It has been reported that when the rat fetus is treated with streptozotocin (STZ) in vivo, islet B cells are destroyed but later recover. To investigate the process of the recovery of B cells after in vitro treatment of the fetal pancreas with STZ and the role of epidermal growth factor (EGF) in the recovery of B cells, we measured the level of insulin released from the cultured fetal pancreas and examined it histologically. As a result, we immunohistologically confirmed the regeneration of B cells in the pancreas that had been cultured for 48 hr after destruction of islet B cells by STZ treatment. An immunohistologic study using proliferating cell nuclear antigen (PCNA) showed that without the addition of EGF, the cell division index was significantly higher in the STZ-treated group (STZ group) than in the untreated group (intact group), whereas with the addition of EGF, the cell division index increased in both groups, but EGF did not have a significant cell division-promoting effect on the pancreas in the STZ group. The addition of EGF caused a significant decrease in the concentration of insulin in culture medium in both groups. These results indicate that EGF has a cell growth-promoting effect on intact fetal pancreas in vitro but has the effect of inhibiting the release of insulin, and thus suggest that EGF does not trigger the regeneration of islet B cells.

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Cranial features (development, individual variation, and sexual dimorphism) were examined from the 23 metrical characters and 2 nonmetrical characters (the degree of closure of the 9 cranial sutures and the presence of sagittal crest) in the two spotted seal specimen groups at the Nemuro Strait, Hokkaido. One specimen group was incidentally taken in the salmon trap nets between 1982 and 1983 (n=70), and the other was randomly sampled by damage control kill between 1997 and 1998 (n=82). The development of morphometrical characters of skulls ceased at 5.6, 10.7, 7.9, and 11.9 yr. old, for 1982-83 male, 1982-83 female, 1997-98 male and 1997-98 female, respectively. The sutures were half ankylosed till approximately 10 yr. old in both sexes. The sagittal crest began from about 5 yr. old in male. Individual variation of skull was large in the feeding, breathing, and facial-expression apparatus. On the other hand, the variation of braincase, and skull concerning to the movement of head/neck tended to be small. Only 1997-98 specimens exhibited a sexual dimorphism in skull characters except for the braincase, whereas the dimorphism was not found in 1982-83 specimens. We could not detect the significant difference between two specimen groups, although there were a few differences in characters related to the rostrum and mandible.

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Chicken interferon-γ (ChIFN-γ) was expressed by baculovirus in a C-terminal truncated form, namely ChIFN-γT, to accelerate the secretion of the expressed protein. It is also expressed as ChIFN-γT bearing poly His tag, ChIFN-γTHis, for easy purification. The expressed proteins were detected by SDS-PAGE analysis with Coomassie brilliant blue staining. The purified ChIFN-γTHis with nickel chelated column showed anti-viral activity in vitro and stimulation of the secretion of nitrogen intermediates such as nitric oxide in chicken peripheral blood mononuclear cells. Antiserum against ChIFN-γTHis recognized the 15 kDa, 16 kDa, and 32 kDa bands that seemed to be an unglycosylated monomer, a glycosylated monomer, and a homodimer of ChIFN-γTHis in the culture supernatant, respectively. The anti-serum also recognized around 14 kDa and 28 kDa bands in the sera of chickens or concanavalin A stimulated spleen cell culture supernatants that seemed to be monomeric and dimeric forms of a natural ChIFN-γ, respectively.

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To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.

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Candida species in clinical urine samples were identified directly by the newly developed method of PCR analysis on 25S ribosomal DNA (rDNA). Two dogs were referred to the Animal Medical Center, Nihon University School of Veterinary Medicine, Fujisawa, Kanagawa, Japan for the examination of chronic cystitis. Microscopic examination of urine samples from these dogs revealed yeast cells. Urine culture on Sabouraud's dextrose agar at 27°C for 5 days produced white to cream colored colonies. The isolates were identifical to Candida albicans and C. parapsilosis by mycological examination, respectively. The nucleotide sequences of 25S ribosomal DNA from these urine isolates showed 99% similarity to those of a reference strain of Candida albicans or C. parapsilosis. The nucleotide sequences of 25S rDNA obtained directly from urine samples were also identical to C. albicans and C. parapsilosis, respectively. Confirming the results on the isolates cultured from the same urine samples. This PCR analysis method could be available for the direct identification of Candida species in urine samples within 2 days.

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The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca²⁺ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB153 medium containing several growth factors and Ca²⁺-free fetal bovine serum, then stimulated with Ca²⁺. Immunoblotting demonstrated strong expression of β1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca²⁺ stimulation, whereas β1 integrin expression was decreased in response to increasing concentrations of Ca²⁺. These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis.

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Expression of 150 kda oxygen-regulated protein, ORP150, was examined in the atheromatous lesions on aortic valves in high-fat diet fed mice. Immunohistochemical staining revealed that ORP150 was expressed on the surface of plaque and was co-localized with phagocytes bearing Mac-3, a mouse macrophage differentiation antigen. These findings suggest that ORP150 is involved in the development of the atheromatous plaque. Titer of autoantibody against ORP150 was gradually elevated in parallel with the length of period of high-fat diet feeding. These results suggest that the deposition of immunocomplex toward ORP150 antigen is involved in atheromatous plaque progression.

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The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T. gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.

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Cryptosporidium parvum (C. parvum) is recognized as a significant pathogen in humans and animals, primarily as a cause of diarrheal illness. Recent genetic and biological studies indicate that C. parvum is not a single species but composed of genetically distinct multiple genotypes. Thus, it is valuable to distinguish between genotypes in the epidemiology of Cryptosporidium infection in humans and animals. Although C. parvum has been detected in humans and animals in Japan, the genotype of isolates remains unclear because identification has been performed only by conventional microscopy. We report herein the genotypes of C. parvum isolates distinguished by the polymerase chain reaction (PCR)-based diagnostic method. C. parvum isolates, originally obtained from a patient and a pet dog, were found to have cattle and dog genotypes, respectively.

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A 14-year-old female cougar died from gastroduodenal adenocarcinomas and rectal adenoma. At necropsy, polypoid tumor masses of various sizes were scattered on the mucosal surfaces of the stomach, duodenum, and rectum. Histologically, the gastric tumor was diagnosed as an intestinal type adenocarcinoma and the tumor cells metastasized to the mesenteric lymph nodes, spleen, and lung. Helicobacter-like organisms were detected in the lumina lined by foveolar epithelium. In the duodenum, the carcinoma cells were localized in the lamina propria and many of them were intensely positive for proliferating cell nuclear antigen (PCNA). In contrast, the rectal adenoma had a lower number of PCNA-positive cells. In the rectum, chronic inflammation with numerous spirochetes was also noted. These results indicated that the occurrence of the gastrointestinal tumors might be associated with the bacterial infection described above.

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A 6-year and 9 month-old, male, Shih Tzu dog showed ataxia and trembling. By MRI examination, a mass (1 cm) was found in the right cerebellum. As the dog did not respond to radiation therapy, and showed a rise of intracranial pressure, he was euthanized. The cerebellar mass was soft and hemorrhagic. Histologically, the mass contained vimentin-positive spindle- or polyhedral-shaped cells arranged in a cord-like pattern. Mucinous materials were observed in the intercellular spaces. Ultrastructural examination revealed cell processes, microtubule-like structures and desmosomes. The case was diagnosed as myxoid type meningioma.

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Ovaries were collected from normal cycling female guinea pigs on each day of the estrous cycle (n=5 per day) for histological analysis of ovarian morphology. Three types of ovarian cysts were observed: serous cysts, follicular cysts and parovarian cysts. The most common were serous cysts (cystic rete ovarii), which were present throughout the estrous cycle with an overall incidence of 63.5% (54 out of 85 animals). Follicular cysts occurred in 22.4% of guinea pigs overall (19 out of 85). Only one parovarian cyst (1 out of 85) was observed in the present experiment. Follicular cysts always coincided with serous cysts and were less common during diestrus. The incidence of serous cysts did not vary significantly across the estrous cycle. In a second experiment, cycling female guinea pigs were arrested in a prolonged luteal phase by a progesterone implant in order to achieve ovarian synchrony. They were then treated with inhibin antiserum (0.5 or 1 ml per animal i.v.; n=6 per group) or normal goat serum (controls; n=6 per group). There was a dose dependent increase in the incidence of serous ovarian cysts following passive immunization against the inhibin α-subunit. These results suggest that serous cysts are a normal component of the cyclic guinea pig ovary and that alterations in the inhibin-follicle-stimulating hormone system appear to modulate the incidence of serous ovarian cysts in this species.

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The first case of human babesiosis was reported in Japan. The epidemiology of this disease in Japanese nature remains unclear. In this study, 97 common field mice captured in Hokkaido, Japan, were examined. Blood specimens absorbed onto filter papers were eluted and tested by nested PCR using specific primers for the B. microti nuclear small subunit rRNA genome. Twenty-three percent (11/47) of Apodemus speciosus and four percent (2/50) of Clethrionomys rufocanus were positive. The 159-bp primary sequences of PCR products tested exhibited 97.5% and 96.8% homology with those of the human isolate in Japan and of U.S. strains of B. microti, respectively.

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A survey of Salmonella was carried out in fecal samples of 887 pigs with diarrhea collected from 235 pig farms between April 1996 and March 2001. Salmonella was isolated from 84 feces (9.5%) of 887 pigs and from 45 (19.1%) of 235 farms. The higher prevalence was found in weaned pigs (12.4%) and fattening pigs (17.3%) than in sows (4.2%) and suckling pigs (4.5%). Isolation rates of S. Typhimurium were higher from weaned and fattening pigs than from the others. Therefore, risk of horizontal infection of S. Typhimurium will increase, if no adequate health managements are practiced when weaned and fattening pigs have diarrhea.

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The effect of two natural retinoids and synthetic retinoids with or without retinoid synergists on the proliferation and differentiation of 3 melanoma cell lines were investigated in vitro. No retinoids showed significant growth inhibitory effect on these cell lines when used alone, however, cell differentiation and significant growth inhibition were observed when treated with a combination of retinoids and a retinoid synergist. This study may suggest that, though the cells showed low susceptibilities when retinoids were treated alone, the combination of retinoids and a retinoid synergist may be effective to control the growth of canine melanoma cell lines.

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To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 μg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17β-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17α-hydroxylase/C_17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3β-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.

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Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P₄), androstenedione (A₄), estradiol-17β (E ₂) and Prostaglandin F_2α (PGF _2α) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P₄ and PGF_{2 α} were similar among the different sizes of follicles. The release of A₄ was observed in middle and large follicles. E₂ concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A₄ release. PGF_2α release was considerably high after LH infusion compared to the control group. Infusion of PGF_2α increased P₄ and A ₄ release but there was no change in E₂ release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF_2α seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.

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