Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset

Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted "-omics" approaches are underutilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program, with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. alpha-2-Glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples.     

Palmitic and Stearic Acids Bind Ca2+ with High Affinity and Form Nonspecific Channels in Black-Lipid Membranes. Possible Relation to Ca2+-Activated Mitochondrial Pores

A mitochondrial hydrophobic component that forms Ca²⁺-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids—mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca²⁺ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca²⁺-binding is inhibited by Mg²⁺ but not by K⁺. For palmitic acid, the K _d for Ca²⁺ was 5 M at pH 8.5 and 15 M at pH 7.5, with the B _max of 0.48 ± 0.08 mmol/g. This corresponds to one Ca²⁺ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca²⁺ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca²⁺, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca²⁺ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A₂ inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.     

Fibrin D-dimer impairs the accumulation and anticoagulant properties of heparan sulphate and stimulates secretion of plasminogen activator inhibitor-1 by rabbit coronary endothelial cells

Fibrin split product D-dimer (DD) is most probably involved in the development of vascular disorders. At 1.5 microM concentration DD inhibited the incorporation of D-[1-(3)H]glucosamine hydrochloride and [2-(14)C]acetate x Na into pericellular heparan sulphate (HS) of rabbit coronary endothelial cells without affecting other groups of glycosaminoglycans (GAGs). At the same time, DD reduced HS ability to bind antithrombin (AT) and suppressed NO production. The effect of DD on pericellular GAGs was similar to that of N(omega)-methyl-L-arginine, the competitive inhibitor of endothelial NO synthase (eNOS). L-Ascorbic acid, eNOS activator, increased the level of endogenous NO in the DD-treated cells, and restored HS accumulation and antithrombin binding. It is suggested that DD influence on endothelial HS may be mediated by NO production. Another effect of DD, namely, stimulation of plasminogen activator inhibitor-1 (PAI-1) secretion did not depend on the NO level. The decreased HS content, reduced anticoagulant properties of HS, and increased PAI-1 secretion disorganized the endothelial matrix, and promoted fibrin formation and vascular damage. This points to DD as an important factor in the development of vascular disorders.     

Properties of Staphylococcus aureus in case of the unfavorable course of burn infection

Clinical and microbiological study of wound discharge from 35 patients demonstrated a relationship between biological properties of S. aureus, the causative agent of burn infection, and the course of the infected burn trauma. The prognostic importance of the antioxidant properties of these staphylococci was established: in cases of the unfavorable course of the burn process they showed essentially greater resistance to peroxinitrite and higher superoxide dismutase activity in comparison with the cultures isolated from patients with uncomplicated wound infection.     

Nonenteric escherichiosis and the problem of human reproductive health

The contribution of nonenteric Escherichia infections (NEI) to the formation of male and female infertility is analyzed. The negative influence of NEI, and particularly pyelonephritis, on the course of pregnancy, parturition and the postnatal period is shown. The role of E. coli in the development of life-threatening meningoencephalitides, pneumonia, sepsis in newborn infants is emphasized, NEI risk factors are systematized with due regard to concrete stages in the pathogenesis of this pathology. Phenotypic differences between the causative agents of NEI and nonpathogenic and diarrhea-inducing E. coli are characterized.     

Taxonomic characterization and antibiotic resistance of pathogens of perinatal infectious inflammatory pathology in infants in Orenburg]

The materials on regional features of the nosologic structure, taxonomic spectrum and antibiotic resistance of the pathogens of perinatal infectious inflammatory pathology (IIP) in infants in Orenburg are presented. From 2002 to 2005 the average level of IIP of 19.0 cases per 1000 living new borns and the dominance of skin inflammatory diseases (vesiculopustulosis in particular) in the nosologic structure among the other forms of IIP (including conjunctivitis and pneumonia) were observed. The prevalence of the gram positive cocci with the leadership of coagulase negative staphylococci among the skin and eye forms of IIP was shown. Their portion reached 62.8%. The portion of the other microorganisms was much lover. Gramnegative bacteria (Neisseria spp., E. coli, K. ozaenae) were isolated from 6.1, 4.3 and 0.6% of the cases. The Candida isolates were detected in 1% of the cases. The regional antibiotic resistance register for the pathogens of IIP was developed. Its use is obligatory in prescribing drugs for empirical therapy of such pathology and prevention of its complications.     

Nonlinear dynamics of the distributed biochemical systems functioning in the dissipative structure formation mode

Nonlinear dynamical biomolecular systems can evidently be considered as prototypes of information processing devices at molecular level capable to solve problems of high computational complexity. Keeping in mind this goal the dynamics of biochemical system based on enzymatic oxidation of uric acid was considered. The system was studied in the version of distributed biomolecular structure having predetermined geometry of enzyme distribution on a porous planar medium. Being in the regime of stepwise dissipative structure formation this system demonstrated complicated modes of behaviour.     

Sputum microflora in acute pneumonia based on the data from a quantitative study

In this work the data obtained in the quantitative investigations of sputum samples from 106 miners having acute pneumonia are presented. These investigations were carried out twice at the peak of the disease to determine the possible infective agent. The virological study of nasal impression smears by immunofluorescence and the serological study of paired sera made it possible to establish the viral and bacterial nature of the disease in 12% of cases. The expediency of the quantitative investigations of sputum, carried out twice, in combination with the study of the biological properties of opportunistic microorganisms was shown. Streptococcus pneumoniae proved to play the most important etiological role in the appearance of acute pneumonia in miners. This infective agent was detected in 82% of patients by the inoculation of sputum samples in "diagnostic" dilutions (10(-5) and higher). The associations of pneumococci with staphylococci, hemolytic bacteria and Neisseria were found to be capable of playing a significant role in the development of acute inflammation in pulmonary tissue, especially in those cases when these associations were isolated from highly diluted sputum (10(-5)).     

Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.