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.     

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.     

Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel,Anguilla japonica,at Completion of Yolk Resorption

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity.     

Effects of exposure to a 50 Hz electric field on plasma levels of lactate, glucose, free Fatty acids, triglycerides and creatine phosphokinase activity in hind-limb ischemic rats

We previously reported that extremely low frequency electric fields (ELF-EFs) affect energy metabolism in stressed conditions. To further confirm this, the effect of exposure to ELF-EFs on the experimental ischemic rat was examined. The test was based on a comparison of rats treated with EF alone, ischemic surgery alone, the combination of EF with ischemic surgery, or no treatment (double sham). The EF condition used in this study was an alternating current of 50 Hz EF at 17 500 V/m intensity for 15 min per day. The exposure to EF in ischemic rats significantly decreased plasma levels of free fatty acids and triglycerides, compared to those of the no treatment or EF alone group. The plasma lactate levels of two ischemic groups peaked on experimental day-4 and gradually decreased until the end of the study. The changes in the lactate levels induced by ischemia did not show any difference between rats treated with ischemia alone or a combination of ischemia with an EF. Any changes in plasma levels of glucose and creatine phosphokinase activity were not influenced by EF treatment. These results indicate that the EF effect on glycolysis parameters, plasma lactate or glucose levels, does not appear in a highly stressed condition and that EF effects varied dependent on the condition of organism but ELF-EF used in this study have impact on lipid metabolism parameter in a hind-limb ischemic rat. However, further studies are needed to elucidate the association of ELF-EF with the lipid metabolism system.     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.     

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.     

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.     

Antioxidative activities of a mycosporine-like amino acid,porphyra-334

Antioxidative activities of porphyra-334, a mycosporine-like amino acid extracted from laver were evaluated. Oxidation of linoleic acid induced by an alkyl-radical 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was successfully suppressed by porphyra-334 (0–200 μM). The simultaneous application of 0.02 μM α-tocopherol and 50 μM porphyra-334 effectively suppressed the AAPH induced oxidation level to approximately 40% of a single application of porphyra-334 after 10 min reaction. Porphyra-334 (0–200 μM) efficiently suppressed the lipid peroxidation induced by singlet oxygen although the antioxidative effect observed was relatively moderate at the initial stage of oxidation. These results suggested that porphyra-334 may function as an antioxidant which influences the storage stability of laver.