• Development and Reproduction
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• v.20 no.2
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.8
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• pp.1201-1207
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• 2007

This study compared the recovery rate of intrajugular-administered allantoin in the urine and saliva between swamp buffaloes and zebu cattle to examine whether it could explain the lower excretion rate of urinary purine derivatives (PD) in the buffaloes. Three male swamp buffalo yearlings, with an average body weight of $349{\pm}40.35$ kg, and three Thai native cattle ($154{\pm}3.26$ kg) of similar age and sex were used in the study. Animals were kept in individual pens and fed at a maintenance energy level with a diet containing 65% monk bean husk (Vigna radiata) as roughage and 35% concentrates. Allantoin solution was infused into the jugular vein in four incremental rates equivalent to 0, 5, 10 and 15 mmol/d and urine was collected daily in acidified form. Daily PD excretion was linearly correlated with intrajugular allantoin infusion in both species. The relationship between daily urinary PD excretion (Y, mmol/d) and intrajugular allantoin infused (X, mmol/d) was $Y=0.75{\pm}0.318X+22.45{\pm}2.98$ ($r^2$ = 0.36, n = 12, MSE = 38.02, CV = 21.9, p<0.01) for swamp buffaloes and $Y=0.96{\pm}0.10X+15.93{\pm}0.92$ ($r^2$ = 0.91, n = 12, MSE = 3.60, CV = 8.27, p<0.01) for zebu cattle. The salivary PD concentration was not correlated with intrajugular allantoin infusion in both species, with values for buffaloes numerically lower than those for cattle. The present study reconfirmed previous studies that buffaloes have a lower plasma PD excretion rate via the renal route and a significant proportion (22%) of the plasma PD loss is via the saliva. However, results of our present and previous studies suggest that differences in purine base (PB) metabolism between buffaloes and zebu cattle occur before the purine compounds reach the plasma pool.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.148-156
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• 1993

Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were $ 2.0 * 10^{-4}$ M for inosine, $2.0 *10^{-3}$ M for deoxyinosine, $ 2.0 * 10^{-5}$ M for guanosine and $2.0 10 ^{-5}$ M for deoxyguanosine. According to the ratio of relative $K_{cat}$Km, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in Lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be $ 6 * 10^{-4}$ M for formycin B as the competitive inhibitor of inosine, $ 9 * 10^{-6}$ M for guanine as the competitive inhibitor of guanosine, $2 * 10^{-4}$ M for hypoxanthine as the non competitive inhibitor of guanosine and $4.5 * 10 ^{-4}$ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values o $f^ 2.0 * 10 {-5}$ M, $2.6 * 10^{-5}$ M and $8.5 * 10 ^{-4}$ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of $1.8 * 10^{-5}$ M and $ 3.0 * 10^{-5}$ M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.e.

• Applied Chemistry for Engineering
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• v.17 no.3
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• pp.317-320
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• 2006

The optimum mobile phase condition for analysis of the six purine derivatives (caffeine, guanine, hypoxanthine, purine, theobromine, and theophylline) were determined by a HCI program. Reversed-phase HPLC system was used with the binary mobile phase, water and methanol. Three retention models (Snyder, Langmuir, and Binary polynomial) were considered to predict the retention factors. The elution profiles were calculated by the plate theory based on the binary polynomial retention model. From the final calculated results, the binary polynomial retention model showed the best agreements between the calculated and experimental data. In the isocratic mode, the optimum mobile phase composition of water/methanol is 93/7(v/v). However, we used step-gradient mode to decrease the run-time ($1^{st}$ mobile phase : water/methanol = 93/7 (v/v), gradient time : 5 min, $2^{nd}$ mobile phase : water/methanol = 75/25 (v/v)). The experimental and simulated profiles of above the two conditions show a good agreement.

• Bulletin of the Korean Chemical Society
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• v.35 no.9
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• pp.2743-2748
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• 2014

Novel 4'-trifluoromethyl-5'-norcarbocyclic purine phosphonic acid analogs were efficiently synthesized from commercially available 1,3-dihydroxy cyclopentane (5). Trifluoromethylation was successfully performed by using the Ruppert-Prakash reaction. The purine nucleosidic bases were efficiently coupled by using the Mitsunobu reaction. The synthesized adenosine phosphonic acids analogs 13 and 16 were screened for antiviral activity against human immunodeficiency virus-1 (HIV-1). Adenine derivative 13 exhibited significant anti-HIV-1 activity.

• Bulletin of the Korean Chemical Society
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• v.32 no.8
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• pp.2689-2694
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• 2011

Novel 5'-norcarbocyclic adenine and guanine phosphonic acid analogues with 6'-electronegative moiety such as unsaturated C-C bond were designed and synthesized from commercially available 2-methylene-propane-1,3-diol (4). Regioselective Mitsunobu reaction successfully proceeded from an allylic functional group (${\pm}$)-12b at low reaction temperature in polar cosolvent (DMF/1,4-dioxane) to give purine phosphonate analogues (${\pm}$)-13 and (${\pm}$)-20. The purine nucleoside phosphonate and phosphonic acid analogues were subjected to antiviral screening against HIV-1. Guanine analogue (${\pm}$)-23 shows significant anti-HIV activity in PBM cell lines ($EC_{50}=8.1\;{\mu}M$).

• Asian-Australasian Journal of Animal Sciences
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• v.4 no.2
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• pp.161-164
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• 1991

A rapid and accurate method is described for the determination of nucleo-bases in rumen micro-organisms. A procedure to satisfactorily hydrolyse the micro-organisms involving reaction with a mixture of readily volatile organic acids (acetic and formic acids) under high pressure, is proposed, and optimal conditions for an analytical procedure with reversed phase HPLC is described. The following nucleobases contents (mmol/kg DM) of rumen micro-organisms were found: Adenine (Ade), 82.62; Guanine (Gua), 61.34; Cytosine (Cyt), 84.61; Thymine (Thy), 35.74; Uracil (Ura), 68.62; Hypoxanthine (Hxn), 13.06; Xanthine (Xn), 8.35. Total purine-N content (g/kg N) of rumen micro-organisms were 99.60. The nucleic acid N content (g/kg N) of microbial isolates were: RNA-N, 109.9; DNA-N, 50.9.

• Journal of Microbiology
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• v.34 no.3
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• pp.270-273
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• 1996

Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30 $^{\circ}C$ the bimolecular rate constant for the modification was 0.015 $min^{-1}mM^{-1}$ in 50 mM $NaHCO_3$ buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope value of 0.9, indicating that modification of one arginine residue was needed to inactivate the enzyme. Preincubation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

• Journal of Microbiology
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• v.34 no.1
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• pp.82-89
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• 1996

Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 * 10$^{5}$ delton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI$_{2}$ or CaCI$_{2}$, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 * 10$^{-3}$ M, 3.0 * 10$^{-3}$ M, 5.0 * 10$^{-4}$ M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

• Korean Journal of Microbiology
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• v.29 no.3
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• pp.172-178
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• 1991

Intracellular purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae was partially purified using ammonium sulfate fractionation, heat treatment, a DEAE-Sephadex A-50 anion exchange chromatography and a Sephadex G-100 gel filtration chromatography. The enzyme was purified 20 fold with 3% recovery. The stability of enzyme was kept by addition of inosine and dithiothreitol. The pH optimum was found to be from 6.3 to 7.3 PNP was sensitive to 10mM of $Hg^{2+}$ , $Cu^{2+}$ , and was inactivated completely by 2 mM of p-chloromercuribenzoate and 5,5'-dithiobis (2-nitrobenzoate). The enzyme was capable of catalyzing the phosphorolysis of inosine, deoxyinosine, guanosine, deoxyguanosine and adenosine.