Ceramide aminoethylphosphonate from jumbo flying squid Dosidicus gigas attenuates the toxicity of cyanotoxin microcystin-LR

We have previously established the method for isolation of ceramide aminoethylphosphonate (CAEP) from jumbo flying squid Dosidicus gigas. In this study, we performed a MTT assay to evaluate the safety of CAEP to the cell lines for the application to health food and supplements. The CAEP did not show any cytotoxicity to various HEK293-transfectant cells. Next, we elucidated the positive function of CAEP to the somatic cells. Recently, we have reported that hepatotoxin microcystin-LR was taken up into the hepatocytes mediated by hepatocellular uptake transporters OATP1B1 and OATP1B3, and the cells were induced cytotoxicity subsequently. Cytotoxicity of microcystin-LR to permanently OATP1B3-expressing HEK293-OATP1B3 cells rather than to HEK293-OATP1B1 cells was preferentially attenuated by CAEP in a concentration-dependent manner. In addition, the enzyme activity of serine/threonine phosphatase, which was inhibited by microcystin-LR, was recuperated by co-exposure to CAEP. Furthermore, microcystin-LR-induced cellular protein phosphorylation were disrupted by CAEP exposure. These results suggested that CAEP is a promising remedy and/or preventive medicine for liver damage with microcystin-LR.     

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.     

The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term

The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro‐matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) (‘ET‐vivo’ embryos), or cultured in vitro for 5 or 6 days (‘IVC’ embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET‐vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.     

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development

We evaluated the effects of polyethylene glycol (PEG) and Supercool X‐1000 (SC) as supplements during the vitrification of immature cumulus‐enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG‐, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG‐ groups; however, all values were lower than those in the non‐vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC‐, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC‐ groups but lower than those in the non‐vitrified control. The percentage of cleavage in the SC‐ group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non‐vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.     

Milking performance evaluation and factors affecting milking claw vacuum levels with flow simulator

Milking performance of milking machines that matches the production capability of dairy cows is important in reducing the risk of mastitis, particularly in high‐producing cows. This study used a simulated milking device to examine the milking performance of the milking system of 73 dairy farms and to analyze the factors affecting claw vacuum. Mean claw vacuum and range of fluctuation of claw vacuum (claw vacuum range) were measured at three different flow rates: 5.7, 7.6 and 8.7 kg/min. At the highest flow rate, only 16 farms (21.9%) met both standards of mean claw vacuum ≥35 kPa and claw vacuum range ≤ 7 kPa, showing that milking systems currently have poor milking performance. The factors affecting mean claw vacuum were claw type, milk‐meter and vacuum shut‐off device; the factor affecting claw vacuum range was claw type. Examination of the milking performance of the milking system using a simulated milking device allows an examination of the performance that can cope with high producing cows, indicating the possibility of reducing the risk of mastitis caused by inappropriate claw vacuum.     

The complete mitochondrial genome of the domestic red deer (Cervus elaphus) of New Zealand and its phylogenic position within the family Cervidae

We determined the complete nucleotide sequence of the mitochondrial genome of the semidomestic red deer (Cervus elaphus) of New Zealand. The genome was 16 357 bp long and contained 13 protein‐coding genes, 12SrRNA, 16SrRNA, 22 tRNAs and a D‐loop as found in other mammals. Database homology searches showed that the mitochondrial DNA (mtDNA) sequence from the New Zealand semidomestic deer was similar to partial mtDNA sequences from the European, Norwegian (C. e. atlanticus) and Spanish red deer (C. e. hispanicus). Phylogenetic analysis of the mitochondrial protein‐coding regions revealed two well‐defined monophyletic clades in subfamilies Cervinae and Muntiacinae. However, red deer and Sika deer were not found to be close relatives. The analysis did identify the red deer as a sister taxon of a Samber/Sika deer clade, although it was more closely related to the Samber than the Sika group.     

Development of SNP markers for individual identification and parentage test in a Japanese Black cattle population

This study describes the development of efficient single nucleotide polymorphism (SNP) markers for individual identification and parentage tests in a Japanese Black cattle population. An amplified fragment length polymorphism method was employed to detect informative candidate markers, and yielded 44 SNP markers from 220 primer combinations. 29 unlinked SNPs were finally selected as diagnostic markers. The allelic frequencies for each marker were estimated by using PCR‐RFLP in the Japanese Black population. Based on the frequency data, the estimated identity power of these markers was 2.73 × 10^?12. Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.96929 and 0.99693, respectively. This panel of SNP markers is theoretically sufficient for individual identification, and would also be a powerful tool for a parentage test in Japanese Black cattle. The markers could contribute to the management of the beef industry in Japan.     

Mutation analysis of barley malt protein Z4 and protein Z7 on beer foam stability

Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins.