Microbial hydrolytic enzyme activity in the sediments of a net cage aquaculture area

Potential microbial extracellular leucine-aminopeptidase (LAP) and β-glucosidase (βGlc) activity in the sediments of a red sea-bream (Pagrus major) aquaculture area were measured. Temporal variations in these enzymatic activities were low and stable from December 1999 to April 2000. They rose but fluctuated widely from May to November 2000. LAP activity in the sediments ranged from 334 to 4461 nmol/g per h in the 0–1-cm layer and from 106 to 258 nmol/g per h in the 4–5-cm layer. βGlc activity ranged from 221 to 4121 nmol/g per h in the 0–1-cm layer and from 18 to 238 nmol/g per h in the 4–5-cm layer. These values were higher than others reported and the organic matter load from the fish farms appeared to promote these enzymatic activities, although the relationship was not linear. The ratio of LAP to βGlc was smaller than other reported values in marine sediments or in water samples from the same aquaculture area, suggesting that not only quantitatively but qualitatively an aspect of organic matter degradation in the sediment might be changed by the heavy supply of organic materials from fish farms.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Prevalence of Dirofilaria immitis infection in living raccoon dogs assessed by hematological examination

The prevalence of Dirofilaria immitis in free-ranging raccoon dogs (Nyctereutes procyonoides viverrinus) was examined in the southeast region of Kanagawa Prefecture, Japan, using a rapid immunomigration (RIM) test kit. Between April 2007 and March 2010, we examined 108 raccoon dogs rescued and housed by the Kanazawa Zoological Garden. D. immitis infection was found in 8 (7.4%) raccoon dogs. This is the first report to reveal the prevalence of D. immitis infection in living raccoon dogs. The prevalence of the infection was lower than previously reported values obtained on postmortem examination. One reason might be that the present study included young raccoon dogs infected with immature worms. Significant high-risk areas of D. immitis infection in the raccoon dogs were not observed.     

A surge-like increase in luteinizing hormone preceding musth in a captive bull African elephant (Loxodonta africana)

This study was conducted to determine the correlation between reproductive hormones and musth in a male African elephant. Changes in circulating luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and immunoreactive (ir-) inhibin and the degree of musth were evaluated for 4 years. LH increased 4 weeks before musth began. The highest concentrations of testosterone and ir-inhibin were observed from April to October. There were positive correlations among testosterone, ir-inhibin and musth behavior. These findings suggested that the surge-like LH in the pre-musth period might stimulate secretion of testosterone and ir-inhibin and thus initiate the musth behavior. This study also suggested that the high LH level before musth might be a useful biomarker for the beginning of the musth season.     

Plasma concentration and cardiovascular effects of lidocaine during continuous epidural administration in dogs anesthetized with isoflurane

The cardiovascular effects of continuous epidural administration (CEA) of lidocaine were investigated in anesthetized dogs. Loading epidural injections of 2, 4, or 6 mg/kg of lidocaine were followed by CEA with 1, 2, or 3 mg/kg/hr lidocaine, respectively, for 2 hr under 2.0% isoflurane anesthesia. Heart rate, direct blood pressure, cardiac index, and stroke volume decreased dose-dependently during CEA, whereas systemic vascular resistance did not significantly differ with dose, and no characteristic changes were observed in any groups. Plasma lidocaine concentration reached a steady state during CEA and increased in a dose-dependent manner. Circulatory suppression caused by lidocaine CEA was not attributable to peripheral vasodilation, but rather to the direct cardiac action of systemic lidocaine absorption from the peridural space.     

Expression analysis of the type I keratin protein keratin 33A in goat coat hair

The coat of a goat, like that of many mammalian species, consists of an outer coat of coarse hairs and an under coat of fine, downy hairs. The coarse guard hairs are produced by primary follicles and the finer cashmere hairs by secondary follicles. We previously reported that hair keratins are components of cashmere hair, and proteomic analysis revealed that their expression is elevated in winter coat hair. To determine detailed characterization, we have cloned keratin 33A gene, a major highly expressed keratin in winter, and then analyzed the expression of goat hair coat. By Western analysis, we detected that keratin 33A protein is expressed only in hair coat among the various goat tissues. Moreover, the expression level in winter has increased in cashmere high‐producing Korean native breed, whereas the expression levels between summer and winter had not changed in cashmere low‐producing Saanen. In addition, by immunohistochemistry we determined that keratin 33A is localized in the major cortex portion of cashmere fiber. These results confirm that keratin 33A is a structural protein of goat cashmere hair fiber.     

Effect of administration of acid‐ and bile‐tolerant lactobacilli on pigs intestinal microbial population

The effects of the administration of four Lactobacillus strains as probiotics on the constitution of microbial populations in the intestine of pigs were investigated by using a litter of pigs. The experimental group was fed a diet blended with the freeze‐dried powder containing lactobacilli at a concentration of 0.2% (W/W). After 4 weeks of probiotics administration, the total viable count of anaerobes in the feces was not significantly different between the pigs fed a diet supplemented with probiotics and the pigs given the non‐supplemented diet. However, viable counts of eubacteria were significantly higher (P < 0.01) in the feces of pigs fed a diet supplemented with probiotics than in the pigs given the non‐supplemented diet. Conversely, viable counts of clostridia were significantly lower (P < 0.01) in the feces of pigs fed a diet supplemented with probiotics than the pigs given the non‐supplemented diet. The total volatile fatty acid concentrations in the feces of pigs that were given a diet supplemented with probiotics were significantly lower (P < 0.05) than those given the non‐supplemented diet. Further, the molar proportion of n‐butyric acids in the feces of pigs that were given a diet supplemented with probiotics, was significantly lower (P < 0.05) than those given the non‐supplemented diet.     

Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3×10⁶ U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.     

Effect of ovary lipid from Skipjack tuna (Katsuwonus pelamis) on brain monoamines in rats

In our previous experiments with rats, ovary lipid from Skipjack Tuna (Katsuwonus pelamis) (OLS) was shown to have a mitigating effect on anxiety and/or fear in elevated T-maze tests. This suggests that OLS has some effect on the central nervous system (CNS) of rats. Thus, we performed experiments to examine the status of CNS in rats given OLS. The effect of OLS on chronic stress was also examined at the same time. The feed for control rats used oil and fat that have the same energy percentages for n-6 and n-3 fatty acids and the same n-6/n-3 ratio as OLS. As a result, rats given OLS for 28 days had lower serotonin levels in various brain areas regardless of stress application, showing that OLS affected the serotonin nervous system. From this, it was inferred that the ability of OLS to mitigate anxiety and/or fear resulted from its action on CNS, especially the serotonin nervous system. Substances other than the essential fatty acids may have been responsible for the action of OLS on monoamines and the metabolites. The effect of OLS on CNS, especially the serotonin nervous system, suggests that OLS may suppress anxiety.     

Oleosome-Associated Protein of the Oleaginous Diatom Fistulifera solaris Contains an Endoplasmic Reticulum-Targeting Signal Sequence

Microalgae tend to accumulate lipids as an energy storage material in the specific organelle, oleosomes. Current studies have demonstrated that lipids derived from microalgal oleosomes are a promising source of biofuels, while the oleosome formation mechanism has not been fully elucidated. Oleosome-associated proteins have been identified from several microalgae to elucidate the fundamental mechanisms of oleosome formation, although understanding their functions is still in infancy. Recently, we discovered a diatom-oleosome-associated-protein 1 (DOAP1) from the oleaginous diatom, Fistulifera solaris JPCC DA0580. The DOAP1 sequence implied that this protein might be transported into the endoplasmic reticulum (ER) due to the signal sequence. To ensure this, we fused the signal sequence to green fluorescence protein. The fusion protein distributed around the chloroplast as like a meshwork membrane structure, indicating the ER localization. This result suggests that DOAP1 could firstly localize at the ER, then move to the oleosomes. This study also demonstrated that the DOAP1 signal sequence allowed recombinant proteins to be specifically expressed in the ER of the oleaginous diatom. It would be a useful technique for engineering the lipid synthesis pathways existing in the ER, and finally controlling the biofuel quality.