Novel electrochemical method to evaluate the antioxidant capacity of infusions and beverages, based on in situ formation of free superoxide radicals.

This work reports a new method to evaluate the antioxidant capacity of infusions and beverages, based on superoxide radicals. Radicals produced by the enzymatic reaction between acetylcholinesterase and hypoxanthine oxidized antioxidant molecules present in commercially available samples or standard solutions, which was monitored by means of cyclic voltammetry using a carbon paste electrode. The Trolox equivalent antioxidant capacity (TEAC) of red wine, coffee and green tea determined using this method were: (1.20 ± 0.06), (0.90 ± 0.02), and (0.65 ± 0.02), respectively. This method suggested TEACred wine > TEACcoffee > TEACgreen tea, which is the same as DPPH, spectrophotometric method. However, the electrochemical one proposed here is rapid and simple.

Effect of corn supplementation on purine derivatives and rumen fermentation in sheep fed PKC and urea-treated rice straw.

This study investigated the effect of different levels of corn supplementation as energy source into palm kernel cake-urea-treated rice straw basal diet on urinary excretion of purine derivatives, nitrogen utilization, rumen fermentation, and rumen microorganism populations. Twenty-seven Dorper lambs were randomly assigned to three treatment groups and kept in individual pens for a 120-day period. The animals were subjected to the dietary treatments as follows: T1: 75.3% PKC + 0% corn, T2: 70.3% PKC + 5% corn, and T3: 65.3% PKC + 10% corn. Hypoxanthine and uric acid excretion level were recorded similarly in lambs supplemented with corn. The microbial N yield and butyrate level was higher in corn-supplemented group, but fecal N excretion, T3 has the lowest level than other groups. Lambs fed T3 had a greater rumen protozoa population while the number of R. flavefaciens was recorded highest in T2. No significant differences were observed for total bacteria, F. succinogenes, R. albus, and methanogen population among all treatment. Based on these results, T3 could be fed to lambs without deleterious effect on the VFA and N balance.

P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

[Studies on HPLC fingerprint of co-hirudo injection].

OBJECTIVE: Using HPLC To determine hypoxanthine in co-hirudo injection for establishing its HPLC fingerprint, and evaluating its internal quality. METHOD: The chromatographic separation was performed on a Kromasil C18 column (4.6 mm x 250 mm,5 microm). A linear gradient elution with A (0.01 mol x L(-1) x KH2PO4) and B (50% methanol) was used, the flow rate was 0.8 mL x min(-1), the detection wavelength was set at 254 nm, and the column temperature was at normal. RESULT: Hypoxanthine was used as the reference substance in the fingerprint of co-hirudo injection, it showed 15 common peaks and theirs similarity threshod was 0.97. CONCLUSION: This method was accurate, repeatable and useful for the quality control of co-hirudo injection.

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP).

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals. The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 micromol min(-1)mg(-1)) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.

Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS.

A high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) analytical method was developed for detection of the nucleosides and nucleobases in two species of Lingzhi, the dried sporophore of Ganoderma lucidum and G. sinense. The method, combining advantages of both DAD and MS, was successfully used to qualitatively identify for six nucleosides namely, adenosine, cytidine, guanosine, inosine, thymidine, uridine and five nucleobases namely, adenine, guanine, hypoxanthine, thymine and uracil in Lingzhi samples. Quantitative analyses showed that uridine was the most abundant nucleoside in these Lingzhi samples and the contents of nine target analytes were found to be different in pileus and stipes of the fruiting bodies and among the different species of G. spp. The established method might apply as an alternative approach for the quality assessment of Lingzhi.

Crystallization and preliminary crystallographic analysis of Bacillus subtilis guanine deaminase.

Guanine deaminase, a key enzyme in nucleotide metabolism, catalyzes the hydrolytic deamination of guanine to xanthine. The first guanine deaminase crystal from Bacillus subtilis was grown in the absence or presence of the inhibitor hypoxanthine in 30% polyethylene glycol 4000, 0.2 M ammonium acetate and 0.1 M sodium citrate pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 84.91, b = 90.90, c = 80.19 angstroms, with one dimer per asymmetric unit. The crystals diffract X-rays to beyond 1.2 angstroms resolution and an initial atomic model has been built based on selenomethionyl multiwavelength anomalous data at 2 angstroms resolution. Unexpectedly, this is the first domain-swapped structure in the cytidine deaminase superfamily.

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings.

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron.

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.

Precursors of the purine backbone augment the inhibitory action of hypoxanthine and dibutyryl cAMP on mouse oocyte maturation.

In this study we have tested the hypothesis that precursors of the purine base backbone--glutamine, glycine, aspartic acid, and formate--promote meiotic arrest when included in medium containing established meiotic inhibitors and that this occurs in glucose-dependent fashion. An initial experiment established that in medium supplemented with 4 mM hypoxanthine and containing no purine precursors, very little meiotic arrest was maintained in cumulus cell-enclosed oocytes after 17-18 hr (90% germinal vesicle breakdown; GVB). Increasing concentrations of glucose reduced the maturation percentage such that only 57% had matured at 0.55 mM. The addition of 2 mM glutamine (Gln) alone reduced the maturation percentage in the absence of glucose (70% GVB), and the further addition of glucose revealed an additive inhibitory effect between these two supplements. Dose response experiments with Gln, glycine (Gly), aspartic acid and formate showed that in medium supplemented with hypoxanthine, very little inhibitory action was observed in the absence of glucose but that upon addition of this hexose, a dramatic decrease in maturation percentage was observed in the Gln and Gly groups. Results of experiments using combinations of precursors showed that when Gln and Gly were added together, greater augmentation of meiotic arrest maintained by either hypoxanthine or dibutyryl cAMP was achieved in the presence of glucose than with either amino acid alone. The addition of purine precursors significantly increased the extent of purine nucleotide production by oocyte-cumulus cell complexes, and this was accentuated by glucose. It is concluded that the presence of purine precursors can augment the meiosis-arresting action of established meiotic inhibitors in glucose-dependent fashion, and that this is due, at least in part, to their incorporation into purine nucleotides via the de novo synthetic pathway.

[Chemical constituents of rhizoma Pinelliae pedatisecta].

Five compounds were isolated from the alkaloid extract of Rhizoma Pinelliae Pedatisecta and their structures were determined as pedatisectine F(I), hypoxanthine (II), erythritol (III), uridine (IV) and pedatisectine G (V), of which I and V are new compounds, while II, III and IV were found in this plant for the first time.

Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates.

Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence. Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic parameters for the 14-mer duplexes were also determined. DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate. Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam methyltransferase is rather tolerant to different substitutions. The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995). The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.