Cucumber mosaic virus isolated from Aconitum spp. in Japan

Viruses were isolated from leaves of plants of Aconitum species with symptoms such as mottling and yellowing in Hokkaido and Gunma prefectures in Japan. These viruses were identified as Cucumber mosaic virus (subgroup II) based on particle morphology, host range, aphid transmission, and serology.     

The contribution of endogenous cellulase to the cellulose digestion in the gut of earthworm (Pheretima hilgendorfi: Megascolecidae)

Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-β-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of midforegut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CMC. In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum.     

Cesium absorption through bark of Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>)

Absorption of radiocesium (¹³⁷Cs and ¹³⁴Cs) through bark, and its subsequent translocation into wood and needles, has been suggested as a potential source of tree contamination, but the process is not well understood. Field experiments were conducted to confirm whether Cs could enter a Japanese cedar tree through the bark and how Cs moves within a tree. Stable Cs (¹³³Cs) was applied to the bark at 1.2-m height on 10- and 26-year-old Japanese cedars. The ¹³³Cs concentrations were determined in the bark, sapwood, and heartwood (for 26-year-old cedar only) of stem disks from several heights, as well as in current-year needles from the canopy. The ¹³³Cs concentrations were considerably higher in the sapwood and heartwood of stem disks from 1.2-m height in treated trees than in untreated trees, suggesting that ¹³³Cs penetrated the bark to enter the wood. The average ¹³³Cs concentrations were higher in the heartwood than the sapwood, indicating ¹³³Cs accumulation in the heartwood. High ¹³³Cs concentrations in the needles of treated trees implied acropetal movement of ¹³³Cs to actively growing organs. Our results demonstrate that Cs can enter Japanese cedar trees through the bark and that Cs is transported radially to the heartwood and vertically to the apex.     

An efficient molecular technique for sexing tiger pufferfish (fugu) and the occurrence of sex reversal in a hatchery population

The tiger pufferfish (fugu) is one of the most important food fishes in East Asia. Since its testes are regarded as a delicacy, sex determination is economically relevant. Previous studies have identified a missense single-nucleotide polymorphism (SNP) in the Amhr2 (anti-Müllerian hormone receptor type II) gene as a strong candidate for a master sex-determining polymorphism. To distinguish genotypic sex efficiently, we developed a high-resolution melting (HRM) assay for this SNP site. By screening 396 fish from two independent crosses reared under controlled conditions, we observed perfect concordance between the SNP genotype and phenotypic sex. Thus, this method holds great potential for use in high-throughput sexing. When analyzing 293 progeny from a third cross reared under unknown conditions, we unexpectedly found that 25 % of phenotypic males exhibited female genotype. These results suggest that environmental factors such as rearing conditions could influence the sex-determination pathway in pufferfish. Alternatively, genetic modifiers might override the signals from Amhr2. This finding raises a concern regarding enhanced stock management of this species, because sex-reversed fish could compromise the sex ratio in subsequent generations. The HRM assay will also be useful for monitoring the degree of sex reversal before release.     

Decontamination of Cs from Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) via kraft cooking

To investigate the possibility of decontaminating ¹³⁷Cs-contaminated Cryptomeria japonica wood, kraft pulping was conducted and the Cs behavior in the reaction process was examined. ¹³³Cs-treated or ¹³⁷Cs-contaminated bark, sapwood, and heartwood chips of Cryptomeria japonica were digested using an aqueous solution of NaOH and Na₂S. The pulp was washed with ultrapure water and filtered, after which the filtrate (black liquor) was collected. The black liquor was acidified to separate the supernatant and precipitation. The Cs (¹³³Cs and ¹³⁷Cs) concentrations in the chip and reaction products were measured. As for wood samples, the majority of Cs was present in black liquor, while only a minor amount of Cs was retained in the pulp (<1%). In the case of bark, although the majority of Cs was present in the black liquor, the proportion of Cs in the pulp was much higher than that in the wood pulp. In addition, the Cs in the precipitation of the bark was higher than that in the wood, possibly because the Cs in the bark was combined with some components, which is insoluble in alkaline solution. Our results suggest that ¹³⁷Cs-contaminated Cryptomeria japonica wood can be used in the pulp and paper industries.     

Effect of temperature decrease on oocyte development,sex steroids,and gonadotropin β-subunit mRNA expression levels in female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17β (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin β-subunit [follicle-stimulating hormone (FSHβ), luteinizing hormone (LHβ)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25°C to 15°C over 39 days, while the control group was maintained at 25°C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSHβ and LHβ mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.     

Determination and quantification of γ-glutamyl-valyl-glycine in commercial fish sauces

It was recently reported that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR). In addition, screening by the CaSR assay and sensory evaluation revealed that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) was a potent kokumi peptide. In this study, the quantities of γ-Glu-Val-Gly in various commercial fish sauces originating from Vietnam (Nuoc Mum), Thailand (Nampra), China (Yu-lu), Korea, Japan (Shottsuru and Ikanago-shoyu), and Italy (Garum) were investigated using a LC/MS/MS method followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses revealed γ-Glu-Val-Gly at concentrations ranging from 0.04 to 1.26 mg/dL, indicating that γ-Glu-Val-Gly is widely distributed among various commercial fish sauces.     

Up-regulation of NOD1 and NOD2 through TLR4 and TNF-alpha in LPS-treated murine macrophages

NOD1 (Card4) and NOD2 (Card15) are thought to be responsible for cytoplasmic defense against bacterial entry. To gain further knowledge about how their expressions are regulated in murine macrophages, we investigated the expression of NOD1 and NOD2 mRNAs after stimulation with various endotoxins, lipopolysaccharide, lipoteichoic acid and peptidoglycan. In macrophage RAW264.7 cells, the first and second rises in NOD1 and NOD2 mRNAs were observed at 2 hr and at 8-12 hr after endotoxin treatment. Increases in NOD1 and NOD2 mRNAs at 2 hr in lipopolysaccharide-treated RAW264.7 cells were reduced with the use of NF-kappaB inhibitor, caffeic acid phenethyl ester. In RAW264.7 cells, lipopolysaccharide-induced increases in NOD1 and NOD2 mRNAs were inhibited with anti-TLR4 antibody, and partially reduced in peritoneal macrophages obtained from TLR4-deficient mice. Furthermore, NOD1 and NOD2 mRNA expressions in RAW264.7 cells were increased by the treatment with proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), or IL-6. In TNF-alpha deficient macrophages, the expression of NOD molecules was minimal at 12 hr, and the second rise in NOD mRNA seen in lipopolysaccharide-treated RAW264.7 cells was inhibited with anti-TNF-alpha, but not with anti-IL-1beta or anti-IL-6 antibody. These observations suggest that immediate response of NODs to endotoxins could result from NF-kappaB activation via TLR signaling, whereas the second rise in NOD mRNAs might have resulted from TNF-alpha production possibly through NF-kappaB, TLR, and/or NOD signalings.