Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC₅₀ values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.     

Atmospheric moisture influences on conidia development in Podosphaera xanthii through host-plant morphological responses

This study investigated the effects of atmospheric moisture on conidia development in cucurbit powdery mildew fungus (Podosphaera xanthii) through host-plant responses. Cucumber (Cucumis sativus L.) seedlings were grown under a high vapour pressure deficit (VPD) (3.8 kPa) or a low VPD (0.4 kPa). When the cotyledons had expanded, spores of P. xanthii were inoculated onto the adaxial surface. Inoculated seedlings for both treatments were then placed in a growth chamber maintained at a VPD of 2.1 kPa. The density of visible P. xanthii colonies on the high-VPD-acclimated cotyledons was 0.46 to 0.85 times that of the low-VPD-acclimated cotyledons 7 days after inoculation. It is likely the post-germination behaviour of P. xanthii such as the infection and consequent hyphal development was affected because spore germination did not differ between the treatments. The percentage of adaxial epidermal leaf cells with haustoria was also lower in the seedlings which had acclimated to a higher VPD. The high-VPD-acclimated cotyledons were thicker and had greater dry mass per area. The water potential of cotyledons did not differ between the treatments, although the stomatal conductance of high-VPD-acclimated cotyledons was lower than that of low-VPD-acclimated cotyledons. From these results, we conclude that the inhibition of P. xanthii conidia development on high-VPD-acclimated cotyledons was mainly caused by changes in leaf morphological properties.     

Major QTLs associated with green stem disorder insensitivity of soybean (Glycine max (L.) Merr.)

Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.     

Resistance levels of cucumber to <Emphasis Type="Italic">Podosphaera xanthii</Emphasis> in a growth chamber are related to haustorial formation and hyphal branching frequency

To clarify relationships between powdery mildew resistance of cucumber and hyphal developmental state of the pathogen, haustorial formation and the hyphal branching frequency were compared among cucumber varieties that differ in resistance levels to powdery mildew pathogen Podosphaera xanthii. Cotyledons of four cultivars were inoculated with P. xanthii. By 2 days after inoculation, secondary haustoria had developed from the first hyphal cell that formed beside the conidium in susceptible cultivars. The fungus on susceptible cultivars also tended to have hyphal branches just after the hyphal cell producing haustoria.     

Studies on the susceptibility to alkaline hydrolysis of inclusion complexes of organophosphorothioate pesticides with β-cyclodextrins

The inhibition of alkaline hydrolysis of organophosphorothioate pesticides such as parathion, parathion-methyl and fenitrothion by the formation of inclusion complexes with β-cyclodextrin and its methylated derivatives was investigated. The inclusion complex/pesticide stability relationship with the series of pesticides and β-cyclodextrins is discussed in connection with the inclusion geometrical parameters, such as those relating to the inclusion orientation and depth of the 4-nitrophenoxy moiety of the included pesticide. These parameters were estimated from the induced circular dichroism of the inclusion complexes by means of the rotational-strength analysis method.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Microhabitat use of wood mice ranging from a reserved belt with evergreen broad-leaved trees to a coniferous plantation

Wood mice Apodemus speciosus and Apodemus argenteus are potentially important seed dispersers and predators of Quercus and Castanopsis in Japan. We investigated the existence of two species of wood mice in warm-temperate forests ranging from a reserved belt of evergreen broad-leaved trees to a coniferous plantation, and analyzed the relationship between wood mouse occurrence and environmental factors to confirm their microhabitat use. We used two-way analysis of variance to analyze differences in the captured number of each wood mouse species in two trapping seasons as well as two stand types to confirm the interaction between the stand type and trapping season. Apodemus speciosus were often captured in the reserved belt, while captures of A. argenteus were independent of season and stand types. It is reasonable to conclude from the results of the trends in occurrence that the two species of wood mice showed different uses of the microhabitat. The result of linear discriminant analysis made it clear that the distance from the reserved belt had much to do with the difference in microhabitat use between the two species in the fruiting season of Quercus and Castanopsis. On average, A. speciosus moved 19.9 m during nonfruiting and 61.3 m during fruiting, while A. argenteus moved 8.1 m during nonfruiting, and 29.0 m during fruiting from analysis of the recapture position. The results indicate that both species of mice move around during the fruiting season more than in the nonfruiting season.     

Extraction of an incisor embedded within the nasal cavity in two guinea pigs

Oral examination of two guinea pigs revealed that the unilateral incisor was absent. On radiographic examination, the incisor was identified within the nasal cavity in both patients. Under anesthesia in both patients, the skin was incised from the nostril to 1.5 cm proximal, and the premaxilla and part of the maxilla were exposed. The bone was removed using a surgical drill, and the incisor was exposed in the nasal cavity. The root was grasped with forceps and carefully extracted as it was degraded and very fragile. Diagnosis was easy using oral and radiographic examination. In guinea pig patients where an incisor is absent on oral examination, this condition should be considered.