Characterization of two putative mechanosensitive channel proteins of Campylobacter jejuni involved in protection against osmotic downshock

Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission.     

Identification of differentially expressed genes during flower opening by suppression subtractive hybridization and cDNA microarray analysis in Eustoma grandiflorum

The forward and reverse suppression subtractive cDNA libraries were constructed in petals of Eustoma grandiflorum at bud stage (stage 1) and anthesis (stage 7). Approximately 1000 clones were isolated from stage 1- (S1) and stage 7-specific (S7) libraries. The clones were sequenced and assembled, which yielded 98 contigs and 444 singletons. BLAST search was conducted on these assembled sequences. Generally, probes isolated from the S7 library exhibited higher expression at stage 7 by microarray analysis, as did those of the S1 library at stage 1. A clone set from the S7 library contained genes from later steps of anthocyanin biosynthesis pathway, terpene synthases, GAST (gibberellic acid-stimulated) family proteins, xyloglucan endotransglucosylase/hydrolase, glycosidases, and stress- and senescence-related proteins. In contrast, the S1 library contained genes associated with flavonol biosynthesis, phenylpropanoid metabolism, terpenoid metabolism, and floral organ development. Gene expression profiling for flavonoid biosynthesis was in accordance with preferential accumulation of flavonols at bud stages and anthocyanins at anthesis.     

Distribution of the introduced cyprinid herpesvirus 3 in a wild population of common carp, Cyprinus carpio L.

Cyprinid herpesvirus 3 (CyHV-3), which causes a lethal disease in common carp, Cyprinus carpio L., and koi, C. carpio koi , first occurred in Lake Biwa, Japan in 2004. To elucidate distribution of CyHV-3 in a wild common carp population, we conducted a PCR survey of CyHV-3 among such fish in Lake Biwa in 2006. Only 6% (1/18) of the common carp smaller than 300 mm were positive with PCR, whereas 31% (18/58) of fish larger than 300 mm were positive. To evaluate their past exposure to CyHV-3 infection based on the presence of antibodies, we also measured the levels of serum anti-CyHV-3 antibodies in the carp, using an enzyme-linked immunosorbent assay. None (0/26) of the fish smaller than 300 mm was positive for the antibodies, whereas 54% (33/61) of fish larger than 300 mm were positive. Of the antibody-positive individuals, 44% (14/32) were also positive by PCR strongly suggesting that wild common carp that survived infection become CyHV-3 carriers. Five individuals were positive by PCR but negative for antibodies indicating that their infection with CyHV-3 had occurred recently. These results suggest that transmission of CyHV-3 from carriers to naïve common carp is still occurring in Lake Biwa.     

P-66 Disseminated histiocytic sarcoma in a dog receiving long-term immunosuppressive therapy

Canine cutaneous histiocytoma is a common tumor of young dogs. Nodules are single or multiple, alopecic and well demarcated. We studied 15 young dogs with cutaneous histiocytoma. Cutaneous nodules were cytologically and histologically investigated using routine techniques. The subject for morphometry was the nuclear area of histiocytes. The figures were statistically interpreted (Student's t- test). Cytologically, histiocytes represented the largest population, being large-sized cells. The nucleus:cytoplasm ratio was large with oval or bean-shaped nuclei and a fine arrangement of chromatin. The cytoplasm was discrete and basophilic, or abundant and foamy. Five regressive stages were histologically described. Stage 1 was represented by dermal and hypodermal histiocyte proliferation, covered by an intact, uninfiltrated and atrophic epidermis. Epidermal ulcers and necrotizing foci with neutrophilic, lymphocytic and plasma cell infiltrations occurred in Stages 2, 3 and 4. Stage 5 looked like a chronic dermatitis. In Stage 1, the nuclear area was the smallest (4615 ± 62 pixels) with a small coefficient of variation (14.28%), proving a homogeneous population. Stage 2 histiocytes had a larger and more heterogeneous nuclear area (6116 ± 130 pixels) with a 21.12% coefficient of variation. In Stage 3, the average nuclear area (6431 ± 115 pixels) was larger than that for Stage 2 and had a 19.18% coefficient of variation. There were no significant differences between the nuclear areas of histiocytes from Stages 2 and 3; however, significant differences occurred between Stages 1 and 2, and between Stages 1 and 3.
Funding: Self-funded .     

Establishment and characterization of a cell line, MCO-Y4, derived from canine mammary gland osteosarcoma

A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39+/-4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Basin-scale distribution pattern and biomass estimation of Japanese anchovy Engraulis japonicus in the western North Pacific

The distribution pattern and biomass of the Japanese anchovy Engraulis japonicus in the offshore region of the western North Pacific (north of 35°N and west of 170°E) were studied using a quantitative echosounder. This is the first attempt at such a study in this region. Data were collected in summer from 2004 to 2007. The biomass was estimated using data collected at 38?kHz. Species compositions in the backscatterings from pelagic fish were assigned based on the results of trawl hauls taking account of sea surface temperature (SST). Japanese anchovy tended to be high density to the west of 153°E and were distributed in an SST range of 9?C24?°C. Although the temporal and spatial coverage of the survey differed each year, at least 1.5?C3.4 million tons of Japanese anchovy were present in the survey area between 2004 and 2007. To take account of the spatial coverage of the survey each year, the most reliable biomass estimate for this region in the time period was 3.4 million tons (coefficient of variation?0.22).     

Color development of proanthocyanidins in vanillin-hydrochloric acid reaction

The influence of proanthocyanidin (PA) structures contained in bark on color development in the vanillin-hydrochloric acid (V-HCl) method used widely as a quantitative method for measuring PA were examined. The maximal absorption wavelength was different in terms of the bark from which the PA was obtained. Phenyl nucleus (resorcinol, phloroglucinol) constituting the A-ring of PA reacts with vanillin to produce the color. The maximal absorption wavelengths of the solutions from synthesized procyanidin and profisetinidin were 500 and 540 nm, respectively, indicating that the color tone differs in the V-HCl method based on the hydroxylation patterns of the A-ring. The colored solution of (+)-catechin with vanillin was dialyzed, and the resulting product (C-VC) was analyzed by gel permeation chromatography and ¹H nuclear magnetic resonance. It was found that C-VC was a polymer complex consisting of 9mol (+)-catechin moieties and 10mol vanillin moieties. It was presumed that the cationized vanillin molecules that do not combine with (+)-catechin play an important role on color development in the presence of C-VC.This study was presented at the 44th and 45th Annual Meetings of The Japan Wood Research Society, Nara and Tokyo, April 1994 and April 1995