Characterization of an endopolygalacturonase Gene cppg1 from Phytopathogenic Fungus Chondrostereum purpureum

Chondrostereum purpureum, a phytopathogenic fungus, produces endopolygalacturonase (endoPG) which has been suggested to have a causal role in the silver-leaf symptom of apple trees. In this paper, we detected C. purpureurn-derived endoPG at the infection sites using ELISA with a polyclonal antibody against endoPG I. A gene encoding endoPG I and its homolog were also isolated from the C. purpureum genome. The endoPG I gene was designated as cppg1. The cppg1 gene is the first fungal endoPG gene reported in the Basidiomycetes. Received 31 May 2000/ Accepted in revised form 13 September 2000     

PCR-based discrimination of Toxoplasma gondii from pigs at an abattoir in Okinawa, Japan

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondii SAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan.     

Identification and cornification-related gene expression of canine keratinocyte differentiation-associated protein, Kdap

The outermost layer of skin, the epidermis, is cornified epithelial tissue composed of keratinocytes. To maintain the structure and function of the epidermis, the regulation of proliferation, differentiation, and cornification of keratinocytes is crucial, and various soluble factors secreted by keratinocytes are involved in these regulations. Previously, work has shown that keratinocytes secreted the protein Kdap (keratinocyte differentiation-associated protein) associated with the formation of cornified cell envelopes, a specialized protective barrier structure on the periphery of terminally differentiating keratinocytes. In the present report, the canine counterpart of human Kdap is identified and an attempt has been made to define its physiological role in canine keratinization. Canine Kdap (cKdap) showed structural features commonly observed in other counterparts and is secreted from transfected cells. The expression profile of cKdap mRNA, which was restrictively expressed in cornified epithelial tissues besides skin has also been determined. These findings indicate that there is a strong association between cKdap expression and cornification, which supports previous observations that Kdap is involved in the synthesis and/or degradation of cornified cell envelopes in humans and mice.     

Anthracnose of three-leaf akebia (<Emphasis Type="Italic">Akebia trifoliata</Emphasis> Koidzumi) caused by <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

In October 2001, anthracnose caused by Colletotrichum acutatum Simmonds ex Simmonds was found on three-leaf akebia (Akebia trifoliata) in Saitama, Japan. This is the first report of anthracnose on three-leaf akebia caused by C. acutatum.     

Molecular epidemiological survey of benign Theileria parasites of cattle in Japan: detection of a new type of major piroplasm surface protein gene

Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.     

Bovine leukaemia virus envelope peptides cause immunomodulation in BALB/c mice

Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection.     

Changes in expression and localization of GPRC5B and RARalpha in the placenta and yolk sac during middle to late gestation in mice

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.     

Infection of bovine immunodeficiency virus and bovine leukemia virus in water buffalo and cattle populations in Pakistan

A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.