Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves.     

TRAIL-decoy receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicles

Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.     

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.     

Bacterial Leaf Spot of Ivy Caused by Xanthomonas campestris pv. hederae

A bacterial leaf spot disease was observed on Hedera helix (English ivy) and H. canariensis (Algerian ivy) in Japan. The causal agent was identified as Xanthomonas campestris pv. hederae (Arnaud 1920) Dye 1978. Received 13 May 2002/ Accepted in revised form 3 July 2002     

Highly regioselective amination of unactivated alkanes by hypervalent sulfonylimino-λ³-bromane

Amination of alkanes has generally required metal catalysts and/or high temperatures. Here we report that simple exposure of a broad range of alkanes to N-triflylimino-λ(3)-bromane 1 at ambient temperature results in C-H insertion of the nitrogen functionality to afford triflyl-substituted amines in moderate to high yields. Marked selectivity for tertiary over secondary C-H bonds was observed; primary (methyl) C-H bonds were inert. Addition of hexafluoroisopropanol to inhibit decomposition of 1 dramatically improved the C-H amination efficiencies. Second-order kinetics, activation parameters (negative activation entropy), deuterium isotope effects, and theoretical calculations suggest a concerted asynchronous bimolecular transition state for the metal-free C-H amination event.     

The Distribution of <Emphasis Type="Italic">Perilla</Emphasis> Species

Perilla (Lamiaceae) contains one tetraploid species, P. frutescens (L.) Britt. and three diploid species, P. citriodora (Makino) Nakai, P. hirtella Nakai and P. setoyensis G. Honda. Tetraploid species have been traditionally cultivated in Asia for their seed oil and for their fragrant leaves that are used as medicine or as a garnish for fish. The center of diversity is still obscure. To conserve the genetic resources, it is important to know the diversity of the tetraploid species. The three diploid species, which are possible parents of the tetraploid species, are all believed to be indigenous to Japan. Their distribution in China and Korea was clarified on the basis of herbarium and field surveys. The tetraploid species is assumed to have originated somewhere around the mid-to downstream area of the Changjiang River. Though Perilla is not cultivated as often in these areas as in northern China, Korea, the Himalayan region, or Myanmar, these areas should also be important for the conservation of genetic resources of tetraploid Perilla crops because of the expected high genetic diversity.     

Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.     

Changes in developmental duration of direct-seeded rice in a well-drained paddy field in response to late planting

We investigated changes in the developmental duration of rice cultivars with a wide range of maturities in response to late planting. Elucidating the relationship between cropping season and the progress of growth stages is important for establishing direct-seeded cultivation and creating growth models. Late planting of the intermediate to late maturing cultivars Akidawara and Hoshijirushi decreased the time from emergence to panicle formation and decreased the cumulative effective temperature (CET) and cumulative effective soil temperature (CEST). In the very early maturing cultivars Ichibanboshi and Fusakogane, the changes in number of days, CET, and CEST from emergence to panicle formation with late planting were small. From emergence to heading and maturity, the number of days, CET, and CEST of many cultivars tended to increase until mid-May and then decreased. The changes in the number of days, CET, and CEST caused by late planting were greater for the intermediate to late maturing cultivars than for the very early maturing cultivars. The differences between cultivars were greatest with early May sowing, and then decreased with later planting. Short-day condition revealed significant differences in the duration of vegetative growth and CET among cultivars, but long-day condition erased these differences. These results demonstrate that the photosensitivity and thermosensitivity of cultivars are especially important in crop planning and for creating growth models of direct-seeded rice.     

Characterization of Bovine herpesvirus type 4 isolated from cattle with mastitis and subclinical infection by the virus among cattle

An outbreak of contagious mastitis occurred among cattle on a farm, and bovine herpesviruses were isolated from the affected mammary tissues, scabs and abscess discharge of the cattle. A bovine herpesvirus type 4 (BoHV-4)-specific fragment was amplified from the isolates by polymerase chain reaction (PCR). Restriction endonuclease analyses demonstrated that the isolates were related to Movar-like European type BoHV-4. To determine the ratio of BoHV-4 subclinical infection in the cattle, a genomic survey was performed by PCR for cattle that were moved to the animal hygiene service station in Ibaraki prefecture. The BoHV-4 genome was occasionally detected in peripheral blood leukocytes, lymph nodes and nervous tissues. The rate of BoHV-4 subclinical infection was relatively high in the cattle.