The impact of nigorobuna crucian carp larvae/fry stocking and rice-straw application on the community structure of aquatic organisms in Japanese rice fields

We examined the effects of predation by Nigorobuna Carassius auratus grandoculis larvae and fry, a crucian carp endemic to Lake Biwa, Japan, on the community structure of aquatic organisms in rice fields. Six experimental plots with three different rice-straw treatments in the presence/absence of stocked larvae were prepared. In each plot, the number of aquatic organisms ranging in size from 30 μm to 5 mm in the water, as well as those from 63 μm to 5 mm in size in the surface sediments, was surveyed 6, 13, 20, 26, 34, and 41 days after the onset of irrigation. Three-day-old fish larvae were released on day 10. Undigested organisms in the gut contents of the larvae or fry were identified on days 20, 26, 34, and 41, respectively. Ten-day-old larvae mainly preyed on Cladocera, but the fry thereafter shifted to Diptera as their main prey. While Cladocera and Podocopida decreased in fish-stocked plots, Euglenales and Halteriida became more abundant there. Top-down or bottom-up effects of fish seemed to control these changes in community structure.     

Culture-independent analysis of the bacterial community during fermentation of pa-som,a traditional fermented fish product in Laos

Pa-som is a traditional salty fermented food made from varieties of freshwater fish with garlic and rice in Laos. A similar product, plaa-som, is made in Thailand. To investigate the fermentation process of pa-som, lactic acid production and pH were monitored during laboratory-scale production of pa-som. Furthermore, the variety of bacteria involved and their succession during fermentation were examined using culture-independent polymerase chain reaction (PCR) denaturing gel gradient electrophoresis targeting the 16S ribosomal RNA (rRNA) gene. d- and l-lactic acid production and a decrease in pH were observed in the samples after 1 day of fermentation. The lactic acid content and pH continued to increase and decrease, respectively, until 4 days of fermentation. Overall, six lactic acid bacteria and eight other indigenous bacteria species were detected during pa-som fermentation. Among the lactic acid bacteria detected, two Lactococcus and one Weissella species were detected in samples after 1–4 days of fermentation, suggesting that these three species play major roles in pa-som fermentation from the initial phase. This is the first report to investigate the fermentation process in pa-som at the microbial level. This research approach should be applicable to a wide variety of pa-som and related fermented fishery products in Laos to help understand their microbial diversity and to identify beneficial bacterial species for improving quality.     

Changes in heart rate and heart rate variability in Thoroughbreds during prolonged road transportation

OBJECTIVE: To determine whether evaluation of heart rate (HR) and HR variability (HRV) during prolonged road transportation in horses provides a sensitive index of autonomic stimulation. ANIMALS: Five 2-year-old Thoroughbreds. PROCEDURE: ECGs were recorded as horses were transported for 21 hours in a 9-horse van. Heart rate, high-frequency (HF) power, low-frequency (LF) power, and LF-to-HF ratio from Fourier spectral analyses of ECGs were calculated and compared with values recorded during a 24-hour period of stall rest preceding transportation. RESULTS: HR, HF power, and LF power had diurnal rhythms during stall rest but not during road transportation. Heart rate was higher and HF power and LF power lower during road transportation than stall rest, and HR, HF power, LF power, and LF-to-HF ratio all decreased with time during road transportation. Heart rate during stall rest was weakly and inversely associated with LF power, but during road transportation was strongly associated with LF power, HF power, and LF-to-HF ratio. Neither LF power nor HF power was correlated with LF-to-HF ratio during stall rest, but LF power was strongly and HF power weakly correlated with LF-to-HF ratio during road transportation. High-frequency power and LF power were significantly correlated with each other during stall rest and road transportation. Heart rate was significantly influenced by LF power and LF-to-HF ratio during stall rest (R(2) = 0.40) and by HF power and LF-to-HF ratio during road transportation (R(2) = 0.86). CONCLUSIONS AND CLINICAL RELEVANCE: HR is influenced by different sympathovagal mechanisms during stall rest, compared with during road transportation; HRV may be a sensitive indicator of stress in transported horses.     

Babesia microti-like parasites detected in Eurasian red squirrels (Sciurus vulgaris orientis) in Hokkaido, Japan

Six Eurasian red squirrels (Sciurus vulgaris orientis), victims of road traffic found during 2002 and 2004 near the Noppro Forest Park in Ebetsu, Hokkaido, Japan, were examined for the presence of Babesia parasites. Three of the six squirrels exhibited positive signals by nested PCRs targeting both the 18S rRNA and beta-tubulin genes. Three squirrels proved to be infected with a B. microti-like parasite as evidenced by sequencing the amplified DNAs and by the morphology of the intraerythrocytic parasites. Genotypically, however, the parasite appeared to be of a new type, as it was clearly distinguishable from any of the known types that have previously been reported in various wild animals. This is the first report showing molecular evidence for the presence of B. microti-like parasites in Sciuridae.     

Occurrence of stilbene oligomers in Cyperus rhizomes

Investigation of the chemical constituents of Rhizoma Cyperi (Cyperus rotundus Linneus) resulted in the isolation of novel enantiomeric and meso-stilbene trimers [i.e., (+)- and (−)-(E)-cyperusphenol A (1, 2 respectively) and (E)-mesocyperusphenol A (3)], a trimer bearing a novel hexacyclic ring system [cyperusphenol B (5)], as well as known stilbenoids (cyperusphenols C (4) and D (6), scirpusins A (7) and B (8), and piceid (9)) and luteolin. HPLC was used for the optical resolution of 1 and 2 as well as for the identification of cooccurrence of enantiomers of 7. The structures of the isolates were established by spectroscopic analyses, including a detailed NMR spectroscopic investigation. The isolates were evaluated in terms of their antiproliferative activity employing the Jurkat cell line (human T-cell leukemia cells), while the IC₅₀ potencies of a racemate of 1 and 2, 3, 5, and 6 were estimated as 27.4, 40.5, 26.4, and 26.3 μM, respectively. The suppression of cell growth by 6 was due to the induction of apoptosis, which was characterized by nuclear changes and PARP-1 cleavage determined by western blotting. We also evaluated the free radical scavenging activity of the isolates.     

Serum cystatin C concentration can be used to evaluate glomerular filtration rate in small dogs

Serum cystatin C levels (CysC) are used in human medicine to document progressive kidney failure. Although CysC are not thought to be useful for the diagnosis of kidney dysfunction in dogs, there has been no specific consideration of body weight as a confounding issue. The aim of this study was to assess that the utility of CysC for the diagnosis of decreased glomerular filtration rate (GFR) in smaller vs. larger dogs. In clinically healthy dogs, serum creatinine (Cre) and CysC correlate directly with body weight; we found that dogs weighing <20 kg had significantly lower CysC than those weighing ≥20 kg (0.27 ± 0.07 vs. 0.34 ± 0.05 mg/l, respectively, P<0.001). In dogs weighing <20 kg, CysC had superior diagnostic accuracy for the detection of mildly decreased plasma iohexol clearance (PCio) (<1.8 ml/min/kg) compared with Cre (sensitivity 100% vs. 80.9% and specificity 100% vs. 85.7%); this was not true for dogs weighing ≥20 kg. Additionally, using a cut-off PCio of <1.8 ml/min/kg, the area under receiver-operating characteristics curve (AUC) of CysC was significantly higher than that of Cre in dogs weighing <20 kg (P<0.05); this was not true for dogs weighing ≥20 kg (P=0.695). In conclusion, CysC is a useful marker for the detection of a mild decreasing GFR compared with Cre in dogs weighing <20 kg.     

Metabolism of diethofencarb (isopropyl 3,4-diethoxyphenylcarbamate) in rats: identification of metabolites in urine

Rats were orally given a diethofencarb (isopropyl 3,4-diethoxyphenylcarbamate) labeled with (14)C, at 300 mg/kg/day, for 4 consecutive days, and 11 metabolites in urine were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Ten of them were newly identified forms. Five of them were S-conjugates: three mercapturic acid conjugates, one S-methyl conjugate, and one SO-methyl conjugate. The others were two phenoxyacetic acids, hydroxyacetanilide, hydroxyisopropyl carbamate, and oxazolinone derivatives. From the results, the existence of the following reactions in rats can be concluded: (1) deethylation of the 4-ethoxy group; (2) conjugation of phenols with glutathione, gamma-glutamyltranspeptidation and depeptidation of the glutathione to form cysteine conjugates, and N-acetylation of the cysteine; (3) cleavage of the C-S linkage of cysteine conjugates followed by methylation; (4) oxidation of the S-methyl group; (5) cleavage of the carbamate linkage; (6) acetylation of the resultant amino group; (7) oxidation of the acetyl group; (8) oxidation of the isopropyl group; (9) cyclization of the oxidized isopropyl carbamate group; and (10) oxidation of the 4-ethoxy group.     

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC₅₀ values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC₅₀ values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.     

DedA protein relates to action-mechanism of halicyclamine A, a marine spongean macrocyclic alkaloid, as an anti-dormant mycobacterial substance

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.