Purification and some properties of alanine racemase from the marine gastropod <Emphasis Type="Italic">Cellana grata</Emphasis>

ABSTRACT:   High levels of free d -alanine were found in the muscle of a marine gastropod Cellana grata that inhabited the intertidal zone, and alanine racemase activity was detected in the muscle. The authors purified alanine racemase from the muscle of C. grata to characterize its enzymological properties. The molecular mass of the enzyme was estimated to be 40.5 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 41.4 kDa by gel filtration, suggesting that the enzyme was monomeric in nature. Kinetic experiments, performed using the purified enzyme, revealed that the Lineweaver-Burk plot for d -alanine as a substrate resulted in a K _m value of 20.4 mM, and the value for l -alanine was 43.0 mM. Of the several types of amino acids tested, alanine was found to be the specific substrate for the enzyme. In the measurement of alanine racemase activity, exogenous pyridoxal 5'-phosphate (PLP) was not required for the enzyme activity; however, aminooxyacetic acid, hydroxylamine and phenylhydrazine, which inhibit PLP-dependent enzymes, strongly inhibited the enzyme activity. These results suggest that the enzyme is PLP-dependent. This is the first report on the purification and some properties of alanine racemase in a marine gastropod.     

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.     

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.     

Chemical constituents ofInonotus obliquus II: a new triterpene, 21,24-cyclopentalanosta-3β,21,25-triol-8-ene from sclerotium

A new lanostane-type triterpene with a cyclopentanol partial structure in the side chain was isolated from the sclerotium of the wood rotting fungusInonotus obliquus along with four known compounds: lanosterol, inotodiol, trametenolic acid, and 3-hydroxy-8,24-dienlanosta-21,23-lactone. The new compound was determined to be 21,24-cyclopentalanosta-3,21,25-triol-8-ene by spectroscopic analyses.An outline of this study was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto     

Thinning effect on height and radial growth of Pinus thunbergii Parlat. trees with special reference to trunk slenderness in a matured coastal forest in Hokkaido, Japan

To evaluate the effect of thinning on a mature coastal Pinus thunbergii Parlat. forest in Hokkaido, northern Japan, we established four study plots with different thinning intensities (control and 20, 40, and 60 % thinned at individual base; each 20 m × 20 m) and monitored them for 10 years. Radial growth of individual trees in the 60 % thinned plot was significantly greater than that in the other plots, whereas height growth in the 60 % thinned plot was significantly lower than in the other plots. Applying the height growth contribution index, which represents the trade-off between relative height growth rate and relative diameter growth rate, revealed that individual trees in the control and weakly (20 and 40 %) thinned plots had invested more in height growth compared to trees in the 60 % thinned plot. Though higher trunk slenderness value (>80) indicates higher susceptibility to meteorological disturbance such as wind storm and heavy snow, trunk slenderness in the control and the weakly thinned plots increased significantly compared to that in the 60 % thinned plot. In this study, 44.3 % of trees in the control plot had trunk slenderness values >80 over a period of 10 years. The number of trees with trunk slenderness values >80 also increased in the weakly thinned plots. In contrast, no increase was observed in the 60 % thinned plot. In Japan, the thinning intensity is legally supposed to be <35 % of stand volume, which is not enough to improve tree architecture in terms of trunk slenderness.     

Preparation of synthetic wood composites using ionic liquids

Synthetic wood composite films containing cellulose, hemicelluloses, and lignin, the three major components of natural wood, were prepared in a room temperature ionic liquid solvent, 1-ethyl-3-methylimidazolium acetate, [EMIM][Ac]. Various synthetic wood composites were obtained by dissolution of individual wood components together with additives, including polyethylene glycol (PEG), chitosan, and multi-wall carbon nanotubes (MWNTs) in [EMIM][Ac]. The addition of water affords a gel that was dried in either a low humidity environment or under vacuum. Synthetic wood films showed smoother surface textures, higher water resistance, and higher tensile strengths than cellulose films formed by the same methods. Tailor-made synthetic wood composites were also prepared having a variety of desirable properties, including antimicrobial activities, controlled hydro-phobicity/philicity, high relative dielectric constant, and a high degree of cohesiveness.     

Pathogenicity of Stemphylium lycopersici isolated from rotted tobacco seeds on seedlings and leaves

A preemergence damping-off of tobacco (Nicotiana tabacum L.) was found in Fukuoka, Japan, in 2009. A fungus repeatedly isolated from the black-rotted seeds reproduced the symptoms in tobacco seeds, and the preemergence damping-off developed after flower and soil inoculation with the fungus. Brown spots and blight developed on leaves of tobacco seedlings after inoculation with the isolate. The fungus was reisolated from the rotted seeds and blighted leaves. The causal fungus was identified as Stemphylium lycopersici based on morphology and phylogenetic analysis. This is the first report to confirm the pathogenicity of S. lycopersici in tobacco in the world.     

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.     

15种杀虫植物叶片对南方根结线虫防治效果及对蔬菜生长影响初报

天然杀虫植物具有分解快、残留低、污染少、毒性小等特点，因此将其利用在南方根结线虫(Meloidogyne incognita)综合防治中具有广阔发展前景。     

Histological Characteristics of the Regression of Corpora Lutea in Wistar Hannover Rats: the Comparisons with Sprague-Dawley Rats

We examined the ovaries of 44 Wistar Hannover (RccHan^TM:WIST) (WH) and 30 Sprague-Dawley (SD) rats at 32-weeks of age to determine whether the ovarian structure and formation/regression of the corpora lutea (CLs) differ between the two strains. The average ovary weight was higher in WH rats. The average number of all CLs, including currently formed and previously formed CLs, was higher in WH rats in all cycles; however, no appreciable difference was detected in the number of newly or currently formed CLs between the two strains. CLs regression characterized by degeneration and necrosis of luteal cells began to appear in diestrus in both strains; however, the distribution of degenerated/necrotic cells in CLs differed. Necrotic cells were scattered in SD rats but were focally observed in the center of the CL in WH rats. The reduction in size of previously formed CLs accompanied by regression started about 2 or more stages later in WH rats than in those of SD rats. In conclusion, the higher number of CLs in WH rats is considered to be due to slow CL regression compared with in SD rats.