Structure-physicochemical function relationships of soybean beta-conglycinin heterotrimers

We purified four single molecular species of beta-conglycinin heterotrimers consisting of the alpha and beta subunits or the alpha' and beta subunits from mutant soybean cultivars lacking the alpha or alpha' subunit, respectively, and examined their structural features and physicochemical functions. The extent of the hydrophobicities of the heterotrimers was related to the number of the alpha or alpha' subunit. The thermal stabilities of the heterotrimers were mainly conferred by the subunit which had lower thermal stability. Solubilities at low ionic strength (mu = 0.08) of the heterotrimers containing the alpha or alpha' subunit were very similar to those of the alpha and alpha' homotrimers, respectively. Emulsifying abilities and heat-induced associations of the heterotrimers containing one beta subunit were similar to those of the alpha or alpha' homotrimer, whereas those of the heterotrimers containing two beta subunits were similar to those of the beta homotrimer.     

Establishment and biological characterization of canine histiocytic sarcoma cell lines

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.     

Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan

The antimicrobial susceptibility of 201 Listeria monocytogenes isolates from foods, environments, animals and human patients in Japan was determined. All isolates were susceptible to ampicillin, the first choice of drug for listeriosis treatment, chloramphenicol, dihydrostreptomycin, erythromycin, enrofloxacin, gentamicin, kanamycin, lincomycin, nosiheptide, salinomycin, vancomycin, and virginiamycin. A human strain was resistant to oxytetracycline. The Minimum Inhibitory Concentration (MIC) for 50% of the strains and the MIC for 90% of the strains were comparable in all the isolates. This is the first investigation to compare antibiotic resistances between isolates from foods and isolates from human patients in Japan. The result showed that most of the isolates were susceptible to antibiotics used in this study.     

A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.     

Nitric oxide induces apoptosis in bovine luteal cells

We previously showed in in vivo and in vitro studies that nitric oxide (NO) is engaged in luteolysis in cattle. Nitric oxide produced locally in the bovine corpus luteum (CL) inhibits progesterone (P4) synthesis and is suggested to be a component of the luteolytic cascade induced by uterine prostaglandin (PG) F2alpha. In the present study, the molecular mechanisms of NO action during structural luteolysis were studied in cultured bovine luteal cells (Days 15-17 of the estrous cycle). The effects of the NO donor (NONOate; 10(-4)M) on DNA fragmentation, cell viability, P4 production and caspase-3 activity were compared with those of PGF2alpha (10(-6)M). Moreover, mobilization of intracellular calcium [Ca2+]i and gene expressions of Fas-L, Fas, bcl-2, bax, and caspase-3 in the cells were determined by semi-quantitative RT-PCR after NONOate treatment. Caspase-3 activity was examined calorimetrically. Contrary to PGF2alpha NONOate decreased cell viability. DNA fragmentation after NONOate treatment increased by more than with PGF22alpha. NONOate increased mobilization of [Ca2+]i in the cells. Although the NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and bax mRNA and caspase-3 expression. The ratio of bcl-2 to bax mRNA level decreased in the cells treated with NONOate. Moreover, NONOate stimulated caspase-3 activity more effectively than PGF2alpha. The overall results suggest that NO is a luteolytic factor that plays a crucial role in regulation of the estrous cycle in structural luteolysis by inducing apoptosis of luteal cells in cattle.     

The occurrence of Listeria monocytogenes in imported ready-to-eat foods in Japan

Quantitative analyses of Listeria monocytogenes in imported ready-to-eat (RTE) foods sold at retail stores in Japan were performed. Of the 77 non-cooked meat products, 6 samples (7.8%) tested positive. The levels of contamination of 4 of the samples were below 100 colony-forming units (CFU)/g, which is the microbiological criterion for L. monocytogenes in RTE foods as determined by Codex. However, Listeria cells at levels of 100 and 400 CFU/g were detected in a salami sample and a raw ham sample, respectively. All of the 70 cheese samples and the 3 samples made from raw ham and cheese showed negative test results. These results suggest that imported RTE foods are potential sources of the causative agent of listeriosis.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.