Effects of structure on radical-scavenging abilities and antioxidative activities of tea polyphenols: NMR analytical approach using 1,1-diphenyl-2-picrylhydrazyl radicals

Tea (Camellia sinensis) leaves contain various antioxidants such as ascorbic acid (1) and polyphenols. This study tries to clarify the molecular mechanisms underlying the antioxidative and radical-scavenging activities of these antioxidants, and the reactivities of each antioxidant have been compared against that of the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, 2) using nuclear magnetic resonance (NMR) analysis. Catechol (3) and (+)-taxifolin (4) were oxidized to o-quinone by 2. However, ethyl protocatechuate (5) and quercetin (6) were not oxidized to o-quinone, even though they possess a catechol structure. The radical-scavenging ability of o-dihydroxyl phenolic compounds with a conjugated olefinic double bond (e.g., 6) was superior to that of compounds without this bond (e.g., 4), whereas the ability of o-dihydroxyl phenolic compounds possessing a conjugated carbonyl bond (5) was inferior to that of compounds lacking this bond (3). Vicinal trihydroxyl phenolic compounds with a conjugated olefinic double bond [e.g., myricetin (7)] had an inferior scavenging ability as compared with compounds lacking this bond [e.g., pyrogallol (8)], but 7 was a better scavenger than compounds with a conjugated carbonyl double bond [e.g., ethyl gallate (9)]. In addition, vicinal trihydroxyl phenolic compounds (e.g., 9) were superior to o-dihydroxyl phenolic compounds (e.g., 6). Finally, 1 scavenged radicals more quickly than 8.     

Genetic diversity and spatial distribution of rhizobial bacteria nodulating on black locust (Robinia pseudoacacia) rhizospheres

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree in Japan. The species is widely used for afforestation because of its symbiotic relationship with nitrogen-fixing rhizobia. In this study, genetic diversity of rhizobia isolated from black locust nodules and spatial distribution of their genotypes were examined. From a coastal forest, six black locust saplings including the whole root systems were collected and positions of nodules on the roots were recorded. Bacteria were isolated from each nodule and then genotyped by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. Dendrogram analysis based on PCR-RFLP and sequencing analysis indicated that Mesorhizobium species dominantly colonized black locust roots in this forest, occupying at least 77% of living nodules. Positions and PCR-RFLP genotypes of the nodules revealed that rhizobia isolated from neighbouring nodules tended to have the same genotype in some cases. Statistical analysis has supported this fact. The clustering distribution of nodules in the same RFLP groups should be considered to contribute to the large genetic diversity of rhizobia.     

Molecular identification of red tide-causing microalga Heterosigma akashiwo strains based on their chloroplast DNA sequences

ABSTRACT:   In order to establish genetic signatures for intraspecific identification, parts of the genes rbcL , rbcS and psbA and their intergenic spacer (IGS) regions were amplified by polymerase chain reaction (PCR) from the chloroplast genomes of Heterosigma akashiwo strains isolated from Japan. A transfer RNA-Leu gene trnL and a hypothetical gene cfxQ , which is related to ribulose bisphosphate carboxylase/oxygenase expression, were found in the upstream region of the rbcL gene and in the rbcS-psbA IGS region, respectively. All the gene-coding regions and the IGS regions between rbcL and rbcS showed the same sequences among the strains tested. In contrast, the rbcL upstream regions and the rbcS-cfxQ IGS regions showed some differences such as nucleotide substitutions, duplications and inversions between NIES-5 and the other strains. Based on these sequence data, five genetic signatures were established and their simple and rapid detection by means of strain-specific PCR primers and PCR-restriction fragment length polymorphism techniques was examined. The results suggested the usefulness of these genetic signatures and techniques for the discrimination of H. akashiwo populations.     

Surface properties of the mushroom Strobilurus ohshimae result in food differentiation by collembolan species

The basidiomycete Strobilurus ohshimae has fruiting bodies covered with prominently projecting hair-like cells called cystidia; it is not consumed by the collembolan Ceratophysella denisana but is consumed by Mitchellania pilosa. To explain this difference, we examined the effects of S. ohshimae cystidia on collembolan survival. In the field, several collembolan species, including C. denisana, were found dead on S. ohshimae, whereas no dead M. pilosa were found on the fungus. Survival of M. pilosa on S. ohshimae was 100%. In the laboratory, cystidium destruction experiments showed that the cystidia of S. ohshimae were capable of killing C. denisana on contact. A cystidium contact time experiment revealed that the cystidia were also capable of killing M. pilosa, although M. pilosa could survive when the contact time was short. In the field, M. pilosa frequently fed on the interior of S. ohshimae. Interior feeding may enable M. pilosa to feed on S. ohshimae by reducing contact time with the cystidia. The deadly surface of S. ohshimae deters the surface feeder C. denisana but not the interior feeder M. pilosa, resulting in food differentiation of these collembolans.     

Biochemical Characterization of Carboxylesterase in the Small Brown Planthopper Laodelphax striatellus (Fallén)

Several forms of carboxylesterase were observed in a malathion-resistant small brown planthopper, Laodelphax striatellus Fallén, by isoelectrofocusing. To study the mechanisms of increased esterase activity, esterases were purified and their biochemical characteristics were investigated in five active esterase isozymes of two resistant strains. These esterases have polymorphic characteristics and their molecular weights ranged from 66 to 70 kDa, due to variations in glycosylation. The pI values of these esterases ranged from 5.3 to 4.7. These esterases were immunologically related and NH₂-terminal amino acids were identical in all isozymes regardless of pI or molecular weight. No differences have been found in kinetic parameters (K_m and V_max) to α-naphthylacetate and specific activity toward α-naphthylacetate and malathion as a substrate in all isozymes. Resistant individuals showed high ali- and malathion carboxylesterase activities and these enhancements were caused by quantitative differences of carboxylesterases with several different pI.     

Detection of Borrelia garinii, Borrelia tanukii and Borrelia sp. closely related to Borrelia valaisiana in Ixodes ticks removed from dogs and cats in Japan

Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.     

Tissue retention and adverse reaction after intravenous injection of hematoporphyrin derivatives in dogs

We evaluated changes in hematology and chemical profile, and the tissue retention of hematoporphyrin derivative (HpD) following the intravenous injection in dogs. HpD at concentrations of 1, 5, 10, and 15 mg/kg was intravenously injected to 16 dogs (n=4 each) and complete blood count (CBC) and blood chemistry were performed on days 1, 3, 5, and 7 after the injection. To examine tissue retention, HpD (5 mg/kg) was administered to 15 dogs and 3 dogs were euthanized on days 1, 2, 7, 14, and 28 after the injection, respectively, to collect the skin, muscle, small intestine, spleen, kidney and liver as tissue samples. There were no significant changes in CBC and blood chemical profile except for an increase in LDH concentrations in dogs given 10 and 15 mg/kg of HpD at day 3. The levels of HpD retention in the tissues were ranked in the following order: liver > kidney > spleen > intestine > muscle > skin.     

Detection of novel gammaherpesviruses in wild animals of South Africa

Herpesviral DNA was detected in 24/261 DNA samples that were extracted from whole blood of 13 wild animal species in South Africa. Herpesviral DNA was shown in 22 impalas (Aepyceros melampus) and 2 springboks (Antidorcas marsupialis). All of DNA sequences detected in impalas were identical, whereas two DNA sequences detected in springboks were different each other. These three sequences showed 44 to 72% homology to the corresponding gene of the Gammaherpesvirinae, indicating that these three viruses should be unrecognized novel gammaherpesviruses. The putative novel herpesviruses were tentatively designated as Aepyceros melampus (impala) herpesvirus 1 (ImHV-1), Antidorcas marsupialis (springbok) herpesviruses 1 (SpHV-1) and 2 (SpHV-2). ImHV-1 seems to be a relatively independent virus. SpHV-1 belongs to a group of ruminant lymphotropic herpesviruses and SpHV-2 is closer to a malignant catarrhal fever virus group. Potential threat of these herpesviruses to domestic animals should be considered.     

A method for extracting carboxy‐myoglobin from beef

The present study was conducted to devise a method for the effective extraction of carboxy‐myoglobin (COMb) from beef without carbon monoxide dissociation. The ratio of COMb to myoglobin was computed at absorptions of wavelengths 541 and 551 nm, which characterize COMb and the isosbestic point between COMb and deoxy‐myoglobin, respectively. The COMb extraction rate was found to vary with temperature, pH and oxygen conditions. The decrease observed in this rate was inversely proportional to the rise in extraction temperature. The COMb extraction rate was also affected by pH, and the stability of COMb in the extract solution was the highest at pH 8.0–9.0. Moreover, the presence of oxygen was found to disturb COMb extraction. According to these results, nearly all COMb could be extracted from carbon‐monoxide‐treated beef under stirring conditions in pH 8.5 deoxidized buffer, at 1°C, and under N₂ flow with the improved extraction method in this study (98.1 ± 2.7%). The decrement of COMb in the extract was accelerated by light, and the COMb was stable for 20 min in the dark, at 1°C. The extraction conditions for COMb described above should allow the accurate evaluation of COMb in meat tissue.