Identification of cortactin molecular forms in human urine and their possible diagnostic value

The protein composition of human urine reflects changes in the biochemical and physiological status of an individual and has an essential diagnostic value. Using precipitation/extraction methods we isolated a protein with Mr ~100 kDa in a human urine. MALDI TOF/TOF mass spectrometry identified this protein as human Src protein kinase substrate cortactin (UniProtKB/Swiss-Prot: Q14247). Screening of urine samples using Western blotting with specific anti-human cortactin antibodies revealed different proteins immunologically related to cortactin in healthy humans and patients with liver cirrhosis and lung cancer diseases. These data suggest that the level of cortacins isoform in urine might serve as a potential marker for testing acute and systemic diseases

Isolation and characterization of peptides from blood serum of patients with multiple sclerosis

In order to find novel molecular markers of multiple sclerosis we developed a scheme of oligopeptides’ isolation including their extraction from blood serum with 10 % trichloroacetic acid, followed by precipitation of soluble substances with acetone in ratio 6:1. Oligopeptides were dissolved in water and their characteristics was determined by gel filtration under HPLC conditions and thin layer chromatography. Obtained data have shown that blood serum of MS patients contains two oligopolypeptides with average molecular masses of 300–500 Da. We also studied biological activity of TCA-soluble peptides toward some eukaryotic and prokaryotic cells in comparison with phosphopeptides isolated from casein hydrolysates. It was found that TCA-soluble peptides are capable of effective inhibiting HeLa cells’ proliferation, while their inhibitory effect was expressed toward Jurkat T-cells and was not detectable toward U373 cells. The casein’s phosphopeptides were capable to stimulate proliferation of Jurkat cells and effectively inhibited growth of cells. Neither antibacterial, nor antifungal activities of these oligopeptides were detected

Influence of Spirocarbone and Derivates of Pyrimidineones on Separate Components of the Oxygen – Transport of blood in vitrol

Rechytskyi, О. Influence of Spirocarbone and Derivates of Pyrimidineones on Separate Components of the Oxygen – Transport of blood in vitrol VII Parnas Conference on Biochemistry and Molecular biology / L. Starykovych, K. Dudok, M. Starykovych, O. Rechytskyi, B. Yeresko, N. Sybirna // Yalta, Crimea, Ukraine, 3-7 October, 2009 The Ukrainian Biochemical Journal. – 2009. – V. 81. – № 4. – Р. 258

Influence of spirocarbone and derivates of pyrrolo pyrimidinediones of survival of leukemia cells

Influence of spirocarbone and derivates of pyrrolopyrimidinediones (1,6-dimethyl-4-phenyl-1,2,3,4,5,7-hexahydropyrolo-[3,4-d]-pyrimidindione-2,5(1Н), and 1,6-dimethyl-4-(2-threefluoromethylphenyl)-1,2,3,4,5,7-hexahydropyrrolo-[3,4-d]-pyrimidinedione-2,5(1Н) on the leukemia cells of L1210 and CEM-T4 lines of mouse and human, res­pectively was studied. Cytotoxic effect of spirocarbone towards L1210 cells accompa­nied by apoptotic fragmen­tation of nuclear DNA

Cytotoxic activity of anti-DNA slgA-antibodies from milk of healthy women

Earlier we have shown that immunoglobulin preparations derived from milk of healthy women by precipitation with 50% ammonium sulfate, possess toxic activity toward several transformed and tumor cells in vitro (Kit Y., and others, Biotechnology 2008). We hypothesized that cytotoxic effect was associated with the presence of cytotoxic anti-DNA sIgA-antibodies in the Ab preparations. To check this hypothesis, we have purified electrophoretic homogeneous anti-DNA sIgAs from human milk and stu­died their affect toward different tumor cells in vitro. Anti-DNA sIgAs were obtained by sequential chromatography on protein A-sepharose, DEAE-fractogel and DNA-cellulose columns. It was found that the sensitivity of studied cells to Ab changed in the order L929> L1210> Jurkat> Namalva. Annexin V – test , as well as DNA-fragmentation test showed that cell death under Abs action occurred by apoptosis

A new mannose-specific lectin from daylily (Hemerocallis fulva L.) rhizome: purification and properties

A new lectin was purified from the daylily (Hemerocallis fulva L.) with the yield of approximately 10 mg per kg of fresh plant rhizome. The purification procedure was based on application of the affinity chromathography on the column with yeast mannan and the ion-exchange chromatography on the column with DEAE-Toyopearl. The lectin possessed low affinity for α-methyl-D-mannopyranoside, D-fructose, D-turanose and 2-acetamido-D-galactopyranose and hight affinity for the yeast mannan. The lectin bound with greatly less affinity for the mannose-containig glycoproteins, such as ovoalbumin, ovomucoid and horseradish peroxidase. According to the results of electrophoresis in 20% DSNa-PAGE, the lectin consists of subunits of 12 kDa molecular weight. According to the results of gel-chromatography on the Toyopearl HW-55, the lectin’s molecular weight is 48 kDa. It agglutinated rabbit erythrocytes very well, while rat and guinea-pig erythrocytes were agglutinated worse, and human erythrocytes were not agglutinated at all. Lectin’s dialysis against 1% EDTA or heating to 60 ºC for 60 min did not stop its hemagglutinating activity

Characteristics of interaction of lectins with carbohydrate determinants of secretory immunoglobulin A (sIgA) isolated from colostrum of parturient women

Secretory immunoglobulin A (sIgA) contains glycoconjugates moieties which can be involved in humoral immunity providing antimicrobial defense. The aim of present work was to characterize carbohydrate moieties of sIgA isolated from human colostrum via their interaction with specific lectins. Preparations of sIgA were obtained from 23 colostrums samples of parturient women by 3-fold precipitation with in ammonium sulphate 50% saturation. In the study a panel of 44 lectins of known carbohydrate specificity was used immobilized onto nitrocellulose membrane. The panel was incu­bated with sIgА preparations and lectin-antibody complexes were detected by protein-A conjugated with horseradish peroxidase. Western-blot analysis was applied to cha­racterize glycosylated polypeptides of sIgA isolated from colostrum of parturient women. sIgA from the colostrum of clinically healthy parturient women contained mainly carbohydrate moieties consisting of N-acetyl-D-glucosamine, sialic acid and D-mannose. Substantial differences in interaction of carbohydrate determinants of slgA isolated from colostrum of individual parturient women with the lectines were found. It is proposed that such characteristics can define sensitivity of mother and child to the action of pathogenic microflora

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity.This work was financial supported by Volkswagen Foundation (grant # VW 90315).Scopu