Changes in the proteomic profile of blood serum in coronary atherosclerosis.

BACKGROUND: Our aim was to study changes in the serum proteomic profile in coronary atherosclerosis. METHODS: The study involved two groups of patients: 1) men with coronary heart disease and coronary atherosclerosis (n = 15); 2) control (n = 15): men without coronary heart disease. The object of this study was blood serum. Separation of proteins for the investigation of differences in serum protein components was performed by two-dimensional electrophoresis. Identification of protein fractions was carried out using peptide mass maps by the matrix-assisted laser desorption ionization method. RESULTS: In blood serum samples from patients with coronary atherosclerosis, protein separation in two-dimensional gels with mass-spectrometric identification revealed an increase of some proteins: hemopexin, transthyretin (monomeric form), retinol-binding protein 4, and components of the complement system: C3 (chain B) and C9. There was a decrease of some proteins: kininogen, zinc finger protein 133, and B-cell CLL/lymphoma 6 member B protein. Comparisons between the experimental and control group were carried out in protein fractions where the protein amount differed more than 1.5-fold (p < 0.05). CONCLUSIONS: Proteome profiling of serum revealed a change in the content of kininogen, hemopexin, transthyretin, retinol-binding protein, and proteins of the complement system (C9, and C3) in coronary atherosclerosis. The contribution to the differential expression of a protein was often made by isoforms of the protein, particularly transthyretin. The change in the concentrations of functionally interacting proteins, such as transthyretin and retinol-binding protein, were noted.

Creation of an Online Platform for Identification of Microorganisms: Peak Picking or Full-Spectrum Analysis.

Identification of microorganisms by MALDI-TOF mass spectrometry is a very efficient method with high throughput, speed, and accuracy. However, it is significantly limited by the absence of a universal database of reference mass spectra. This problem can be solved by creating an Internet platform for open databases of protein spectra of microorganisms. Choosing the optimal mathematical apparatus is the pivotal issue for this task. In our previous study we proposed the geometric approach for processing mass spectrometry data, which represented a mass spectrum as a vector in a multidimensional Euclidean space. This algorithm was implemented in a Jacob4 stand-alone package. We demonstrated its efficiency in delimiting two closely related species of the Bacillus pumilus group. In this study, the geometric approach was realized as R scripts which allowed us to design a Web-based application. We also studied the possibility of using full spectra analysis (FSA) without calculating mass peaks (PPA), which is the logical development of the method. We used 74 microbial strains from the collections of ICiG SB RAS, UNIQEM, IEGM, KMM, and VGM as the models. We demonstrated that the algorithms based on peak-picking and analysis of complete data have accuracy no less than that of Biotyper 3.1 software. We proposed a method for calculating cut-off thresholds based on averaged intraspecific distances. The resulting database, raw data, and the set of R scripts are available online at https://icg-test.mydisk.nsc.ru/s/qj6cfZg57g6qwzN.

A Proteomic Study of Atherosclerotic Plaques in Men with Coronary Atherosclerosis.

BACKGROUND: To study the changes in protein composition of atherosclerotic plaques at different stages of their development in coronary atherosclerosis using proteomics. METHODS: The object of research consisted of homogenates of atherosclerotic plaques from coronary arteries at different stages of development, obtained from 15 patients. Plaque proteins were separated by two-dimensional electrophoresis. The resultant protein spots were identified by the matrix-assisted laser desorption ionization method with peptide mass mapping. RESULTS: Groups of differentially expressed proteins, in which the amounts of proteins differed more than twofold (p < 0.05), were identified in pools of homogenates of atherosclerotic plaques at three stages of development. The amounts of the following proteins were increased in stable atherosclerotic plaques at the stage of lipidosis and fibrosis: vimentin, tropomyosin ß-chain, actin, keratin, tubulin ß-chain, microfibril-associated glycoprotein 4, serum amyloid P-component, and annexin 5. In plaques at the stage of fibrosis and calcification, the amounts of mimecan and fibrinogen were increased. In unstable atherosclerotic plaque of the necrotic-dystrophic type, the amounts of human serum albumin, mimecan, fibrinogen, serum amyloid P-component and annexin were increased. CONCLUSION: This proteomic study identifies the proteins present in atherosclerotic plaques of coronary arteries by comparing their proteomes at three different stages of plaque development during coronary atherosclerosis.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Identification of Bacillus strains by MALDI TOF MS using geometric approach.

Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html.

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta.

X-ray analysis of enzyme-DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9-10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (DeltaG degrees approximately -8.7 to -9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1-DNA interactions provide only one order of magnitude (DeltaG degrees approximately -1.1 to -1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.