Immunoblot analysis of specific antigen bands predictable for Dirofilaria immitis infection in cats

Serial sera from four mongrel cats experimentally inoculated with infectious larvae of Dirofilaria immitis were analyzed by immunoblot patterns against a phosphate buffered saline-extract of D. immitis. Antigen-specific protein bands detected indicate that the low molecular weight bands of 36, 32, 22, 19 and 14 kDa, are predictable for positive adult worm infection, suggesting diagnostic usefulness for adult D. immitis infection in cats.     

Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia

More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.     

Whey acidic protein (WAP) depresses the proliferation of mouse (MMT) and human (MCF-7) mammary tumor cells

We previously reported that the enforced expression of exogenous whey acidic protein (WAP) significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 and EpH4/H6 cells). This paper presents the first evidence that WAP also depresses the proliferation of mammary tumor cells from mouse (MMT cells) and human (MCF-7 cells). We established WAP-clonal MMT and MCF-7 cell lines, and confirmed the secretion of WAP from the WAP-clonal cells into culture medium. The enforced expression of WAP significantly inhibited the proliferation of MMT and MCF-7 cells in in vitro culture. FACScan analyses revealed that G0/G1 phase cell-cycle progression was disordered and elongated in the WAP-clonal MMT and MCF-7 cells compared to that of the control cells. The expression of cyclin D1 was significantly decreased in the WAP-clonal MMT and MCF-7 cells, suggesting that progression from the G1 to the S phase was delayed in the WAP-clonal cells. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary tumor cells via a paracrine mechanism.     

The effect of replacing brewers' grains with barley tea grounds in total mixed ration silage on feed intake, digestibility and ruminal fermentation in wethers

Four wethers were used in a 4 × 4 Latin square to study the feed intake, apparent digestibility, nitrogen balance and ruminal fermentation characteristics when fed total mixed ration (TMR) silages which included wet barley tea grounds (WBTG). The TMR silages were prepared using compound feed including wet brewers' grains (WBG), corn, oat hay, alfalfa hay, dried beet pulp, salt and vitamin-mineral supplement in a ratio of 30.7:15:8:24:10:12:0.15:0.15, respectively, on a dry matter (DM) basis. The WBTG and soybean meal mixture (7:3 on DM basis) were substituted for WBG at ratio of 0% (Control), 5% (LTG), 10% (MTG), and 15% (HTG) on DM of TMR. WBTG addition to the TMR silages increased lactic acid concentration, decreased pH, acetic acid and ammonia-N ( P  < 0.001). Feed intakes and digestibilities for LTG and MTG (except ether extract (EE) digestibility) treatments were not different from the control ( P  > 0.05). However, EE and neutral detergent fiber (NDF) intake, crude protein, EE and NDF digestibility was lower, but the DM and gross energy digestibility was higher for the HTG treatment compared to control ( P  < 0.01). With progressive increases in WBTG concentrations, nitrogen intake, fecal nitrogen and retention nitrogen did not differ, but the urinary nitrogen for MTG and HTG treatments were lower than that of the control ( P  = 0.001). The ruminal total volatile fatty acid concentration and the molar ratios of propionate and butyrate were higher, but the acetate, ratio of acetate to propionate and ammonia-N content were lower for the HTG treatment compared with the control ( P  < 0.05). Therefore, the possible proportion of replacing WBG with WBTG for TMR silage can be 10% or less of the diet DM.     

ISSR variations in eggplant (Solanum melongena L.) and related Solanum species

Inter-simple sequence repeat (ISSR) analysis was performed in eight cultivars of eggplant (Solanum melongena) and 12 accessions in eight related Solanum species to evaluate the applicability of this analysis for assessing the phylogenetic relationships and identifying cultivars. A total of 552 polymorphic amplified bands were obtained from 34 of the 100 primers tested, and the percentage of polymorphisms was 99.1%. Cluster analysis based on the ISSR markers classified the Solanum species into seven groups: (i) S. melongena; (ii) S. aethiopicum and S. anguivi; (iii) S. incanum; (iv) S. violaceum and S. kurzii; (v) S. macrocarpon; (vi) S. virginianum and (vii) S. torvum. Combining the ISSR markers obtained by a few of the 34 primers was enough for distinguishing of the eight cultivars of eggplant. This ISSR analysis was demonstrated to be available for the phylogenetic study and the cultivar identification.     

The mechanism by which mouse spermatozoa are injured during freezing

To improve the cryopreservation protocol for mouse sperm, we attempted to estimate the type and extent of cryoinjury at various steps of the process. First, we demonstrated that mouse sperm are sensitive to chilling at -15 C and that the sensitivity is dependent on the length of exposure. To estimate cryoinjuries, sperm suspensions were ice-seeded at -5 or -15 C, frozen with liquid nitrogen (LN(2)) gas and then frozen in LN(2). In one experiment, sperm seeded at -5 C were cooled slowly to -15 C before deep freezing. At various steps of the cryopreservation process, the sperm were warmed and their viability was assessed based on motility and the integrities of the plasma membrane and acrosome. The motility of frozen-thawed sperm was higher on seeding at -5 C (28%) than at -15 C (9%). The motility did not decrease when the sample was transferred from LN(2) gas to LN(2). To estimate cryoinjury of sperm, we presumed the viability of frozen sperm to be decreased by chilling, hypertonic stress and formation of intracellular ice. When the sperm suspension was cooled and seeded at -5 C, the motility decreased by 25% due to hypertonic stress. When the sperm were cooled in LN(2) gas, the motility decreased by 17% with the formation of intracellular ice. When the sperm were cooled to -15 C, the motility decreased by 51% from chilling. After seeding, the motility decreased by 18% due to formation of intracellular ice and by 7% due to hypertonic stress. Considering the results, it would be preferable to seed samples at a higher temperature to prevent intracellular ice from forming and to cool seeded samples rapidly enough to minimize chilling injury and hypertonic stress, but not too rapidly to allow intracellular ice to form.     

Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.     

Molecular evidence of the hybrid origins of leyland cypress (×Cupressocyparis leylandii)

Leyland cypress (×Cupressocyparis leylandii) has been regarded as an intergeneric hybrid between Monterey cypress (Cupressus macrocarpa) and Alaska cypress (Chamaecyparis nootkatensis) in the Cupressaceae. Each partial sequence of the 18S-rDNA from nuclear and therbcL from chloroplast DNA genomes was determined using above three species. At the 59th site from the 5′ end of the 152 bp of the determined 18S-rDNA sequence, one base difference was identified between Monterey cypress (T) and Alaska cypress (A). However, Leyland cypress showed either nucleotide A or T at this site. Since nuclear DNA is inherited by biparental mode, this result shows genetic evidence that the nucleic genome of Leyland cypress originates from Monterey cypress and Alaska cypress and that this species is of hybrid origin.     

Evaluation of antixenosis in soybean against Spodoptera litura by dual-choice assay aided by a statistical analysis model: Discovery of a novel antixenosis in Peking

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd–5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd–5th instar larvae than for 2nd instar.     

Composition and Regiodistribution of Fatty Acids in Triacylglycerols and Phospholipids from Red and Black Rices

Regiospecific profiles of fatty acids (FA) of triacylglycerols (TAG) and phospholipids (PL) isolated from red and black rices were investigated. The lipids extracted from red and black rices were separated by thin‐layer chromatography (TLC) into eight subfractions, respectively. With a few exceptions, the major lipid components were TAG (76.4–80.5%), free FA (7.2–9.8%), and phospholipids (3.5–3.6%), while hydrocarbons, steryl esters, diacylglycerols (1,3‐DAG and 1,2‐DAG), and monoacylglycerols were present in minor proportions (0.1–4.1%). The PL components isolated from the two cultivars were phosphatidyl choline (52.3–53.7%), phosphatidyl ethanolamine (22.3–23.1%), phosphatidyl inositol (20.6–21.3%), and others (<3.4%). No significant differences (P < 0.05) in FA distribution were found when these cultivars were compared. The principal characteristics of the FA distribution in the TAG and PL were evident in the rices between the two cultivars: unsaturated FA were predominantly located at the sn‐2 position (77.3–96.8%), while saturated FA primary occupied the sn‐1 or the sn‐3 position (35.0–78.7%) in these lipids. The results of this study could provide useful information to both consumers and producers during manufacture of traditional rice foods in Japan.