The growth of malignant tumors is accompanied by an increase in plasmin activity, one of the key enzymes in oncogenesis. IgG proteolysis by plasmin yields fragments with exposed C-terminal lysine residues that can bind to plasmin heavy chain. Highly purified plasminogen (plasmin precursor) and heavy chains containing lysine-binding sites were isolated. The formation of IgG fragments as a result of their proteolysis by plasmin in solution was shown. Using ELISA with immobilized heavy chains, we found increased concentration of IgG fragments in the sera of patients with esophageal cancer in comparison with healthy donors. The test had high sensitivity (91%) and specificity (85%).

Introduction. Prostate cancer (PC) leads the world structure of male cancer mortality. Understanding the prognosis for PC patients is vitally important and requires diagnostic enhancements such as developing biomarker panels. The studyaimed to estimate sensitivity, specificity, and prognostic significance of the level of immunoglobulin G (IgG) proteolysis products in the blood serum depending on PC Gleason score and detect how the levels of IgG proteolytic fragments were associated with the diagnostic marker expression. Materials and methods. The study included 90 PC patients. We used the Gleason score to evaluate tumor grade. The level of the IgG proteolysis products and total prostate-specific antigen (PSA) in the serum was detected with ELISA. We performed an immunohistochemical assay with Ki-67, plasminogen-binding protein enolase-1 (ENO-1), and prostate-specific membrane antigen (PSMA). Results. ROC analysis demonstrated 100% specificity of the IgG proteolysis products test regardless of Gleason score, while the sensitivity reached 72.2%, 57.5%, 52.6%, and 46.2% for ISUP stages 1, 2, 3, and 4, respectively. An intergroup comparison showed significant differences between patient groups according to Gleason score, preoperative PSA and IgG proteolysis product serum levels and Ki-67 index. We detected a positive correlation between the level of IgG proteolysis products in the PC patients’ serum and ENO-1 expression and a negative correlation with the Gleason score and PSMA expression. Conclusion. Preoperative detection of high levels of proteolytic IgG fragments in the serum of PC patients even with low Gleason score and PSMA expression may help predict a more aggressive disease course and choose the management strategy. Keywords: prostate cancer, plasminogen activation system, proteolytic fragments of immunoglobulin G, oncomarkers

For elucidation of the mechanisms of IgG binding with human plasminogen in prostate cancer patients, we propose an original ELISA on polystyrene plates with immobilized heavy and light plasminogen chains. The level of IgG bound to plasminogen heavy chain in the serum of prostate cancer patients significantly exceeded that in healthy volunteers. IgG treated with plasmin more actively (by more than 2 times) bound plasminogen heavy chain than intact IgG. These findings indicate the involvement of lysine-binding sites of plasminogen heavy chain in the interaction with the C-terminal lysine of IgG and their fragments. ROC analysis of ELISA data showed significant differences between serum samples from patients with prostate cancer and benign prostatic hyperplasia. It is hypothesized that IgG in the tumor region undergo proteolysis and their products appear in the circulation.

A method of ELISA for measuring the binding of different samples of immunoglobulin (IgG) and its fragments to human plasminogen (Pg) has been developed. Instead of plasminogen, the heavy chain of plasminogen (Pg-H) containing five ligand-binding kringle domains, immobilized on the surface of the plate, was used in this method as a detector. It was found that IgG treated with plasmin (IgG Pm-t ) binds to the immobilized Pg-H 2.84 times more strongly than intact IgG. Both IgG samples showed a weak nonspecific binding to the immobilized light chain of plasminogen (Pg-L). It was shown that 0.2 M L-lysine inhibits the binding of IgG Pm-t and does not affect the nonspecific binding of intact IgG to the immobilized Pg-H, indicating the involvement of lysine-binding regions of Pg-H in binding to IgG Pm-t . A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgG Pm-t and did not affect the nonspecific binding of intact IgG to the immobilized Pg-H, which indicates a key role of the С-terminal lysine of IgG Pm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgG Pm-t on the rate of activation of Glu- and Lys-forms of Pg (Glu-Pg and Lys-Pg) by a tissue activator of Pg (tPA) and urokinase (uPA) in buffer showed that intact IgG completely inhibited the activation of Glu-Pg and Lys-Pg with both tPA and uPA. Presumably, the inhibitory effect of intact IgG is due to steric hindrances that it creates for protein–protein interactions of the activators with the zymogen. IgG Pm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgG Pm-t on the activation of Glu-Pg under the action of tPA was ∼25% higher than on the activation of Lys-Pg, which is explained by more significant conformational changes in the Glu-Pg molecule compared with the Lys-Pg molecule after their binding to IgG Pm-t . The results suggest that the specific cleavage of IgG by plasmin may be one of the ways by which the plasminogen/plasmin system is involved in various physiological and pathological processes.

INTRODUCTION:The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors.AIM:To identify IgG degraded by plasmin in the plasma of cancer patients.MATERIALS AND METHODS:Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis.RESULTS:The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg-sepharose.CONCLUSIONS:C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer.

Plasma level of IgG autoantibodies to plasminogen was measured by ELISA in patients with benign prostatic hyperplasia ( n =25), prostatic cancer ( n =17), lung cancer ( n =15), and healthy volunteers ( n =44). High levels of IgG to plasminogen were found in 2 (12%) of 17 healthy women, in 1 (3.6%) of 27 specimens in a healthy man, in 17 (68%) of 25 specimens in prostatic cancer, in 10 (59%) of 17 specimens in lung cancer, and in 5 (30%) of 15 specimens in benign prostatic hyperplasia. Comparison of plasma levels of anti-plasminogen IgG by affinity chromatography showed 3-fold higher levels in patients with prostatic cancer vs. healthy men.

One of the problems of plasma proteomics is a presence of large major components. In this work, we use the thermostable fraction as a way to deplete these major proteins. The thermostable fraction of serum samples from patients with ovarian, uterus, and breast cancers and benign ovarian tumor was analyzed using two-dimensional electrophoresis combined with MALDI-TOF(-TOF)-mass spectrometry. Of them, α-1-acid glycoprotein and clusterin are expressly down-regulated in breast cancer, whereas transthyretin is decreased specifically in ovarian cancer. Apolipoprotein A-I forms have decreased spot volumes, while haptoglobin α1, in contrast, is elevated in several tumors. These data are partly consistent with previous art studies on cancer proteomics, which involve mass-spectrometry-based serum profiling techniques. Serum thermostable fraction may be recommended as a good tool for medium and small protein proteome investigation, in particular, by 2D-electrophoresis.

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.

Literature data summarizing new approaches and importance of early ovary cancer diagnostics have been reviewed. Alpha-feta-protein (AFP) and SA125 were the most reliable markers for determination of early ovary cancer stages. Nevertheless, these markers don't reflect the disease stage, malignance and they don't possess sufficient specificity. New methodical approaches have recently been introduced. They include combination of 2-D electrophoresis with mass-spectrometry. These methods allow to inventory and identify almost all proteins of various tissues. Using these methods for scanning proteins from biopsies of ovary cancer tissues new markers have been discovered.

The gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. A widely distributed H. pylori infection is associated with chronic gastritis, gastric and duodenal ulcers, and malignant neoplasms. In this study proteome maps of four H. pylori clinical isolates derived from patients of two Russian regions (Moscow/Moscow Region and Novosibirsk) were obtained using 2D-electrophoresis and MALDI-TOF-mass-spectrometry. Variability of some H. pylori proteins and the level of their expression have been evaluated. These four isolates could be easily subdivided into two equal groups characterized by the close proteome profiles and the isolate from Moscow Region and the isolate from Novosibirsk constituted one group. The present study demonstrates the potential of proteome technology, which can be employed together with genome and transcriptome studies for the multiparameter typing of clinical isolates of pathogenic microorganisms.

Structural sex-depended differences were revealed in 7 out of 10 hypothalamic paraventricular nuclei under study in DB/DB C57 BL-K56 mice. Sex-dependent differences were also found in the responses to diabetes mellitus in 6 of these nuclei. The data obtained seem to corroborate the sex dependence of the paraventricular-vagal neuronal system involved in the control of carbohydrate homeostasis.

The formation of estradiol-17β is one of the pathways of the metabolism of T in the tissues of various regions of the brain of 21-day old rat fetuses (PVN, MPA, ACr, ACc, DVC).

According to the data on reconstruction of the serial sections of the cerebral hypothalamic area, using morphometric and electron microscopical methods, the structure of the paraventricular nuclei (PVN) has been studied in white rats (Wistar strain) hypothalamus. Ten subnuclei have been revealed. Each of them has individual characteristics. Owing to the analysis of the groups revealed, it is possible to divide the subnuclei into 3 main groups: magnocellular, parvicellular and subnuclei consisting of middle size neurons. The coordinated interaction of these subnuclei are supposed to ensure possible participation of PVN in regulation of the vegetative nervous and endocrine systems.

An attempt was made to elucidate whether the hypothalamic paraventricular nucleus (PVN) is involved in carbohydrate homeostasis regulation in rats. The cellular responses to various disturbances of homeostasis were studied in the neuronal subdivisions (subnuclei) of the PVN. The responses were assessed in terms of changes in the sizes of the cell nuclei, nucleoli, and cytoplasm, and by the ultrastructural changes evident in cellular organelles. Significant cellular responses were observed in several subnuclei, with the nature of the responses being determined by the type of homeostatic alteration. The results indicate the possible involvement of the PVN subnuclei in the regulation of carbohydrate homeostasis.

Experiments in vitro with further thin-layer chromatography of steroids were staged to investigate changing of 3H-testosterone into 3H-estradiol-17 beta and 3H-5 alpha-reduced metabolites in a fraction of 1000 g of homogenates of the medial preoptic area (MPA), the paraventricular nuclei (PVN), the dorsal vagal complex (DVC) of the lower cerebral trunk, the rostral and caudal parts of the amygdaloid complex (ACr and ACc) of the brain of 21-day male and female rat fetuses. The activity of aromatase (AA) and 5 alpha-reductase (AR) was detected in all investigated areas. AA was maximum in PVN of females and minimum in ACc of males and females. The production of 5 alpha-dehydrotestosterone prevailed among 5 alpha-reduced products in PVN, DVC and ACr, 5 alpha- and rostane-3 alpha and 17 beta-diol--in MPA and ACr, AR in ACr was much higher than in ACc. AR in DVC of females was slightly higher than in males. Sex differences in the other cerebral areas were undetectable. The results of the investigation suggest that the above cerebral structures are dependent on sex hormones.

The structural correlates of the cell responses in subnuclei of the hypothalamic paraventricular nuclei (PVN) in fasting and carbohydrate loaded adult male rats were studied using light microscopic morphometric methods. The data obtained revealed difference in responsiveness of the subnuclei studied to alternative shifts in the carbohydrate homeostasis: histophysiological features in five of ten cell-divisions, distinguished in the rat RVN, demonstrated their dependence on impaired carbohydrate homeostasis.

The radioisotope technique and thin layer chromatography (TLC) of steroids have been used to measure the aromatase activity levels (AA) in the paraventricular nuclei (PVN) of the hypothalamus, as well as in the rostral and caudal portions of the amygdaloid complex (ACr and ACc), and in the medial preoptic area (MPA) and the dorsal vagal complex (DVC) of the lower brain stem of the 21 day old rat foetuses