Free radical scavenging reactions and antioxidant activities of silybin: Mechanistic aspects and pulse radiolytic studies

Silybin (extracted from Silybum marianum) is the major active constituent of silymarin which possesses a wide range of medicinal properties. These properties may be, in part, due to the potent scavenging capacity of oxidizing free radicals. In this context, scavenging radicals (hydroxyl, azide, dibromide anion radicals, nitrite, carbonate, etc.) of silybin have been studied to understand the mechanistic aspects of its action against free radicals. The transients produced in these reactions have been assigned and the rate constants have been measured by pulse radiolysis techniques. Reduction potential determined both by cyclic voltammetry gave a value 0.62±0.02 V vs NHE at pH 9. Quantum chemical calculations have been performed to further confirm the different activities of individual hydroxyl groups with the difference of heat of formation. Moreover, silybin also protected plasmid pUC18 DNA from soft X-ray radiation which induced strand breaks. These results are expected to be helpful for a better understanding of the anti-oxidative properties of silybin.     

Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.     

Antibody responses of Macaca fascicularis against a new inactivated polio vaccine derived from Sabin strains (sIPV) in DTaP-sIPV vaccine

Antibody responses of Macaca fascicularis against a new tetravalent vaccine composed of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, and inactivated poliovirus derived from Sabin strains (sIPV) was investigated to predict an optimal dose of sIPV in a new tetravalent vaccine (DTaP-sIPV) prior to conducting a dose-defined clinical study. Monkeys were inoculated with DTaP-sIPVs containing three different antigen units of sIPVs: Vaccine A (types 1:2:3 = 3:100:100 DU), Vaccine B (types 1:2:3 = 1.5:50:50 DU), and Vaccine C (types 1:2:3 = 0.75:25:25 DU). There was no difference in the average titers of neutralizing antibody against the attenuated or virulent polioviruses between Vaccines A and B. The average neutralizing antibody titers of Vaccine C tended to be lower than those of Vaccines A and B. The sIPV antigens did not affect the anti-diphtheria or anti-tetanus antibody titers of DTaP-sIPV. Furthermore, the average neutralizing antibody titers of Vaccine A against the attenuated and virulent polioviruses were comparable between M. fascicularis and humans. These results suggest that M. fascicularis may be a useful animal model for predicting the antibody responses to sIPVs in humans, and that it may be likely to reduce the amount of sIPVs contained in DTaP-sIPVs, even for humans.     

Seroepidemiology of herpes simplex virus in Yogyakarta, Indonesia

Sera from 487 individuals in Yogyakarta, Indonesia, were tested for antibodies to herpes simplex virus (HSV) by a passive hemagglutination method. Age-specific incidence rates for antibodies to HSV were calculated. For sera from persons other than prostitutes, in the age group from 10 to 19, the positive rate was 48% but in the age group higher than 20, it was more than 87%. Fifty of 59 pregnant women (85%) were positive. The positive rate and the distribution of antibody levels in prostitutes were higher than in the general population.     

Dipeptide synthesis by L-amino acid ligase from Ralstonia solanacearum

Despite its utility, dipeptides have not been widely used due to the absence of an efficient manufacturing method. Recently, a novel method for effective production of dipeptides using l-amino acid α-ligase (Lal) is presented. Lal, which is only identified in Bacillus subtilis, catalyzes dipeptide synthesis from unprotected amino acids in an ATP-dependent manner. However, not all the dipeptide can be synthesized by Lal from B. subtilis (BsLal) due to its substrate specificity. Here, we attempted to find a novel Lal exhibiting different substrate specificity from BsLal. By in silico screening based on the amino acid sequence of BsLal, RSp1486a an unknown protein from Ralstonia solanacearum was found to show the Lal activity. RSp1486a exhibited different substrate specificity from BsLal, and preferably synthesized hetero-dipeptides where more bulky amino acid was placed at N terminus and less bulky amino acid was placed at C terminus in opposition to those synthesized by BsLal.     

Photoirradiation products of flavin derivatives,and the effects of photooxidation on guanine

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.