The influence of N-stearoylethanolamine (NSE) on the content of lipid peroxidation products, activity of antioxidant enzymes and the nitric oxide level in the liver and blood plasma of rats with insulin-resistance (IR) state was investigated. IR state was induced in rats by prolonged high-fat diet (58% of energy derived from fat) for 6 months combined with one injection of streptozotocin (15 mg/kg of body weight). The existence of IR state was estimated by results of glucoso-tolerance test and blood plasma insulin content. The level of lipid peroxides products was shown to be higher in the liver of insulin resistant animals as a result of reduced superoxide dismutase and catalase activity, however, glutathione peroxidase activity was increased. The increase of nitric-oxide content in the liver and blood plasma of high-fat diet rats compared with healthy control animals was also observed. The administration of the NSE suspension per os in a dose of 50 mg/kg during 2 weeks to the rats with induced insulin-resistance state contributed to the increase of superoxide dismutase, catalase and glutathione peroxidase activity. In consequence of antioxidant enzymes activation the intensity of POL process was decreased. The NSE administration caused normalization of nitric oxide level, restoring pro-/antioxidant balance in the liver and blood plasma of rats with IR state. In conclusion, the NSE administration to the rats with insulin-resistance state restored pro-/antioxidant balance and enhanced the content of nitric oxide, therefore, improving insulin sensitivity.
A catalytic fragment of soluble NTPase has been isolated from bovine kidneys.The 236-fold purification was carried out to obtain the preparation with a specific activity of 37.7 U/mg of protein. The purification scheme included the enzyme extraction followed by four column chromatography steps. The catalytic fragment was activated with divalent metal ions, had a pH optimum of 7.0, and possessed specificity for ITP, GTP, UTP and XTP. The apparent K(m) for Mg-ITP, Mg-GTP and Mg-UTP complexes were calculated from Hanes plots to be 1.70 mM, 0.93 mM and 0.48 mM, respectively. As estimated by gel filtration and SDS-PAAGE, the catalytic fragment has Mw 54.7 kDa being composed of two identical polypeptide chains. Our results suppose soluble NTPase from bovine kidney to consist of regulatory and catalytic structural units.
Oral administration of antituberculosis drugs to rats for 60 days in doses that are equivalent to clinical ones, causes changes in mRNA levels expression of liver cytochrome P-450 isoforms CYP3A2, CYP2C23, CYP2E1 and pro- and antioxidant state. Experimental composition "Metovitan" given with anti-TB drugs provided a correction of these abnormalities, that is evidenced by modulation of the level of CYP3A2, CYP2C23, CYP2E1 gene expression and antioxidant activity, inhibition of lipid peroxidation. "Metovitan" normalizes the enzymatic activity and content of total billirubin in the blood serum, shows high hepatoprotective properties, exceeding the efficiency of methionine.
In experiment on laboratory rats the models of acute and chronic pancreatitis were developed to study the changes of lipoperoxidation-antioxidant protection system depending on morphological changes of the pancreas. The acute and chronic pancreatitis is accompanied with intensification of lipoperoxidation and gradual inhibition of antioxidant system due to development of subsequent chronization of the pathological process.
It is known that the addition of methanol to the culture medium stimulates the photosynthetic productivity of some species of microalgae, but its influence on the biochemical composition of the biomass has not been investigated. The aim of the present work is to determine the effect of methanol (50 mM) on the content of free amino acids, soluble proteins and reduced nicotinamide coenzyme NAD(P)H in C. reinhartdii cells. It is shown that in case of illumination of C. reinhardtii methanol raises intracellular content of NAD(P)H four times more efficiently than in the darkness. Total content of free amino acids is increased and their ratio is changed. The concentration of glutamic acid, glutamine, alanine, serine and tyrosine also increases. The concentration of valine and methionine is reduced. Growth of culture with methanol is followed by an increase in the content of intracellular protein by 30% after 20 hours of cultivation. The obtained data indicate that methanol stimulates growth of C. reinhardtii, not only as a result of additional carbon utilization, but also due to improved nitrogen assimilation and the impact on the energy metabolism of cells.
No liver and colon alterations in rats, caused by cytostatic compounds 5-amino-4-(1,3-benzothyazol-2-yl)-1-(3-methoxyphenyl)-1,2-dihydro-3H-pyrrol-3-one (D1) and 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2,5-dione (MI-1) when administered over a long time were found, as evidenced by the histopathological data and the data of activity of transaminases, alkaline phosphatase and lactate dehydrogenase in the blood serum. D1 and MI-1 in vivo decrease the total area of DMH-induced colon tumors in rats by 46-60%. Furthermore, D1 and MI-1 partially protect the liver and colon mucosa from toxic effects caused by 1,2-dimethylhydrazine (DMH) reducing DNA oxidative modifications, as evidenced by urine 8-hydroxydeoxyguanosine level. The effects of both compounds are similar, but MI-1 is less toxic for the liver and colon of intact animals possessing more pronounced antitumor activity and protective properties in the setting of chemically induced carcinogenesis.
The aim of the paper was to describe the biochemical effects of Paclitaxel (Ptx), gamma-irradiation (IR) and their combination in undifferentiated thyroid cancer cells (ATC). IR activated common DNA damage-induced signaling and manifested certain mitogenic effect by inactivation of retinoblastoma protein (pRb). There was clear antagonism between Ptx and IR relative to cell cycle regulators--tumor suppressor p53, pRb, CHK2 and c-Abl as well as proapoptotic Bax expression, but combined action of both agents enhanced caspase-3 and, especially, caspase-8 activation. The Ptx at low (1-25 nM) concentrations caused noticeable radioprotective effect. Thus, in ATC cells the ionizing radiation and Ptx exhibited competitive effects upon phosphorylation of cell cycle controllers: p53, pRb, CHK2, cAbl and expression of Bax. At the same time, the combined effect of radiation and Ptx enhanced antiapoptotic Bcl-2 phosphorylation, caspases activation and survivin expression. The net effect of these events during the first 48-72 h of cells incubation can be considered as antiapoptotic--Ptx attenuated cytotoxic effect of IR.
The presented research involves the integral assessment of biochemistry indexes of natural populations of voles under conditions of environmental pollution by heavy metals. The raised content of mobile forms of Pb, Cd, Cr, Ni and Co in soils was revealed for a distance of 500 m to the south-west of Tripillya Thermal Power Plant (TPP) (Kyiv region, Ukraine). It considerably (up to 3-5 times) exceeds the levels in the territory of Kaniv Nature Reserve (Cherkassy region, Ukraine). The territory of National Nature Park "Holosiivsky" (Kyiv, Ukraine) is characterized by rather increased content of active form of investigated heavy metals, especially Pb. The increase of the concentration of diene conjugates (up to 7-10 times) and thiobarbituric acid (TBA) active compounds (up to 2-3 times) in the liver of bank vole (Myodes glareolus) polluted by heavy metals has been found. The insignificant increase of the content of Schiff bases in liver homogenate of voles in the region of impact of the Tripillya TPP (2 times in spring and summer, 3 times - in autumn) was detected. Seasonal dynamics of the maintenance of lipid peroxidation products has been revealed. The registered changes of biochemical indicators evidence for availability of biochemical stress in the bank vole organism in the region of influence of the Tripillya TPP.
The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 +/- 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation me- dium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.
In an experimental model of acute pancreatitis (AP) in rats no alteration in leukocyte's viability was found by flow cytometry as compared to control. After 1 day of AP production of reactive oxygen forms in granulocytes was increased more than 5 times, but after 3 days their level was decreased. Alterations of pro/antioxidant status and specific changes in the fatty acid composition in the pancreas were established. With the development of AP, the processes of lipids peroxidation were intensified while antioxidant system was altered, that was evidenced by inflammation in the pancreas. In these conditions, the increase of phospholipase A2 activity was accompanied by significant changes of fatty acid composition of the total lipids in the pancreas. This increased relative total content of saturated fatty acids, in particular myristic, palmitic and stearic acid increased, while the total content of polyunsaturated essential fatty acids omega-3 (linolenic, eicosapentaenoic, dokozapentayenoic, docosahexaenoic) decreased. The preparation containing omega-3 polyunsaturated fatty acids partially normalized the lipid and fatty acids composition as well as prooxidant-antioxidant system.
At the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory it was established for the first time, that the Löwdin's G*.C* DNA base pair formed by the mutagenic tautomers can acquire, as the A-T Watson-Crick DNA base pair, four biologically important configurations, namely: Watson-Crick, reverse Watson-Crick, Hoogsteen and reverse Hoogsteen. This fact demonstrates rather unexpected role of the tautomerisation of the one of the Watson-Crick DNA base pairs, in particular, via double proton transfer: exactly the G.C-->G*.C* tautomerisation allows to overcome steric hindrances for the implementation of the above mentioned configurations. Geometric, electron-topological and energetic properties of the H-bonds that stabilise the studied pairs, as well as the energetic characteristics of the latters are presented.
The dependence of respiration rate of rat permeabilized acinar pancreacytes on oxidative substrates concentration was studied at various [Ca2+] - 10-8-10-6 M. Pancreacytes were permeabilized with 50 microg of digitonin per 1 million cells. Respiration rate was measured polarographically using the Clark electrode at oxidation of succinate or pyruvate either glutamate in the presence of malate. Parameters of Michaelis-Menten equation were calculated by the method of Cornish-Bowden or using Idi-Hofsti coordinates and parameters of Hill equation - using coordinates {v; v/[S]h}. In the studied range of [Ca2+] the kinetic dependence of respiration at pyruvate oxidation is described by the Michaelis-Menten equation, and at oxidation of succinate or glutamate - by Hill equation with h = 1.11-1.43 and 0.50-0.85, respectively. The apparent constant of respiration half-activation (K0.5) did not significantly change in the studied range of [Ca2+] while at 10-7 M Ca2+ it was 0.90 +/- 0.06 mM for succinate, 0.096 +/- 0.007 mM for pyruvate and 0.34 +/- 0.03 mM for glutamate. Maximum respiration rate Vax at pyruvate oxidation increased from 0.077 +/- 0.002 to 0.119 +/- 0.002 and 0.140 +/- 0.002 nmol O2/(s.million cells) due to the increase of [Ca2+] from 10-7 to 5x10-7 or 10-6 M, respectively. At oxidation of succinate or glutamate Ca2+ did not significantly affect Vmax Thus, the increase of [Ca2+] stimulates respiration of mitochondria in situ of acinar pancreacytes at oxidation of exogenous pyruvate (obviously due to pyruvate dehydrogenase activation), but not at succinate or glutamate oxidation.
The effect of mitochondrial ATP-dependent K(+)-channel (K+(ATP)-channel) opener diazoxide (DZ) on the oxygen consumption, functional state and the opening of cyclosporine-sensitive pore in the rat liver mitochondria has been studied. It has been established that K+(ATP)-channel activation results in the increase of the oxygen consumption rate (V4(s)) and the uncoupling due to the acceleration of K(+)-cycling, the decrease in state 3 respiration rate (V3) and the respiratory control ratio (RCR). Under K+(ATP)-channel activation an inhibition of oxidative phosphorylation takes place which reduces the rate of ATP synthesis and hydrolysis as well as ATP production and consequently results in the seeming increase of P/O ratio. It has been shown that the increase in ATP-dependent K(+)-uptake accompanied by the opening of mitochondrial permeability transition pore (MPTP) leads to dramatic uncoupling of the respiratory chain due to simultaneous activation of K(+)- and Ca(2+)-cycling supported by MPTP and Ca(2+)-uniporter as well as K(+)-channels and K+/H(+)-exchange. K+(ATP)-channel activation leads to the partial inhibition of MPTP, but insufficient for the restoration of mitochondrial functions. Elimination of Ca(2+)-cycling after MPTP opening is necessary to return mitochondrial functions back to the control level which shows that MPTP could serve as the mechanism of reversible modulation of bioenergetic effects of K+(ATP)-channel activation.
It was established in experiments on the rats in the acute period of modeling pituitrin-isadrin myocardial infarction the formation of nitrogen monoxide decreases along with its accelerated transformation into peroxynitrite. It was evidenced by more than double inhibition of NO synthase activity in the myocardium and by decreasing the amount of nitrates on the background of the increasing level of peroxynitrites' marker--nitrotyrosine by 246.6% at an average. Experimental therapy of rats by ademol which is a derivate of adamantan (1-adamantiloxy-3-morpholino-2 propanol hydrochloride) better than by corvitin normalizes the processes of synthesis of nitric oxide. At the same time ademol probably exceeded the reference drug in ability to increase NO synthase activity and amount of nitrate, and promoted a decrease of the level of nitrotyrosine in the myocardium on the average by 36.3; 50.6 and 12.7%, respectively. Corrective influence of ademol on indicators of metabolism in NO system under the conditions of acute cardiac ischemia indicates to promicing development of domestic cardioprotector on its base.
In blood plasma of 8 healthy volunteers with resuspended lymphocytes incubated with 5-fluorouracil (5-FU) the drug distribution between cells and liquid was assessed by means of HPLC. Rapid accumulation of 5-FU in lymphocytes was proved (the drug concentration on the 3-rd minute is 2.5-fold higher than in plasma) as well as the absence of temporal changes of 5-FU content both in lymphocytes and blood plasma during 30 minutes of experiment.
The role of inositol-1,4,5-trisphosphate of (IP3)-sensitive Ca2+ channels in Ca2+ homeostasis maintenance under activation of M-cholinergic receptors and P2Y receptors in the secretory cells of the rat lacrimal gland was investigated. The study was carried out on intact and permeabilized secretory cells of exorbital lacrimal glands of rats. The cells were isolated using the modified Herzog, Sides, Miller method (1976) and permeabilized with digitonin (50 mg per 0.5 million cells). The functioning of the Ca(2+)-transport systems was estimated by changes of Ca2+ content in the studied cells, which was determined by the spectrophotometric method using arsenazo III. It was shown that IP3-sensitive Ca2+ channels (IP3Rs) of investigated cells are directly inhibited by 2-APB (10 microM/l). On the other hand, the channels are activated by IP3, cholinomimetic (carbacholine) and purine receptor agonist (ATP). When both M-cholinergic receptors and P2Y receptors were activated Ca2+ was released from the same IP3-sensitive store because the effects of ATP and carbacholine at high concentrations (1mM/l and 10 microM/l, respectively) on the Ca2+ content were non-additive. The presence of the store-operated Ca(2+)-channels in secretory cells of the lacrimal gland is confirmed by the observed increase of cellular Ca2+ content as a result of Ca2+ mobilization from the store by carbacholine or thapsigargin and following restoration of Ca2+ concentration in the extracellular solution.
The use of glyphosate as a herbicide in agriculture can lead to the presence of its residues and metabolites (aminomethylphosphonic acid) in food for human consumption and pose a threat to health. The effect of these herbicides on the fish organism at the biochemical level has been insufficiently studied. We studied changes in the content of adenine nucleotides, enzyme activity, quantitative indexes of energy metabolism substrates in carp under the action of glyphosate. It has been found that proteins are the major energy substrate under the influence of glyphosate in the liver, brain, white muscle of carp yearlings. Glyphosphate decreases energy metabolism in the brain of carp and increases it in the white muscles. The growth of activity of catabolic enzymes in the liver under the influence of glyphosate can be attributed to the adaptive remodelling of metabolic pathways for homeostasis and enantiostasis in response to herbicides.
Using the methods of non-empirical quantum chemistry at the MP2/6-311++G(2df,pd)// B3LYP/6-311++G(d,p) level of theory it was established for the first time, that Hoogsteen, reverse Hoogsteen, Watson-Crick and reverse Watson-Crick configurations of the A.T and G.C DNA base pairs are isoelectronic and isomorphic structures with similar dynamic properties. Based on these results, non-ionisation mechanism of the Hoogsteen <"breathing" of the G*.C* DNA base pair, namely transformation of the tautomerised (Lowdin's) G-C base pair with Watson-Crick geometry into the Hoogsteen electroneutral G*.C* H base pair stabilized by the three O6H...N4, N3H...N7 and C8H...02 H-bonds, was postulated. It is suggested that such scenario activates only in those cases, when DNA is not located in aqueous solution, but works together with proteins and cytosine protonation at the N3 atom is precluded.
Plasma membrane Ca2+,Mg2+-ATPase is an important element of general myometrium tonus control mechanism, which also makes a contribution to muscle tension relaxation after its contraction. Expiriments were done on the myometrial cell plasma membrane suspension, which was treated with 0.1% digitonin solution. The authors have investigated the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(trifluor) methyl(phenylsulphonylimino)-methylamino-25,26,27,28-tetra propoxi-calix[4]arene) on the Ca2+,Mg2+-ATPase activity (the magnitude of 10.5 was 20.2 +/- 0.5 mkM). The inhibitory action of calix[4]arene C-90 on the activity of Ca2+, Mg2+-ATPase is explained as cooperative action of four trifluormethyl(phenylsulfonylimino)methylamino groups that are spatially oriented on the calix[4]-arene base rather than with the action of tetra-phenol macrocycle or separate pharmacophore sulphonilamidin groups. Considering established kinetic pattern of calix[4]arene C-90 inhibitory action on the plasma membrane Ca2+,Mg2+-ATPase activity, stationary kinetical model of basal calcium concentration control in unexcited uterus myocytes was developed. It is assumed that obtained results may be promising for creation of new generation ("supramolecular") pharmacological agent - uterus basal tonus stimulator - on the base of calix[4] arene C-90.
Glucocorticoid therapy is accompanied by development of processes typical of steroid osteoporosis. Indirect effects of glucocorticoids on the bone tissue are due to changes in mineral metabolism, which is regulated by vitamin D3. In this connection, we studied the influence of prednisolone on cholecalciferol metabolism. The study has shown that prednisolone action causes impairment of cholecalciferol metabolism in hepatocytes due to inhibiting vitamin D3 25-hydroxylase activity. Microsomal (CYP2R1) and mitochondrial (CYP27A1) isoenzymes of vitamin D3 25-hydroxylase were found to function at different concentrations of the substrate. The relative protein contents of the isoenzymes greatly differed in the liver with the prevalence of CYP27A1 over CYP2R1. Prednisolone administration resulted in the lowering of both mitochondrial and microsomal isoenzymes of vitamin D3 25-hydroxylase. The inhibition of vitamin D3 25-hydroxylating system in hepatocytes contributed to a significant reduction in blood serum 25OHD3.
