[Relationship between hypercholesterolemia and osteoarthritis (preliminary results)].

AIM: To evaluate the relationship of hypercholesterolemia (HCE) with clinical, instrumental, and laboratory parameters in osteoarthritis (OA) in a multicenter, cross-sectional study. MATERIALS AND METHODS: The study included 183 patients aged 40-75 years, with a confirmed diagnosis of stage I-III OA (ACR) of the knee joints, who signed an informed consent. The mean age was 55.6±10.7 years (40 to 75), body mass index was 29.3±6.3 kg/m2, and disease duration was 5 [1; 10] years. For each patient, a case record form was filled out, including anthropometric indicators, medical history, clinical examination data, an assessment of knee joint pain according to VAS, WOMAC, KOOS and comorbidities. All patients underwent standard radiography and ultrasound examination of the knee joints and laboratory tests. RESULTS: HCE was detected in 59% of patients. Depending on its presence or absence, patients were divided into two groups. Patients were comparable in body mass index, waist and hip measurement, and disease duration but differed significantly in age. Individuals with elevated total cholesterol levels had higher VAS pain scores, total WOMAC and its components, an overall assessment of the patient's health, a worse KOOS index, and ultrasound findings (reduced cartilage tissue). HCE patients showed high levels of cholesterol, low-density lipoproteins, triglycerides, STX-II, and COMP (p<0.05). However, after stratification by age, many initial intergroup differences became insignificant, and differences in the WOMAC pain score persisted. CONCLUSION: The results of the study confirmed the high prevalence of HCE in OA patients (59%). Patients with OA and increased total cholesterol have more intense pain in the knee joints.

[SNAI1 and SNAI2 - transcriptional master-regulators of epithelial-mesenchimal transition].

Epithelial-mesenchymal transition is a result of cellular epigenetic reprogramming. During this process differentiated epithelial cells lose specific markers of epithelial phenotype and gradually start displaying qualities of poorly differentiated mesenchymal cells, resistant to apoptosis and capable of local invasion. Despite their obvious importance for biology and medicine, many aspects of epithelial-mesenchymal transition, especially those related to its genetic regulation, remain poorly characterized. In this review we analyze molecular structure and mechanisms of regulation of two closely-related transcription factors SNAI1 and SNAI2, which play an important role in induction and progression of epithelial-mesenchymal transition during both normal development and carcinogenesis. Special attention is paid to the role of SNAI1 and SNAI2 and their active co-reeressors in initiation of epigenetic repression of epithelial differentiation marker genes.

[Disturbance of the radial system of interphase microtubules in the presence of excess serum in cell culture medium].

The role of the orderliness of microtubules in the cell has been studied. For this purpose, a population of Vero cells with chaotically arranged microtubules was used, which was obtained after the addition of excess serum to cell culture medium. An increase in the total area and a slight dispersion of the Golgi apparatus were found; however, the rate of culture growth as a whole remained normal. Thus, the radial arrangement of microtubules is not vital even for cells where it is usually well pronounced.

Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady-state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.

Synthesis and biological evaluation of boronated nucleosides for boron neutron capture therapy (BNCT) of cancer.

Several N-3 substituted carboranyl Thd analogs were synthesized. These agents as well as some non-boronated nucleosides were evaluated in phosphoryl transfer assays with recombinant human TK1 and TK2. For some carboranyl thymidine analogs, TK1 phosphorylation rates approached 38% that of thymidine. Their in vitro cytotoxicty appeared to correlate with the TK1 levels in the tested cells. In some cases increased uptake in tumor cell nuclei compared with the surrounding cytoplasm was detected in vitro.

[Secondary structure of the M2 protein of influenza type A virus and its role in forming resistance to rimantadine and deitiforin]. / Vtorichnaia struktura belka M2 virusa grippa tipa A i ego rol' v formirovanii rezistentnosti k remantadinu i deitiforinu.

Studies of the molecular aspects of resistance of influenza virus A to drugs (rimantadine, deytiforine, amantadine) allow a purposeful design of new compounds with a broad spectrum of antiviral activity and evoking no resistance. In this work the nucleotide sequence of rimantadine- and deytiforine-resistant influenza A strain Leningrad/156/83 (H3N2) was compared with that of A/Victoria/35/72. The influence of aminoacid substitutions in the M2 protein on its secondary structure in the membrane and its role in resistance development was shown.

[Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha]. / Okislenie 17alpha,20beta- i 17alpha,20alpha-digidroksipregn-4-en-3-onov--pobochnykh produktov biotransformatsii progesterona rekombinantnymi mikroorganizmami, ékspressiruiushchimi tsitokhorm P45017alpha.

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Syndrome of alcoholic embryopathy]. / Sindrom alkogol'noi émbriopatii.

The paper discusses 8 cases with the syndrome of alcoholic embryopathy (5 boys and 3 girls aged from 8 to 14 years). All children examined had specific craniofacial abnormalities, oligophrenia, small height and weight at birth, microcephaly, combined in some cases with different defects of development (defect of interatrial septum, cleft palate, spina bifida). Two cases of alcoholic embryopathy are given in detail. The significance of different factors in the pathogenesis of this syndrome is discussed.

[Effect of steroid biosynthesis modificators on the progesterone biotransformation by a recombinant yeasts expressing cytochrome P450c17].

Using the recombinant microorganisms S. cerevisiae GRF18 YEp5117alpha, expressing cytochrome P450c17 from bovine adrenal cortex, we investigated the influence of the various modificators of steroids biosynthesis on the relationship between the 17alpha-hydroxylation of progesterone and 20alpha-reduction. Dexamethasone and metirapon had no effect on the reaction of progesterone 17alpha-hydroxylation and on the reaction of 17alpha-hydroxyprogesterone 20alpha-reduction. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450c17 or its heme group under the conditions of the biotransformation of progesterone by recombinant yeasts. Ketokonazol, mifepriston and danazol acted as low-affinity competitive inhibitors, but the 20-dihydro derivatives of progesterone were mixed type inhibitors for the cytochrome P450c17. All modifiers that we used did not influence the functional properties of the yeast analog of 20alpha-hydroxysteroid dehydrogenase. According to the influence on the catalytic parameters of the cytochrome P450c17, the modifiers used can be arranged in the following order: 20beta-dihydroprogesterone (maximum effect) > mifepriston = ketokonazol > 20alpha-dihydroprogesteron > danazol > dexamethasone, metirapon (without effect).

The effects of high salt concentrations on the regulation of the substrate specificity of human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the pET 9d vector system with a histidine tag-sequence and a thrombin cleavage site fused to the N-terminus of the dCK coding sequence. The His-tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2-0.4 M NaCl during the dCK reaction caused a stimulation of 2'-deoxycytidine (dCyd), and the antileukemic analog 2-chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2'-deoxyguanosine (dGuo) phosphorylation, both with His-tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis-Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.

Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017alpha.

The cDNA encoding cytochrome P-45017alpha from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.