta26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fab-fragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bbeta15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbetaSARGHRPLDKKREEA(12-26), BbetaLDKKREEA(19-26), BbetaAPSLRPAPPPI(26-36), BbetaAPSLRPAPPPISGGGYRARPA(26-46) and BbetaGYRARPA(40-46), which imitate the various sequences in the N-terminal region of the fibrin Bbeta-chain, have been investigated. Peptides Bbeta12-26 and Bbeta26-46, but not Bbeta40-46, Bbeta19-26, and Bbeta26-36, proved to be specific inhibitors of fibrin polymerization. The IC(50) values for Bbeta12-26 and Bbeta26-46 were 2.03 x 10(-4) and 2.19 x 10(-4) m, respectively. Turbidity and electron microscopy data showed that peptides Bbeta12-26 and Bbeta26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bbeta12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aalpha17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bbeta12-46 of one fibrin desAA molecule and the D-domain of another has been constructed.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Quantification of D-dimer and soluble fibrin in blood plasma of people with ischemic heart disease and hypertension

The method of D-dimer quantification in the human blood plasma has been developed using monoclonal antibodies 111-3b and II-4d. The method has been verified on the blood plasma of the patients with ischemic heart disease with and without stenocardia and with hypertension. The results showed that at ischemic heart disease with and without stenocardia and at hypertension the quantities of D-dimer in the blood plasma were generally less than the highest normal level 500 ng/ml (64.3%, 76.2% and 95%, correspondingly). The semiquantitative measurements of soluble fibrin levels in blood plasmas of the patients with ischemic heart disease and hypertension have been performed. It has been shown that the quantity of soluble fibrin at these diseases range greatly from < 0.03 mg/ml to 0.15 mg/ml. There was no correlation between the quantities of D-dimer and soluble fibrin in blood plasmas of the patients. Electrophoresis in PAAG with SDS showed that the soluble fibrin at these diseases had the mo- lecular mass of the fibrin (ogen). Thus the soluble fibrin in blood plasmas analysed consisted mainly of fibrin desAA oligomers (may be with fibrinogen incorporation) which are not stabilized by the factor XIIIa.     

Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

Dopamine system genes and personality traits of extraversion and novelty seeking

Dopamine neurotransmissin is thought to play a relevant role in behavioral reinforcement system. Polymorphism of the genes involved in dopamine system has been reported for association with psychological traits related to impulsive and sensation seeking behaviors. The study was aimed at a search for association of catechol-O-metyltransferase (COMT) and dopamine receptor D4 (DRD4) gene polymorphism with personality traits in Russian population. A sample comprised 130 subjects. It was found that carriers of the Met/Met COMT genotype had higher scores of novelty seeking as compared to those with the Val/Val and Met/Met genotypes. The association was observed in women only. In the presence of the C allele of the DRD4 gene, females with the Met/Met genotype demonstrated higher scores on extraversion and hypomania. The results are consistent with the current theoretical concepts on the regulation of dopamine neurotransmission in the brain.     

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.