Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Hypercalcemia in a dog with resolution of iatrogenic Cushing's syndrome

A six-year-old spayed Pug was presented with crust formation and ulcer on the skin. The patient had received long-term glucocorticoid therapy for treatment of tentatively diagnosed panniculitis. Severe calcification and pyoderma was observed and the patient was diagnosed with iatrogenic Cushing's syndrome and predonisolone was gradually withdrawn. After the withdrawal, the patient developed marked hypercalcemia (15.3 mg/dl) and finally died from renal failure. It is postulated that the eluted calcium from the calcified lesions may have contributed to the high serum calcium level as the underlying disease was not identified on necropsy.     

Phylogenetic analysis of the hemagglutinin (H) gene of canine distemper viruses isolated from wild masked palm civets (Paguma larvata)

Hemagglutinin (H) gene of two CDV isolates, the Haku93 and Haku00 strains, from masked palm civets was molecularly analyzed. H genes of both two CDVs contained one open reading frame encoding 607 amino acids. Nucleotide and predicted amino acid sequences of H gene of the CDV Haku93 and Haku00 revealed high similarity to those of recent field isolates such as the Yanaka and Tanu96, while they showed limited identity to those of old vaccine strains. Potential N-linked glycosylation sites in both Haku93 and Haku00 were identical to other recent CDV isolates. Phylogenetic analysis revealed that the CDV strains derived from masked palm civets were classified into the group of recent Japanese CDV isolates.     

Follicular microvasculature and angiogenic factors in the ovaries of domestic animals

The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate the process of thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation. A recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the process of thecal angiogenesis during follicular development. We are developing a novel technology for the induction of follicular development using the technique of in vivo gene administration. Here, we summarize the recent progress of our research.     

Melanin biosynthesis as a prerequisite for penetration by appressoria of Colletotrichum lagenarium: Site of inhibition by melanin-inhibiting fungicides and their action on appressoria

Melanin biosynthesis by appressoria was studied in relation to their penetrating ability using tricyclazole [5-methyl-1,2,4-triazolo(3,4-b)benzothiazole], pp 389 [4,5-dihydro-4-methyltetra-zolo(1,5-a)quinazolin-5-one], and pyroquilon [1,2,5,6-tetrahydropyrrolo(3,2,1-i,j)quinolin-4-one], and color mutants of Colletotrichum lagenarium. Tricyclazole at 100 μM inhibited melanin biosynthesis by appressoria of C. lagenairum 104-T, and caused accumulation of 3,4-dihydro-4,8-dihydroxy-1(2H)naphthalenone (DDN) in the culture medium. By contrast, DDN was not detected in culture media of tricyclazole-treated mutant 8015, which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene (1,3,8-THN). Vermelone restored melanization of appressoria of albino mutant 79215 and of the parent strain 104-T treated with tricyclazole, pp 389, and pyroquilon; however, scytalone restored melanization only in appressoria of albino mutant 79215. These results indicate that tricyclazole, pp 389, and pyroquilon inhibit the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway of appressoria of C. lagenarium. Colorless appressoria formed in the presence of the three melanin-inhibiting chemicals germinated laterally on nitrocellulose membranes and rarely penetrated the membranes. On the other hand, when pigmented appressoria were restored by application of vermelone in the presence of the three chemicals, lateral germination of the appressoria was largely suppressed, and the membranes were effectively penetrated. From these results, it is concluded that the major effect of tricyclazole, pp 389, and pyroquilon on appressoria of C. lagenarium, causing failure of penetration, is the inhibition of melanization. Effects of the chemicals on other metabolic functions can be precluded as significant factors affecting the penetration process.     

Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells

Dendritic cells are specialized antigen‐presenting cells with immuno‐modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)‐stimulated adherent cells (PHA‐Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA‐Ad cells enhanced interferon‐γ from autologous PBMC in vitro. PHA‐Ad cells also stimulated antigen‐independent proliferation of peripheral blood lymphocytes. These results suggest that PHA‐Ad cells from PBMC possess a stimulatory function to evoke anti‐tumour immunity and that they demonstrate potential for therapeutic applications in dogs.     

Neutrophils contact to plasma membrane of keratinocytes including desmosomal structures in canine pemphigus foliaceus

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that affects certain mammals including dogs. In canine PF, neutrophils are infiltrated intensely into pustular lesions including acantholytic cells, although neutrophilic infiltration is not characterized in human PF. The roles of the neutrophils in the cutaneous lesions of canine PF have not yet been understood. The purpose of this study was to characterize the ultrastructural features underlying the acantholysis with pustule formation in canine PF. Four dogs diagnosed as PF on the basis of clinical signs, histopathological findings, and direct and indirect immunofluorescence examinations were performed. Electron microscopy revealed that the acantholytic cells were adjacent to multiple neutrophils in the pustules. At the contact points between neutrophils and acantholytic keratinocytes, half-desmosomes of acantholytic keratinocytes with intact attachment plaques were observed within invaginations of neutrophils. Furthermore, on the surface of acantholytic cells in the pustules, neutrophil granules seemed to be secreted to the surface of acantholytic cells and to degenerate the half-desmosome structures. Neutrophils were also observed within the epidermis adjacent to the pustule. At the intercellular gap between two dissociated keratinocytes, neutrophils inserted its pseudopodia into the gap between the two half-desmosomes of keratinocytes. These findings taken together suggested that, at least in the areas where we analyzed ultrastructurally, neutrophils contact desmosomal structures and seem to play some parts in separation of keratinocytes and degeneration of split-desmosomes in pustules of dogs with PF.     

A Possible treatment strategy and clinical factors to estimate the treatment response in Bebesia gibsoni infection

The effectiveness of combination therapy using clindamycin, metronidazole and doxycycline against canine babesiosis, and the usefulness of platelet count and the plasma C-reactive protein (CRP) concentration as an estimation factor for treatment, were evaluated in four dogs experimentally infected with Babesia gibsoni. The combination therapy successfully eliminated B. gibsoni in peripheral blood in 3 of 4 dogs, however the remaining dog showed obvious uncontrolled relapse after a temporary recovery. In addition, it was shown that CRP levels decreased in an inverse relationship to the recovery of packed cell volume and therefore CRP levels could be used as an optional clinical marker to estimate the response to treatment.     

Pyridinoline concentrations in muscular and skin collagen of fish and relationship between collagen solubility and pyridinoline concentration in fish muscular collagen

Pyridinoline (Pyr), one of the mature crosslinks of collagen, was determined in muscular collagen of three species of fish. The amounts of muscular Pyr in red sea bream, yellowtail, and tiger puffer were 3.4, 8.8, and 50.3 mmol/mol collagen, respectively, indicating that the Pyr concentration in muscular collagen differs greatly among fish species. The Pyr concentration in tiger puffer muscular collagen was the greatest, but it was only one-fourth that in rabbit muscle. As in mammalian skin collagen, Pyr was not detected in skin collagen of red sea bream and yellowtail. However, tiger puffer skin contained Pyr (3.75 mmol/mol collagen). The presence of Pyr would have a relationship to some features of tiger puffer skin, such as mechanical strength and thickness. Pyr concentrations in acid-soluble collagen (ASC), pepsin-solubilized collagen (PSC), and insoluble collagen (ISC) in muscles of three species of fish were determined. Pyr was found in ISC > PSC > ASC, from the highest to the lowest concentration, and the concentration in ISC was 45–200 times that in ASC. Therefore, Pyr crosslinks that are formed between collagen molecules would have a close relationship to collagen solubility.