Pleiades Publishing Ltd

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.

Sự phát triển của apoptosis đi kèm với sự giảm xuống của các bản sao của NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), và cũng có sự thay đổi về các đặc tính xúc tác của enzyme gan chuột so với nhóm đối chứng. Việc bổ sung acid thioctic đã làm tăng mức độ biểu hiện về giá trị bình thường. Khối lượng phân tử của các chế phẩm đồng nhất của NADP-ICDH được tinh chế từ gan của chuột đối chứng và chuột thí nghiệm là giống nhau 112 ± 5.8 kDa, tuy nhiên, các giá trị K m và pH-tối ưu đã thay đổi trong quá trình kích thích apoptosis. Sự điều chỉnh hoạt động của NADP-ICDH bởi một số trung gian của chu trình axit tricarboxylic cũng khác biệt giữa các nhóm này.

The inhibitory effects of compositions of α-tocopherol (α-TP) and salicylic acid derivatives on the process of initiated oxidation of methyl oleate have been investigated. α-TP and the salicylic acid derivatives exhibited the synergistic effect, which was demonstrated by the methods of UV-spectroscopy and high-performance liquid chromatography (HPLC). Kinetics of α-TP utilization during methyl oleate oxidation was investigated under conditions of its independent use as well as using its binary mixture with the synthetic antioxidants.

Cardiovascular diseases (CVD) remain the leading cause of death in industrialized countries. One of the most important risk factors for atherosclerosis is hypercholesterolemia; its diagnostics is mainly based on regular analysis of the lipid profile, including the determination of total cholesterol, low and high density lipoprotein cholesterol, and triglycerides. However, in recent years, much attention has been paid to the crosstalk between metabolic pathways of cholesterol and sphingolipid biosynthesis. Sphingolipids are a group of lipids that include a molecule of sphingosine aliphatic alcohol. These include sphingomyelins, cerebrosides, gangliosides, ceramides, sphingosines, and sphingosine-1-phosphate. It has been found that sphingolipid catabolism is associated with cholesterol catabolism. However, the exact mechanism of this interaction still remains unknown. Ceramide attracts particular attention as a CVD inducer. Aggregated lipoproteins isolated from atherosclerotic zones were found to be enriched with ceramides. Ceramide and sphingosine levels increase after ischemia/reperfusion of the heart, in the infarction area and in the blood, as well as in hypertension. Sphingosine-1-phosphate (S-1-P) exhibits pronounced cardioprotective properties. Its quantity sharply decreased during ischemia and myocardial infarction. S-1-P predominated in the structure of high-density lipoproteins (HDL), where it has a significant impact on their multiple functions. An increase in ceramide and sphingosine and a decrease in S-1-P levels during progression of coronary heart disease may be an important factor in the development of atherosclerosis. It is proposed to use determination of sphingolipid levels in the blood plasma as markers for early diagnostics of cardiac ischemia and in hypertension in humans. Recently, intensive studies have been undertaken to create drugs that can correct S-1-P metabolism. The most successful developments include agents targeted to the S-1-P receptor, which mediates all S-1-P effects. Chromatography-mass spectrometry is proposed as the main method for testing these lipids.

Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations.

Using the L-phenylalanine secreting strain of Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via the ribulose-5-monophosphate (RuMP) cycle of carbon assimilation, as an example, we have continued studies on the use of methylotrophic bacteria for the preparative microbial synthesis of amino acids labeled with stable isotopes, including deuterium (2H), suitable for biomedical applications and clinical diagnostics. Here we demonstrate the data on adaptation of the methylotrophic bacterium B. methylicum to the maximal concentration of deuterium in the growth medium with 98% (v/v) 2H2O and 2% (v/v) [2H]MeOH, and biosynthesis of deuterium labeled L-phenylalanine with different levels of isotopic enrichment. The strain was adapted to 2H2O by means of plating of initial cells on solid (2% agarose) minimal growth media M9 with an increasing gradient of 2H2O concentration from 0, 24.5, 49.0, 73.5 up to 98% (v/v) 2H2O and subsequent selection of individual colonies stable to the action of 2H2O, which were capable to produce L-phenylalanine. L-phenylalanine was extracted from the growth medium with isopropanol followed by subsequent crystallization in ethanol (output 0.65 g/L). Using the developed method of microbial synthesis it is possible to obtain deuterated L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2H2O in growth media, from 17% (the growth medium with 24.5% (v/v) 2H2O) right up to 75% (the growth medium with 98% (v/v) 2H2O) of deuterium as evidenced by results of the electron impact (EI) mass-spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfonyl chloride (dansyl) phenylalanine isolated from growth media under different experimental conditions.

Metabolic activity of innate immune cells infected by various doses of Gram-negative (Yersinia pseudotuberculosis, Salmonella enteritidis) and Gram positive (Staphylococcus aureus, Listeria monocytogenes) bacteria has been investigated. Using various animal models we found that in during the initial period (up to 2 days) the changes in cellular responses depend on the type of the pathogen. In response to infection caused by Gram-negative bacteria predominant of neutrophil accumulation in the foci of inflammation was observed, while Gram-positive bacteria induced preferential accumulation of macrophages. The study of metabolism of these cells showed that the response of terminally differentiated primed phagocytes to pathogen appearance was higher than in cells circulating in blood. In addition to the priming state the phagocyte reactivity is influenced by the bacterial load. At a low phagocyte/microbe ratio the cells reaction is almost undetectable, while an excess of microorganisms causes (despite of the increase of the phagocytic parameters) the hyperactivation of cell metabolism and production of maximal amounts of bactericide agents, which exhibit a damaging effect on the cell itself.

Oxidative stress plays an important role in cardiovascular diseases and atherosclerosis. Fibrinogen (FB), a central protein of the plasma coagulation cascade, is an independent risk factor of atherosclerosis. Importantly, it can be readily oxidized during oxidative stress and in various pathological conditions. Since endothelial dysfunction plays a key role in atherosclerosis it is interesting to investigate the effect of oxidized fibrinogen (ox-FB) on human umbilical vein endothelial cells (HEC). Here, we have investigated the effect of ox-FB on the development of programmed death of HEC incubated in vitro for 24 h under two different conditions: (1) at low serum level (0.1%) and in the absence of growth factors (“starvation”); (2) in full medium (5% human fetal serum) with growth factor supplement. Apoptosis was evaluated using analysis of nuclear morphology, phosphatidylserine externalization on the HEC surface and caspase-3 activation. Under starvation conditions characterized by significant cell death and activation of apoptosis addition of unoxidized FB significantly improved cell survival and prevented caspase-3/7 activation. In the presence of ox-FB caspase activity was 1.5 times higher than in the presence of FB, nevertheless, ox-FB demonstrated significant protection of HEC. Under optimal cultivation conditions FB caused a 3-fold decrease in the rate of apoptosis, while ox-FB improved cell survival but it was less active than FB. Thus, FB promoted HEC survival under stress conditions (in starvation), however, oxidative modification of this protein decreased its antiapoptotic activity.

Concentration of thyroid hormones in the serum of the rats after 14-day injections of potassium iodide (1, 3, 10, 100, and 500 physiological daily doses) did not differ from the control values. Excessive administration of potassium iodide increased the total iodide content in the rat thyroid tissue by 60–121% (35–108% and 94–128% for the protein-bound and free iodide, respectively), indicating the activation of the uptake and organification of iodide. The long-term injection of both low and high doses of potassium iodide increased the activity of catalase by 8–18% and SOD by 33–50% and enhanced the level of toxic LPO products reacting with thiobarbituric acid by 15–38%. It is suggested that reactive oxygen species and the excessive iodination of proteins (particularly thyroglobulin) induced by the long-term administration of high doses of potassium iodide can play an important role in the development of thyroid dysfunctions and autoimmune diseases.

The development of the metabolic syndrome (MS) involves many genes. Certain evidence exists in the literature on the association of polymorphism in the mitochondrial DNA (mtDNA) oriB site, also known as the polycytosine pathway, with the development of insulin resistance, type 2 diabetes mellitus (T2DM) and other metabolic disorders in various ethnic populations. It is suggested that certain polymorphisms at this site are associated with mtDNA copy number in the cell. In this study, using capillary sequencing, we have identified various allelic variants of the mtDNA oriB site in patients with MS (n = 106) and conditionally healthy donors (n = 71). The mtDNA copy number in blood leukocytes was determined by the droplet digital polymerase chain reaction (ddPCR). It has been shown that the variant of the continuous polycytosine tract is significantly more frequent in MS patients with T2DM (p < 0.01). In general, the mtDNA copy number of blood leukocytes was lower in MS patients than in controls. We did not find any correlations between the oriB site variability and the mtDNA copy number.