Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).     

Comparison between old-growth stands and secondary stands regenerating after clear-felling in warm-temperate forests of Yakushima, southern Japan

In order to infer successional changes in structure, species composition and diversity of warm-temperate forest, we compared secondary stands regenerating after clear-felling (41–64-years old) with old-growth stands at altitudes between 300 and 800 m on Yakushima Island, southern Japan. Stem density and maximum stem diameter differed between secondary and old-growth stands, but basal area and aboveground biomass did not. At lower altitudes, the dominant species in old-growth stands with a strong sprouting capacity (Castanopsis cuspidata) also dominated secondary stands, and species composition of secondary and old-growth stands was similar. At higher altitudes, by contrast, the dominant species in old-growth stands (Distylium racemosum) had little sprouting capacity and was poorly represented in diverse secondary stands, which were dominated by Castanopsis or other less abundant species. Secondary stands had greater species diversity (Shannon–Wiener index) than old-growth stands, particularly at higher altitudes. This was due to greater species richness resulting from higher stem density per area, but not to greater evenness. We grouped the component species that share ecologically similar traits into four guilds (fagaceous, primary evergreen, secondary evergreen and deciduous species). Secondary stands were characterized by greater numbers of deciduous and secondary evergreen species. We concluded that different sprouting capacities of dominant species and different regeneration traits among guilds are responsible for the change in species composition and diversity during succession.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.     

Oryzacystatin-II, a cystatin from rice (Oryza sativa L. japonica), is a dimeric protein: possible involvement of the interconversion between dimer and monomer in the regulation of the reactivity of oryzacystatin-II

We examined the biochemical and structural properties of oryzacystatin-II, a phytocystatin in rice (Oryza sativa L. japonica), under heat-stress conditions. The enzyme inhibitory reactivity of oryzacystatin-II was enhanced by heating in a temperature-dependent manner and reached a maximum level by heating at 65 degrees C for 10 min. Size-exclusion chromatography showed that oryzacystatin-II forms a homodimer at ambient temperature and that the enhancement of inhibitory reactivity is due to the conversion of the dimeric to a monomeric form. The monomeric form of oryzacystatin-II reverted to the dimer during storage at 4 degrees C, suggesting that dimerization is an intrinsic property of oryzacystatin-II. The affinity of the monomer for cysteine proteinases was significantly higher than that of the dimer. This is the first paper to describe the noncovalent dimerization for a cystatin under nonstress conditions.     

Specific developmental stages of gametogenesis for radiosensitivity and mutagenesis in rice

Summary Developmental stages during gametogenesis of rice were histologically examined in the period from differentiation of reproductive organs to anthesis. Plants were exposed to acute X-rays of 20 Gy. Radiosensitivity and mutation frequency were investigated in relation to the developmental stages of reproductive organs. The most radiosensitive stage, as measured by reduction of the M1 pollen-and seed-fertilities, was the last premeiotic interphase. Mutations induced at different developmental stages were scored in M3 strains. Sterility mutants and short-culm mutants were most frequently observed. Grain shape, panicle morphology, heading-date and endosperm character mutants were induced at a relatively low frequency. The overall mutation frequency varied with the developmental stage at the time of irradiation. The highest overall mutation frequency was observed when radiation was applied 10 days before anthesis, the late tetrad stage of microspores. Radiation exposure of florets at the late tetrad stage was found to be a more efficient method of inducing a large number of mutations than radiations applied to seeds and fertilized egg cells.     

Endothelin receptor mediating contraction of isolated bovine coronary artery

We studied endothelin (ET) receptors and their subtypes on isolated bovine coronary arteries. Endothelin receptors that mediated contraction of isolated bovine coronary artery were characterized by the use of antagonists and agonists. Contractions induced by the nonselective agonist ET-1 (10-10-10-7 M) were not affected by the removal of the endothelium (pEC50: 8.52, maximal contraction: 105% of that induced by 60 mM KCl). BQ-123 (3 x 10-7 M) antagonized contractions of endothelium-denuded coronary rings induced by low concentrations of ET-1 (10-10 or 10-9 M), but potentiated the contractions induced by higher concentrations of ET-1 (3 x 10-8 and 10-7 M). BQ-788 (10-6 M) potentiated contractions induced by ET-1 (3 x 10-10 and 10-7 M). In the presence of BQ-788 (10-6 M), BQ-123 (3 x 10-8-3 x 10-6 M) concentration - dependently inhibited contractions induced by ET-1 (3 x 10-10 and 10-7 M) (pA2: 6.61). Sarafotoxin S6b (10-9-3 x 10-7 M) evoked contractions in the denuded coronary artery (pEC50: 8.49, maximal contraction: 139% of 60 mM KCl). The BQ-123 caused a concentration-dependent rightward shift of contractions induced by sarafotoxin S6b (pA2: 7.89). The present study indicates that ET-1 and sarafotoxin S6b contract the isolated bovine coronary artery by stimulating ETA receptors on smooth muscle cells, and that ETB receptors might suppress the ET-1-induced contractions.