Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The development of rating of perceived exertion-based tests of physical working capacity

The purpose of the present study was to use ratings of perceived exertion (RPE) from the Borg (6-20) and OMNI-Leg (0-10) scales to determine the Physical Working Capacity at the Borg and OMNI thresholds (PWC(BORG) and PWC(OMNI)). PWC(BORG) and PWC(OMNI) were compared with other fatigue thresholds determined from the measurement of heart rate (the Physical Working Capacity at the Heart Rate Threshold: PWC(HRT)), and oxygen consumption (the Physical Working Capacity at the Oxygen Consumption Threshold, PWC(VO2)), as well as the ventilatory threshold (VT). Fifteen men and women volunteers (mean age +/- SD = 22 +/- 1 years) performed an incremental test to exhaustion on an electronically braked ergometer for the determination of VO2 peak and VT. The subjects also performed 4 randomly ordered workbouts to exhaustion at different power outputs (ranging from 60 to 206W) for the determination of PWC(BORG), PWC(OMNI), PWC(HRT), and PWC(VO2). The results indicated that there were no significant mean differences among the fatigue thresholds: PWC(BORG) (mean +/- SD = 133 +/- 37W; 67 +/- 8% of VO2 peak), PWC(OMNI) (137 +/- 44W; 68 +/- 9% of VO2 peak), PWC(HRT) (135 +/- 36W; 68 +/- 8% of VO2 peak), PWC(VO2) (145 +/- 41W; 72 +/- 7% of VO2 peak) and VT (131 +/- 45W; 66 +/- 8% of VO2 peak). The results of this study indicated that the mathematical model used to estimate PWC(HRT) and PWC(VO2) can be applied to ratings of perceived exertion to determine PWC(BORG) and PWC(OMNI) during cycle ergometry. Salient features of the PWC(BORG) and PWC(OMNI) tests are that they are simple to administer and require the use of only an RPE scale, a stopwatch, and a cycle ergometer. Furthermore, the power outputs at the PWC(BORG) and PWC(OMNI) may be useful to estimate the VT noninvasively and without the need for expired gas analysis.     

Synthesis of the nucleoside moiety of liposidomycins: elucidation of the pharmacophore of this family of MraY inhibitors

Tunicamycins (TCMs) and liposidomycins (LPMs) are naturally occurring inhibitors of the bacterial translocase (MraY). Based on structure-activity relationship (SAR) studies, a molecular model has been proposed for their inhibitory mechanism. This study points out the importance of the nucleoside moiety of liposidomycins in the inhibition of MraY. A simplified molecule (I) based on the liposidomycin core structure has been synthesised and tested on MraY. The compound displayed a moderate inhibitory activity (IC50 = 50 microM). The validation of the molecular model was then performed by synthesising higher homologues of I, containing an additional stereocentre in the 5' position (XIV and XV). In agreement with the prediction, only the (S) isomer XV showed significant activity against MraY (IC50 = 5 microM).     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

Analysis of reproductive parameters of urban and rural population of Chuvashia

Genetic and demographic characteristics for urban and rural population of the Chuvash Republic (Chuvashes and Russians) were calculated based on 1122 questionnaires. The sibship sizes for Chuvashes were 2.05 (urban) and 2.78 (rural). For Russians these indices were 1.75 (urban) and 2.00 (rural), respectively. Crow's index and its components were I(m) = 0.04; I(f) = 0.18; and I(tot) = 0.22 for urban, and I(m) = 0.07; I(f) = 0.27; and I(tot) = 0.36 for rural Chuvashes, respectively; and I(m) = 0.04; I(f) = 0.30; and I(tot) = 0.36 for urban, and I(m) = 0.03; I(f) = 0.29; and I(tot) = 0.33 for rural Russians, respectively.     

Mechanism of reaction of myeloperoxidase with nitrite

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.     

Compatibility of osmolytes with Gibbs energy of stabilization of proteins

This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25 degrees C (DeltaG(D) degrees ) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385-1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for T(m), the midpoint of the denaturation and DeltaH(m), the enthalpy change of denaturation at T(m). Values of DeltaG(D) degrees were determined with Gibbs-Helmoltz equation using known values of T(m), DeltaH(m) and DeltaC(p), the constant-pressure heat capacity change. It has been observed that T(m) increases with the osmolyte concentration, whereas DeltaG(D) degrees remains unaffected in the presence of the osmolyte. This observation on DeltaG(D) degrees in the presence of osmolytes has been considered in the physiological context.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.     

Synthesis of some carbamates of myo-inositol.

The syntheses are described of the 1-O-carbamoyl (11), 1-O-carbamoyl-2-O-stearoyl (10), 1-O-(acetylcarbamoyl)-2-O-stearoyl (12), 1-O-(heptylcarbamoyl) (13), 2-O-(heptylcarbamoyl) (14) 1,2-di-O-(heptylcarbamoyl) (15), and 1-O-(octadecylcarbamoyl) (16) derivatives of myo-inositol. None of these compounds had significant activity against phospholipase C.     

蹄叶橐吾根的化学成分研究

从蹄叶橐吾（Ligularia fischeri）的根中分离得到13个化合物,通过波谱学等方法鉴定为2-hydrox-platyphyllide（1）,liguhodgsonal（2）,nsujapone（3）,6’-亚油酰基田-胡萝卜苷（4）,Luped（5）,6,7-二羟基香豆素（6）,3-谷甾醇（7）,胡萝卜苷（8）,（25,3S,4R,12E）-N-[2’-（R）-羟基二十二碳酰基]-1,3,4-三羟基-2-氨基-二十碳-12-烯（9）,单硬脂酸甘油酯（10）,α-羟基二十四烷酸（11）,1,3-二棕榈酸甘油酯（12）,二十七烷醇（13）。其中化合物1—6为首次从该种植物中分离得到。     

Analysis of a mathematical model of the effect of inhibitors on the growth of tumors

In this paper, we study a model of tumor growth in the presence of inhibitors. The tumor is assumed to be spherically symmetric and its boundary is an unknown function r=R(t). Within the tumor the concentration of nutrient and the concentration of inhibitor (drug) satisfy a system of reaction-diffusion equations. The important parameters are Lambda(0) (which depends on the tumor's parameters when no inhibitors are present), gamma which depends only on the specific properties of the inhibitor, and beta; which is the (normalized) external concentration of the inhibitor. In this paper, we give precise conditions under which there exist one dormant tumor, two dormant tumors, or none. We then prove that in the first case, the dormant tumor is globally asymptotically stable, and in the second case, if the radii of the dormant tumors are denoted by R(s)(-),R(s)(+) with R(s)(-)infinity)R(t)=R(s)(-), provided the initial radius R(0) is smaller than R(s)(+); if however R(0)R(s)(+) then the initial tumor in general grows unboundedly in time. The above analysis suggests an effective strategy for treatment of tumors.     

Flora of planktonic microalgae of Amursky Bay, Sea of Japan

Long-term studies of the species composition of phytoplankton in Amursky Bay (Sea of Japan) carried out during the period from 1991 to 2006 revealed a total of 357 species of planktonic microalgae from eight divisions: Cyanophyta (8 species), Chrysophyta (8), Bacillariophyta (157), Cryptophyta (5), Dinophyta (143), Raphidophyta (3), Euglenophyta (11), and Chlorophyta (22 species). An annotated checklist of species is presented. In comparison with the late 1960s and early 1970s, the species richness of phytoplankton increased markedly; a greater number of bloom-forming species was recorded.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

Enzymology of repair of etheno-adducts

Etheno(epsilon)-adducts such as 1,N(6)-ethenoadenine (epsilon A), 3,N(4)-ethenocytosine (epsilon C), N(2),3-ethenoguanine (N(2),3-epsilon G), and 1,N(2)-ethenoguanine (1,N(2)-epsilon G) are produced in cellular DNA by two independent pathways: (i) by reaction with oxidised metabolites of vinyl chloride, 2-chloroacetaldehyde and 2-chloroethylene oxide; (ii) by endogenous processes through the interaction of lipid peroxidation (LPO)-derived aldehydes and hydroxyalkenals. They have been found in DNA isolated from human and rodent tissues. However, the levels of adducts were significantly increased by cancer risk factors contributing to lipid peroxidation and oxidative stress.The highly mutagenic and genotoxic properties of epsilon-adducts have been established in vitro by analysing steady-state kinetics of primer extension assays and in vivo by site-specific mutagenesis in mammalian cells. Therefore, the repair processes eliminating exocyclic adducts from DNA should play a crucial role in maintaining the stability of genetic information. The epsilon-adducts are eliminated by the base excision repair (BER) pathway, with DNA glycosylases being the key enzymes of this pathway. They remove epsilon-adducts from DNA by hydrolysing the N-glycosidic bond between the damaged base and deoxyribose, leaving an abasic site in DNA. The ethenobase-DNA glycosylases have been identified and their enzymatic properties described. They are specific for a given epsilon-base although they can also excise different types of modified bases, such as alkylated purines, hypoxanthine and uracil. The fact that ethenoadducts are recognised and excised with high efficiency by various DNA glycosylases in vitro suggests that these enzymes may be responsible for repair of these mutagenic lesions in vivo, and thus constitute important contributors to genetic stability.     

Investigation of the mechanism of nicotine-induced relaxation on the sheep sphincter of Oddi

Possible mechanisms for nicotine-induced relaxation were investigated in the isolated sheep's sphincter of Oddi. Sheep's sphincter of Oddi rings were mounted in tissue bath with modified Krebs-Henseleit solution and aerated with 95% oxygen and 5% carbon dioxide. Tension was measured with isometric force transducers, and muscle relaxation was expressed as percent decrease of precontraction induced by carbachol. Nicotine (1 x 10(-5) to 3 x 10(-3) mol/L) produced concentration-dependent relaxation on sphincter of Oddi precontracted by carbachol (10(-6) mol/L). Nicotine-induced relaxation was 72.8 +/- 4.2% of precontraction with carbachol (10(-6) mol/L) (mean pD2 value, 3.76 +/- 0.05 mol/L). Nicotine-induced relaxation was not affected by N(w)-nitro L-arginine methyl ester (L-NAME) (3 x 10(-5) mol/L), methylene blue (10(-5) mol/L), indomethacin (10(-5) mol/L), hexamethonium (10(-5) mol/L), glibenclamide (10(-5) mol/L), 4-aminopyridine (10(-3) mol/L), tetraethylammonium (3 x 10(-4) mol/L), clotrimazole (10(-6) mol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (10(-6) mol/L), and anthracene-9-carboxylate (9-AC) (10(-6) mol/L), but potentiated by bupivacain (10(-5) mol/L). A calcium-antagonizing effect of nicotine was not observed. The results suggest that nicotine-induced relaxation of the sheep's sphincter of Oddi is not mediated by the release of prostaglandins, nitric oxide (NO), or a related substance; by the activation of potassium channels or chloride channels; or by the stimulation of nicotinic cholinoceptors. Potentiation of the nicotine-induced relaxation by bupivacain indicates that blockade of sodium channels may play a role in this relaxation.     

Mechanism of chaperone-like activity. Suppression of thermal aggregation of betaL-crystallin by alpha-crystallin

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.     

Study of some epidemiological aspects of 253 cases of cryptococcosis.

Some epidemiological characteristics of 253 cases of cryptococcosis (CRY) diagnosed between 1981 and 1993 in the Mu?iz Hospital (MH) of Buenos Aires City, were studied. The incidence of CRY associated with AIDS (CRY+AIDS) in the MH during 1983-1993, could be divided into 3 periods: between 1983 and 1988 1-3 cases a year were diagnosed; during 1989-91, the number of cases dopubled annually and in 1992-93 the annual increment was lower. CRY associated with predisposing causes other than AIDS (CRY+non AIDS) exhibited an annual incidence of 0-3 cases during the whole period studied. CRY was more frequent in males (86%). The difference between sexes was more evident in CRY+AIDS patients (88% males) than CRY+non AIDS ones (65% males). The median age (MA) of the studied population was 28 (range 10-71) years; 27 (10-48) in women and 29 (17-71) in men. CRY+AIDS and CRY+non AIDS patients exhibited a MA of 29 (17-51) and 40 years (10-71), respectively. AIDS was the predisposing factor in 92% of patients; 65% of them were intravenous drug abusers and 22% homosexual males, with a MA of 27 (17-40) and 33 (23-55) years, respectively. Cryptococcus neoformans var. neoformans was isolated from all CRY+AIDS and 79% of CRY+non AIDS patients and the gattii variety (Serotype B) produced 4 (21%) cases of CRY+non AIDS.     

Effectivity of expression of mature forms of mutant human apolipoprotein A-I.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.     

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte-cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100mM) for 24h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO(3)(-)/NO(2)(-)) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose-response inhibitory effect on cumulus expansion (P<0.05). Addition of 1 and 10mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates (P>0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes (P<0.05), and increased NO(3)(-)/NO(2)(-) concentration when compared to controls (P<0.05). To evaluate if this effect was reversible, 10(-5)M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100mM AG that inhibited the nuclear maturation. However, association of 10(-5)M SNP to 100mM AG did not reverse the effects of AG, but increased NO(3)(-)/NO(2)(-)concentration (P<0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1mM AG (83.9+/-6.2%) did not differ from control oocytes (83.6+/-8.2%; P>0.05), but 10mM partially inhibited migration (3.8+/-6.4%) and 100mM totally inhibited migration (P<0.05). SNP (10(-5)M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10mM AG to IVM medium reduced (73.0+/-8.1%, 15.0+/-8.9%; P<0.05) cleavage and blastocyst development, respectively when compared with controls (89.1+/-3.4%, 37.6+/-7.3%, respectively), but did not differ, (P>0.05), from the group treated with 1mM AG (80.9+/-8.4%, 41.5+/-10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.