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.     

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.     

Histopathological Changes in the Pancreas from a Spontaneous Hyperglycemic Cynomolgus Monkey

Morphological and immunohistochemical examinations were carried out on the pancreas of a hyperglycemic 5-year-old male cynomolgus monkey. Body weight gradually decreased from 6 months before termination, accompanying a slight reduction in food consumption and anorexia for the last 2 days. The blood glucose level was markedly elevated when examined at termination. Histopathologically, in the exocrine pancreas, diffuse hyperplasia of centroacinar and intercalated duct cells and diffuse atrophy of acinar cells with sporadic apoptosis were observed, although most centroacinar and intercalated duct cells were proliferating cell nuclear antigen (PCNA)-positive in both the present case and age-matched control animals. In the endocrine pancreas, the islets tended to be hypertrophic, with an increase in insulin-positive cells in comparison with the age-matched control animals. PCNA-positive cells also tended to increase in the islets, although positive cells for phospho-histone H3, a marker for mitotic cells, were not detected in the endocrine and exocrine pancreas. Moreover, neither inflammation nor amyloidosis was noted in the islets. In conclusion, the present case probably suffered from early-stage type 2 diabetes mellitus, and it provides fundamental information concerning pancreatic histopathology under insulin-related derangement in monkeys.     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

Effects of some indole-related compounds on the infection behavior of Magnaporthe grisea

Based on their effect on the infection behavior of Magnaporthe grisea, indole-related compounds were classified into three groups. The first group, including tryptophan, indole-3-butyric acid, indole-3-pyruvic acid, and indole-3-acetamide, did not inhibit infection behavior such as spore germination, appressorium formation, or infection hypha formation in M. grisea. The second, including indole acetic acid, indole-3-acetonitrile, oxindole, and tryptamine inhibited all stages of infection behaviors in a dose-dependent manner. The third, including gramine and indole, did not inhibit spore germination or appressorium formation, whereas it did inhibit infection hypha formation in a dose-dependent manner. These results suggest that endogenous or exogenously applied indole-related compounds in the second and third groups may contribute to protection in blast-susceptible plants such as rice and barley.     

Molecular cloning of canine Mcl-1 gene and its expression in tumor cell lines

The canine Mcl-1 gene was cloned and sequenced. Canine Mcl-1 clone was 2694 base pairs in length and encoded 350 amino acids. The predicted amino acid sequence was 87.7%, 77.1% and 75.7% homologous to predicted human, mouse and rat Mcl-1, respectively. RT-PCR analysis revealed that canine Mcl-1 mRNA was expressed in PBMCs (peripheral blood mononuclear cells), bone marrow cells, MDCK (Madin-Darby canine kidney) and GL-1 (canine B cell leukemia) whereas undetectable in CL-1 (canine T cell lymphoma) cell line.     

Spatial kernel models capturing field heterogeneity for accurate estimation of genetic potential

According to Fisher’s principles, an experimental field is typically divided into multiple blocks for local control. Although homogeneity is supposed within a block, this assumption may not be practical for large blocks, such as those including hundreds of plots. In line evaluation trials, which are essential in plant breeding, field heterogeneity must be carefully treated, because it can cause bias in the estimation of genetic potential. To more accurately estimate genotypic values in a large field trial, we developed spatial kernel models incorporating genome-wide markers, which consider continuous heterogeneity within a block and over the field. In the simulation study, the spatial kernel models were robust under various conditions. Although heritability, spatial autocorrelation range, replication number, and missing plots directly affected the estimation accuracy of genotypic values, the spatial kernel models always showed superior performance over the classical block model. We also employed these spatial kernel models for quantitative trait locus mapping. Finally, using field experimental data of bioenergy sorghum lines, we validated the performance of the spatial kernel models. The results suggested that a spatial kernel model is effective for evaluating the genetic potential of lines in a heterogeneous field.     

颗粒物料深床降速干燥过程的解析

通过对颗粒物料的干燥特性分析,给出单一粒体降速干燥具有二段性的物料在深床干燥过程各降速阶段的解析式;讨论诸干燥参数及物性参数的变化和相互关系,建立数学模型,采用计算方法,示出解析结果以及这一解析方法的应用。