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.     

Experimental approach to prezygotic chromosome screening using only a single pair of gametes in mice

During in vitro embryo production, chromosome screening is essential to prevent pregnancy losses caused by embryonic chromosome aberrations. When the chromosome screening is completed before fertilization, gametes are effectively utilized as genetic resources. The aim of this study was to investigate whether chromosome screening of gametes accompanied by fertilization would be feasible using a single mouse spermatozoon and oocyte. Metaphase II oocytes were divided into a cytoplast and a karyoplast. For genome cloning of the gametes, androgenic and gynogenic embryos were produced by microinjection of sperm into cytoplasts and parthenogenetic activation of karyoplasts, respectively. Pairs of blastomeres from androgenic and gynogenic embryos were fused electrically to produce diploid embryos, which were transferred into pseudopregnant surrogate mothers to examine fetal development. Blastomeres from androgenic and gynogenic embryos were individually treated with calyculin A—a specific inhibitor of type 1 and 2A protein phosphatases—for 2 h to induce premature chromosome condensation. Thereafter, chromosome analysis of blastomeres, reflecting the genetic constitution of individual spermatozoa and oocytes, was performed, and we confirmed that most of the androgenic and gynogenic 2-cell embryos had a haploid set of chromosomes in their sister blastomeres. The reconstructed embryos from blastomeres of androgenic and gynogenic 2-cell embryos could be implanted and develop into live fetuses, albeit at low efficiency. This study indicates that prezygotic chromosome screening and embryo production using a single pair of gametes may be practicable.     

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.     

Absorption of color additives and settling volume in water of blue-green alga,ishikurage (Nostoc commune)

The properties of ishikurage (Nostoc commune) and other algae were compared. Ishikurage and suizenji nori (Aphanothece sacrum) absorbed much more amaranth than others. They have a high settling volume (SV) in water. The high SV of ishikurage is attributable to its neutral detergent fiber (NDF). The acid detergent fiber (ADF) from the alga had a high amaranth absorption. However the absorption by the ADF did not explain the absorption by ishikurage.The results suggest that ishikurage may have a nutritional significance as a source of dietary fiber.     

Abundance of sulphate-reducing bacteria in fish farm sediments along the coast of Japan and South Korea

The abundance of sulphate-reducing bacteria (SRB) was investigated in the coastal marine sediments along the coast of Japan and South Korea. Sediment samples were collected from fish and shellfish farms between 2006 and 2008. As non-fish farming reference sites, sediments were also collected from highly eutrophic bays, a highly sulphidogenic saline lake, and the deep sea. A quantitative real-time PCR analysis that targeted the gene coding for a portion of the α-subunit of dissimilatory sulphite reductase (dsrA) was performed to assess the abundance of the SRB in the sediments. Between 2.8 × 10⁷ and 2.5 × 10⁹ copies of the dsrA gene per gram dry sediment were detected. There was no relationship between dsrA gene copy number and total bacterial count in the sediments, whereas organic matter contents (particulate organic carbon and nitrogen, ignition loss and chemical oxygen demand) and acid-volatile sulphide contents were significantly correlated with the dsrA copy number. The data presented demonstrate that organic enrichment of sediment may influence the abundance of SRB communities in coastal marine sediments and that the cell density of SRB may be used as a biological indicator for assessing pollution levels in sediments of marine fish farms.     

Construction of a linkage map and identification of DNA markers linked to Fom-1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon

Resistance to Fusarium oxysporum f.sp. melonis race 2 is conferred by a single dominant gene, Fom-1 in melon. Here, we identified DNA markers tightly linked to Fom-1 that could be used for marker assisted selection in breeding programs. First, we developed 125 F₂ plants derived from the cross between melon lines P11 (fom-1fom-1) and MR-1 (Fom-1Fom-1). Using the F₂ population, we constructed a linkage map including 14 SSR markers which had not been mapped previously. Fom-1 was confirmed to be allocated to linkage group 7. Then, we identified four AFLP markers using bulked segregant analysis. The AFLP marker TAG/GCC-470 was completely linked to Fom-1 and other three markers were mapped near Fom-1. TAG/GCC-470 and TCG/GGT-400 were respectively converted to STS and CAPS markers. Usefulness of DNA markers was confirmed in the analysis with several melon cultivars and lines.