Molecular phylogeny and diversity of Myanmar and Bhutan mithun based on mtDNA sequences

The mithun (Bos frontalis), synonymous with mithan and gayal, is considered to be a domesticated form of gaur (B. gaurus). However, there has been a controversy concerning its origin. In an effort to address this issue, the mitochondrial cytochrome b (cytb) genes of 20 mithun from Myanmar and 13 from Bhutan were sequenced to trace its maternal origin. Seven cytb haplotypes were found in the 33 mithun, and the phylogenetic tree for these haplotypes clearly showed three embranchments involving five gaur types, a B. indicus type, and a B. taurus type. Sixteen Myanmar and 12 Bhutan mithun had gaur haplotypes, while a B. indicus haplotype was found in three Myanmar and one Bhutan mithun. The B. taurus haplotype was detected in a single Myanmar animal. These results demonstrated that the principal maternal origin of mithun was gaur and suggested that it was directly domesticated from gaur. However, some introgression of domestic cattle existed in current mithun populations. The presence of cattle mtDNA raised the question of how many cattle nuclear genes might have been integrated into the gene pool of mithun.     

Effects of reactive radicals and heat on <Emphasis Type="Italic">trans</Emphasis>-isomerization of eicosapentaenoic acid

Trans geometric isomers of eicosapentaenoic acid (TEPA) have been found as minor components in human platelets. However, there is little information on the mechanism of trans-isomerization of eicosapentaenoic acid (EPA) in vitro and in vivo. The effects of reactive radicals and heat on trans-isomerization of EPA were examined. Trans-isomerization occurred when EPA ethyl ester reacted with nitrogen dioxide radical (NO₂) but not with 2,2′-azobis(2,4-dimethylvaleronitrile). TEPA was also produced from EPA ethyl ester heated at 200°C for 60 h. No TEPA, however, was detected in sardines Sardinops melanostictus after boiling, roasting, or microwave heating. These results suggest that EPA is trans-isomerized by NO₂ in vivo while trans-isomerization of EPA does not occur during conventional cooking.     

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

ABSTRACT: The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.     

Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs

Obtaining sufficient transgenic cells via selective cultivation of genetically manipulated somatic cells is difficult due to the limited number of cell divisions. Additionally, if irreversible mutations in a cell's chromosomes occur during selective cultivation and the cell is used as the nuclear donor, somatic cell nuclear transfer (SCNT) embryos often exhibit abnormal development. On the other hand, a SCNT method in which fetal cells derived from SCNT embryos are used as the nuclear donor (recloning method) is an effective technique for obtaining large quantities of transgenic cells. In this study, we compared the in vivo development rate of SCNT embryos produced from porcine alpha1-3 galactosyltransferase gene knockout (GTKO) cells by a recloning method with that of SCNT embryos produced without recloning from porcine GTKO cells (direct method). In the direct method, 557 and 462 cloned embryos were produced using two types of activation methods, the two-step activation (TA) method and the delayed activation (DA) method, and then transferred into 6 and 4 recipients, respectively, but no piglets were born from these recipients. In the recloning method, 956 and 1038 cloned embryos were produced using the TA and DA methods, respectively, and then transferred to 8 and 7 recipients, respectively. Two piglets were born from one recipient in the TA group and 6 piglets were born from 3 recipients in the DA group. This report indicates that the recloning method improved the developmental capacity of SCNT embryos reconstructed with gene-targeted somatic cells.     

Flight Distance and Avoidance Score in Thoroughbred Foals and Yearlings and the Relationship with Handling Frequency in the Young Ages

Flight and avoidance reactions from human were examined using 168 postweaning Thoroughbred foals in 22 breeding farms. Further 114 yearlings of 168 foals were tested in the following summer. The foal handlings by the stabler were asked in questionnaire. The relationship between the behavioural reactions and the foal handling frequencies was analyzed. The flight reaction was estimated as the distance from the animal to a stranger when the animal began to flight away from his approach. The avoidance scores were set up from (1) (not resistant) to (5) (touch rejection) from human touching. In the stabler questionnaire, handling frequencies of “body brushing”, “rectal temperature measurement”, “hoof cleaning”, and “stall cleaning” in the early nursing period were asked. The handling frequencies were scored from (1) (not done) to (5) (every day). In the preliminary test, a measurement reliability of the flight distance and the avoidance score was confirmed. The mean flight distances were 0.56 m and 0.27 m in the postweaning foals and the yearlings, respectively. Touch-avoidance scores of the highest frequency were (3) and (2) in the postweaning foals and the yearlings, respectively. As the results of Spearman’s rank-correlation analysis, “body brushing” showed highly negative relationships with “flight distance” (ρ=–0.31, P<0.001) and “avoidance score” (ρ=–0.37, P<0.001) in the postweaning foals. In the yearlings, “hoof cleaning” also showed significantly negative relationships with these behavioural indices (ρ=–0.24, P<0.01; ρ=–0.22, P<0.01, respectively).     

Correlation of phytoplasma concentration in Hydrangea macrophylla with green-flowering stability

The interaction between phytoplasma concentration and green-flowering stability was studied in hydrangea cultivars. Three green and 18 nongreen cultivars were subjected to polymerase chain reaction (PCR) analysis to determine Japanese hydrangea phyllody (JHP) phytoplasma infection. The results showed that JHP-phytoplasma was detected only in ‘Midori’ plants, which have green sepals. ‘Midori’ plants were propagated, and from 29 rooted cutting plants, they were grouped into three types on the basis of sepal color, that is, green (75.9%), blue-green (13.8%) and blue (10.3%) sepals. To clarify the variability in the sepal color of ‘Midori’ plants, JHP-phytoplasma concentration in the sepals and leaves of green-, blue-green- and blue-flowering plants was determined by PCR analysis. The semiquantitative PCR comparisons of 370 bp DNA fragments showed that the JHP-phytoplasma concentrations in green sepals were 16 times higher than that in blue-green sepals. JHP-phytoplasma could not be identified by PCR analysis in blue sepals and leaves. These results showed that JHP-phytoplasma concentration correlated with green sepal stability in ‘Midori’ plants. A histological observation of sepals showed that epidermal cells of blue and blue-green sepals had a dome shape. Otherwise, green sepals were leaflike with flat epidermal cells, and palisade parenchyma cells with numerous chloroplasts.     

Structural identification of anthocyanins and analysis of concentrations during growth and flowering in buckwheat (Fagopyrum esculentum Moench) petals

The anthocyanin profiles and variety/breeding-line differences of anthocyanin concentrations in petals of common buckwheat flowers have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-rhamnoside, and cyanidin 3-O-galactosyl-rhamnoside were isolated from the petals of common buckwheat (Fagopyrum esculentum Moench), separated using high performance liquid chromatography and identified using reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry techniques. In every variety/breeding line tested, cyanidin 3-O-rutinoside was detected as the major anthocyanin and the next is cyanidin 3-O-glucoside whereas cyanidin 3-O-rhamnoside and cyanidin 3-O-galactosyl-rhamnoside were trace or not detectable in white and pink flowered buckwheat. Of all the varieties/breeding lines tested, Gan-Chao, a Chinese variety, contained the highest amount of anthocyanins. The largest part of cyanidin moiety was presented as a proanthocyanidin form (PAs-Cy). Anthocyanins and PAs-Cy in petals were increased along with increase of flower development stages. Therefore, fully developed petals of red flowered buckwheat, especially Gan-Chao, are promising as a new anthocyanin-rich material for food processing.     

Seroepidemiology of Toxoplasma gondii and Neospora caninum in seals around Hokkaido, Japan

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.     

Ascophyllan Purified from Ascophyllum nodosum Induces Th1 and Tc1 Immune Responses by Promoting Dendritic Cell Maturation

Marine-derived sulfated polysaccharides have been shown to possess certain anti-virus, anti-tumor, anti-inflammatory and anti-coagulant activities. However, the in vivo immunomodulatory effects of marine-derived pure compounds have been less well characterized. In this study, we investigated the effect of ascophyllan, a sulfated polysaccharide purified from Ascophyllum nodosum, on the maturation of mouse dendritic cells (DCs) in vitro and in vivo. Ascophyllan induced up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in bone marrow-derived DCs (BMDCs). Moreover, in vivo administration of ascophyllan promotes up-regulation of CD40, CD80, CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen cDCs. Interestingly, ascophyllan induced a higher degree of co-stimulatory molecule up-regulation and pro-inflammatory cytokine production than fucoidan, a marine-derived polysaccharide with well-defined effect for promoting DC maturation. Ascophyllan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in the presence of DCs in an IL-12-dependent manner. Finally, myeloid differentiation primary response 88 (MyD88) signaling pathway was essential for DC maturation induced by ascophyllan. Taken together, these results demonstrate that ascophyllan induces DC maturation, and consequently enhances Th1 and Tc1 responses in vivo. This knowledge could facilitate the development of novel therapeutic strategies to combat infectious diseases and cancer.