The anatomical location of testes in mammals ranges from a location close to that observed in the embryo to a lower position usually involving a pendant scrotum. In scrotal mammals, the abdominal position of the cryptorchid testis, which elevates its temperature, is detrimental to spermatogenesis and causes infertility. Spermatocytes are sensitive but late spermatids are relatively resistant to thermal stress suggesting that the latter might be protected in some way. In general, most organisms express Hsp70 proteins, which play a crucial role in the protection of cells against thermal stress. We have found previously that the Hsc70t protein, a member of the Hsp70 family of proteins, is constitutively expressed in the late spermatids of mice. Here, we have utilized immunohistochemistry with anti-mouse Hsc70t antiserum to examine the expression of the spermatid-specific Hsp70 antigen in the testes of several mammalian species with different degrees of testes migration. Our data indicate that the antigen is conserved in the mammals including marsupials. We also examined whether antigens of Hsp70-related proteins were expressed in non-mammalian vertebrates including not only homoiothermal but also poikilothermal animals. The spermatid-specific Hsp70 antigens were not detectable in the testes of the animals examined. From results of immunohistochemistry with BRM22 monoclonal antibody which reacts broadly with Hsp70 family proteins, however, we revealed constitutive expression of antigens of Hsp70-related proteins in spermatogenic cells of the vertebrates. These results suggest that the expression of spermatid-specific Hsp70 protein may be involved in the developmental pathway during spermiogenesis in mammals rather than in thermotolerance.

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The variable region in the VP2 gene of twenty-three infectious bursal disease virus (IBDV) isolates, collected in Vietnam in 1997 and 1998, was amplified as cDNA by using the reverse transcription-polymerase chain reaction and sequenced. Analysis of amino acid substitutions and phylogenetic relationships of the deduced amino acid sequences (residues 206-350) showed that the nineteen Vietnamese vv IBDVs clustered with the European vv IBDVs, Japanese vv IBDVs and Chinese vv strains, and that the four Vietnamese virulent strains were closely related to European virulent strain 52/70. These results suggest that Vietnamese vv IBDVs, European vv IBDVs, Japanese vv IBDVs and Chinese vv strains have the same origin.

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To determine the extent of genetic diversity among isolates of Salmonella enteritidis obtained from outbreaks in Fukuoka Prefecture, Japan, from 1989 to 1994, we analyzed a total of 55 isolates of S. enteritidis obtained from 13 distinct outbreaks with pulsed-field gel electrophoresis. These isolates showed three different patterns in pulsed-field profile with Bln I digestion. The groups A, B and C consisted of three outbreaks isolates (Dice coefficient, F=1), of seven outbreaks (F= 0.56-0.94) and of three outbreaks (F=0.65-0.78), respectively. This result suggests that a few limited clonal lines of S. enteritidis were successively causing outbreaks in this area from 1989 to 1994.

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Brown adipose tissue (BAT) is the major site of non-shivering thermogenesis in rodents. Rapid angiogenesis is induced in association with adaptive hyperplasia of this tissue when the animal is exposed to cold. We demonstrated previously adrenergic activation of mRNA expression of vascular endothelial growth factor (VEGF) in rat BAT and its possible contribution to the cold-induced angiogenesis in this tissue. In the present study, we examined the effect of cold exposure on mRNA expression of other two angiogenic factors, VEGF-B and basic fibroblast growth factor (bFGF), in rats. Conventional Northern blot analysis revealed abundant mRNA expression of VEGF-B as well as VEGF, but not bFGF, in BAT. When rats were exposed to cold at 4°C, the VEGF mRNA level was increased by 2.7-fold in 1-4 hr and returned to the basal level within 24 hr. In contrast, the VEGF-B mRNA level did not change throughout the course of cold exposure. A significant expression of bFGF mRNA was detected in BAT by reverse transcription-polymerase chain reaction (RT-PCR). To evaluate the tissue bFGF mRNA level quantitatively, a competitive RT-PCR method was developed using a shorter RNA fragment as a competitor. The bFGF mRNA level in BAT was found to increase by 2.3-fold in 4 hr and decreased to the basal level within 24 hr after cold exposure. These results suggest that cold exposure leads to induce VEGF and bFGF rapidly and transiently in BAT, which in turn stimulate the proliferation of vascular endothelial cells in this tissue.

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CD45 is cell surface glycoprotein and expressed on all haematopoietic cells except mature erythrocytes and platelets. Eight isoforms of CD45 are generated by alternative splicing of exons 4-6. B220 including all three exons is expressed specifically on pan-B cell lineage. Recently, it was reported that B220 was expressed on apoptotic T cells induced by staphylococcal enterotoxin B (SEB). In the present study, we investigated the expression of B220 on murine thymocytes after whole-body X-irradiation. We used the forward light scattering of flow cytometry as a parameter of cell size, and defined two populations; FSC^high (normal cell size) and FSC^low (correspond to apoptotic cell in size) fraction. B220⁺ cells in FSC^high fraction reached a maximum value (35%) at 18 hr after irradiation. In FSC^low fraction, 40-60% cells were positive for B220 at any time points. These results suggest that B220 is expressed on thymocytes in the pre-apoptotic stage, because B220 was expressed on not only FSC^low cells but also FSC^high cells.

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The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK^-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.

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To investigate the structure of porcine genes involved in the beta-oxdation of fatty acid, we isolated the short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) genes from the pig. The cDNA of SCAD, MCAD and LCAD genes were 1899 bp, 1835 bp and1704 bp long and coded for 413-aa, 422-aa and 430-aa precursor proteins, respectively. Three genes, SCAD, MCAD and LCAD were mapped to 14p16.2-23.2, 6q32.4-33, and 15q24.2-26.3, respectively.

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Rats heavily infected with larval Taenia taeniaeformis show hyperplasia of the gastric mucosa accompanied by mucous cell proliferation, increase in the level of intragastric pH and hypergastrinemia. Sixty one rats were divided into 2 groups designed as infected (36 rats) and control (25 rats) group. These rats were examined with time course of the infection histopathologically and physiopathologically, during 14-112 days postinfection (DPI). In the infected rats, gastric mucosal hyperplasia began to be observed at 56 DPI, and the structural disturbance of zymogenic units in the corpus and mucous units in the antrum had increased with time. However, the degree of these changes in the antrum was weaker than those in the corpus. Alcianblue and/or PAS-positive cells increased in their numbers with time, and 4 types of cells other than typical surface mucous cell and mucous neck cell were observed by electron-microscopy. However, zymogenic and parietal cells decreased in their number after 56 DPI. Further, the infected rats showed changes in the serum concentration of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, glucose and total protein. Some similarities with Menetrier's disease were discussed.

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Adult Thai Setaria worms collected from cattle which were bred, housed and slaughtered in Thailand were morphologically identified as Setaria digitata. Furthermore, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) adult Thai S. digitata had the same protein profiles as adult Japanese S. digitata, but did not possess the protein with a molecular size of 69 kDa which was confirmed in adult S. marshalli. In addition, there were no differences in the protein profiles between male and female S. digitata. In point of the distribution pattern of the proteins ranging from 73 to 64 kDa revealed by 2D-PAGE, there were no differences between Thai and Japanese S. digitata, and between male and female worms of the species.

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Localization of metallothionein (MT) -I & -II and MT-III and its significance in the brain aging in dogs were examined using immunohistological and molecular pathological techniques. MT-I & -II immunohistochemistry showed positive staining in the hypertrophic astrocytes throughout the aged dog brains; these MT-I & -II immunoreactive astrocytes were predominant in the cerebral cortex and around the blood vessels in the brain. These findings dominated in the brain regions with severe age-related morphological changes. In situ hybridization using MT-I mRNA riboprobes also demonstrated signals for MT-I mRNA in these hypertrophic astrocytes. Immunohistochemistry using a guinea pig antiserum against a synthetic polypeptide of canine MT-III demonstrated positive MT-III immunoreactivity predominantly in neurons in the Zn-rich regions such as hippocampus and parahippocampus. The findings were supported by in situ hybridization using MT-III mRNA riboprobes. Both MT-III immunoreactivity and signals for MT-III mRNA were demonstrated in neurons in the brain regardless of the intensity of the age-related changes. These results suggest, first, MT-I & -II may be induced in relation to the progress of the age-related morphological changes in the brain, playing an important role in the protection of the brain tissue from the toxic insults responsible for the brain aging, and second, MT-III may play a role in maintenance of Zn-related essential functions of the brain.

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Nasal nocardiosis was found in a female Japanese Black calf, 11 months of age. Macroscopically, the posterior half of the left nasal passage was completely obstructed by yellowish brown caseous substance and the mucosa was irregularly thickened. In the brain, a few soft brown foci were present in the olfactory bulb and frontal lobe. Microscopically, there were closely packed granulomas in the nasal cavity and brain. The lesions were characterized by a center of cellular debris surrounded by epithelioid macrophages, neutrophils, lymphocytes and multinucleated giant cells of the Langhans type. Special stains revealed the presence of a large number of filamentous branching gram-positive, partially acid-fast organisms in these epithelioid cells and giant cells, and in cellular debris.

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The present study examined effects of diazepam (DZP) alone or in combination with δ9-tetrahydrocannabinol (THC) on feeding behavior as well as body weight in male ddY strain mice at 5 weeks of age. Because we saw no hyperphagic effect of DZP with or without THC in mice, we explored the hyperphagia elicitable by DZP. THC [2 (THC2) or 4 (THC4) mg/kg/day s.c.] was given daily for 7 days. For the last day the mice were starved and injected i.p. with DZP (2 mg/kg) 10 min prior to a food or maze test. Controls received vehicle injections. Feeding behavior was measured after giving food for 2 hr. THC4 significantly reduced body weight gain. DZP, with or without THC, induced hyperphagia. THC4 alone also induced hyperphagia that was not significantly affected by DZP. Time taken to find food was extended by DZP and further with THC. Both DZP and THC can therefore interact on food ingestion but synergize on food seeking in mice through different mechanisms.

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We have previously shown that interleukin-1β relaxes vascular smooth muscle by the NO-dependent and independent mechanisms (Takizawa et al.: Eur. J. Pharmacol. 330: 143-150, 1997). In this study, we investigated the mechanism of NO-independent relaxation. Treatment of the rat aorta with interleukin-1β for 24 hr inhibited the high-K⁺ induced contraction by decreasing cytosolic Ca²⁺ level ([Ca²⁺]_i). The relationship between [Ca²⁺]_i and tension in intact muscle and the pCa-tension curves in permeabilized muscle suggested that Ca²⁺ sensitivity of contractile element was not changed after the interleukin-1β-treatment. After a treatment with interleukin-1β for 24 hr, contractile effects of phenylephrine (1 μM-10 μM) were markedly inhibited in the presence of L-NMMA (100 μM) applied to inhibit NO synthesis. A blocker of ATP-sensitive K⁺ channel, glibenclamide (1 μM), partially recovered the interleukin-1β-induced inhibition. In contrast, a blocker of Ca²⁺-activated K⁺ channel, charybdotoxin (0.1 μM), was ineffective. These results suggest that membrane hyperpolarization due to activation of ATP-sensitive K⁺ channels may partly be responsible for the NO-independent mechanism of interleukin-1β-induced inhibition of vascular smooth muscle contraction.

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Extracorporeal membrane oxygenation (ECMO) is frequently used for treatment of patients with severe hypoxemia due to life-threatening respiratory failure. Due to this hypoxemia, the myocardium of these patients is insufficiently provided with oxygen, and consequently their cardiac function commonly deteriorates. But veno-arterial (V-A) ECMO provides oxygenated blood to the coronary arteries from ECMO circuit insufficiently. To increase the coronary blood flow distributed from ECMO, we placed the arterial cannula 1 cm above the aortic valve and evaluated the regional blood flow from the proximal arterial cannula in comparison with the distal cannula. Eight neonatal dogs weighting 1.8-2.5 kg were supported by V-A ECMO. The regional blood flow from the arterial cannula was measured by injection of colored microspheres into ECMO circuit. The site of the arterial cannula was changed under fluoroscopy. The bypass flow was maintained at either 50 or 100 ml/min/kg. We found that the coronary blood flow distributed from the proximal arterial cannula was significantly higher than that from the distal cannula. The proximal arterial cannula appears necessary to provide sufficient oxygenated blood to the coronary circulation during V-A ECMO. Therefore, it is expected that the increased cardiac function may improved, and that the survival rate of the patients with retarded cardiac function due to severe hypoxemia may increase by proximal placement of the arterial cannula during V-A ECMO.

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Open heart surgery was performed on two groups of dogs under extracorporeal circulation with or without hypothermia to investigate hemodynamic changes during extracorporeal circulation. During hypothermic cardiopulmonary bypass (CPB), arterial O₂ tension and postoperative blood pressure were favorably maintained, indicating that hypothermic extracorporeal circulation can be performed for a long period of time. On the other hand, during normothermic CPB, the average surgical duration was significantly shorter, and marked shifts in the concentrations of various enzymes were suppressed. However, due to reductions in arterial O₂ tension, the length of cardiac arrest time was restricted, demonstrating that this method is suitable for performing extracorporeal circulation for CPB of relatively short duration. If circulation circuitry can be improved, such as through the development of a surpassing oxygenator, normothermic CPB would incur less stress on the body, thus making it preferential to hypothermic CPB in most cases.

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Highly lung metastasizing model of canine osteosarcoma in nude mice was established from five subcutaneous implantation cycles of lung tumor deposits. The selection of cells with increased metastatic properties from the parent POS canine osteosarcoma cell line recovered medium sized and polygonal Highly Metastasizing POS cells (HMPOS). The doubling time of HMPOS and POS in culture averaged 30 ± 1.2 hr and 32 ± 1.3 hr respectively, and their cell growth patterns in vitro were comparable to their in vivo growth patterns. HMPOS cells produced more tumor deposits (>20 nodules, >1-mm in diameter) of various sizes with replacement of lung tissues at 12 weeks after implantation. POS cells produced fewer and smaller lung deposits (<10 nodules, 1-mm in diameter). Tumor size and number of metastatic tumor deposits showed a regular association. HMPOS cells developed an osteoblastic type of cellular differentiation subcutaneously and in the lungs. HMPOS micrometastasis along the alveolar walls and blood vessels at 4 weeks averaged 6-7 small tumor locus. Each micrometastatic locus contained an average of 5-7 tumor cells, and developed a pleomorphic osteoblastic type of cellular differentiation. An average of 4 macrometastatic nodules could be seen at 6 weeks, composed of an average of 23 tumor cells, 10 nodules at 8 weeks, 12 nodules at 10 weeks and 20 nodules at 12 weeks. These model provides an opportunity for the evaluation of new treatments against canine lung metastatic osteosarcoma in a nude mice model.

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A 3-month-old, female Japanese Black calf that showed signs of neurological dysfunction soon after birth was twice examined by magnetic resonance imaging (MRI). Survey MR images showed changes in a hydrocephalus from mild to severe and the existence of a mass above the brain stem that could be distinguished from the surrounding cerebral parenchyma. Contrast MRI examinations using Gd-DO3A-butriol showed the mass to have a doughnut-like form. As the mass changed, the clinical signs aggravated. We diagnosed a brain stem abscess, which we confirmed pathologically. To our knowledge there are no other reports of the use of contrast MRI to examine cattle.

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Twelve pregnant gilts were assigned to a completely randomized block design with two treatments in two blocks (2 farrowing groups). The treatments were a feeding amount of 6 kg or 2 kg/day provided during lactation. The lactation diet contained 18.6% crude protein, 1.0% lysine, and 3.27 Mcal/kg metabolizable energy (as-fed basis). Litters were weaned at 2100 on day 21 after farrowing. Blood samples for luteinizing hormone (LH) measurements were taken at 15-min intervals for 8 hr on day 12 of lactation, and samples for glucose and insulin were collected at 1-hr intervals for 3 hr on day 12. The effects of feed intake treatments on LH pulse frequencies (2.9 vs 0.7) and insulin concentrations (15.0 vs 8.9 IU/mL) were found (P<0.05) on day 12 of lactation. In regression analysis, greater cumulative feed intake from 1 to 12 days was associated with higher insulin concentrations (P=0.04), greater LH pulse frequencies (P=0.01) on day 12 of lactation, and shorter weaning-to-estrus intervals (WEI) (P=0.03). Furthermore, an association between insulin concentrations and LH pulse frequencies was found on day 12 of lactation (P=0.01). Using regression models for weaning-to-estrus interval, when each cumulative feed intake from 4 to 21 days was used as an independent variable, the R² values increased from 0.24 to 0.37. These results suggest that feed intake during early and mid-lactation influences LH secretion as early as day 12 after farrowing, and is associated with shorter WEI. This research also indicates that feed intake from 4 to 12 days of lactation is more important than that during the first few days after farrowing.

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Canine deciduoma could be induced in the diestrous uterus by an intrauterine inoculation of a culture suspension of E. coli originally isolated from naturally occurring canine pyometra. These deciduomas had the same histological findings as those of naturally occurring canine pyometra with so called “Swiss cheese endometrium”. This suggests a possibility that the canine pyometra is a kind of naturally occurring decidual reaction (deciduoma) induced by one of several triggers such as bacterial infection.

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The mechanism of onion-induced hemolytic anemia in ruminants was investigated. The ether-extract obtained from the mixture of rumen fluid and onion juice incubated at 38.5°C for 9 hr induced oxidative damage in sheep erythrocytes in vitro, indicating the production of certain oxidants in the mixture. The increase of the oxidative effect in the mixture was inhibited completely by the removal of rumen microorganisms and partly by treatment with antibiotics and by oxygen gas. The sheep fed onions (50 g/kg body weight/day) for 15 days developed more severe Heinz body hemolytic anemia than did the sheep fed the equivalent amount of onions with 5 g/day ampicillin sodium salt. The results indicated that certain rumen bacteria appear to be involved in the onset of onion-induced hemolytic anemia in sheep.

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To determine the site of latent infection of canine herpesvirus (CHV), tissues from dogs convalescent from acute infection with CHV were examined for the presence of viral genome DNA by the nested polymerase chain reaction. CHV DNA was detected in the trigeminal ganglia and the retropharyngeal lymph nodes. In situ hybridization study of the tissues revealed that CHV genome persisted in the nuclei of ganglionic neurons and lymphocytes.

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DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 詰 aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase α, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase.

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We have developed a new quantitative method for rabies virus (RV) detection using enzyme-linked immunosorbent assay (ELISA). The method named N-ELISA was based on the quantitation of nucleoprotein (N) in RV virions captured by RV-specific polyclonal antibodies on an ELISA plate. Both infective and defective interfering (DI) particles of RV could be detected by this method. When viruses were propagated in a medium of pH 7.4 adjusted with 7% NaHCO₃, N-ELISA could detect them with titers of more than 10⁶ pfu/ml, though the result did not correlate highly with that of the infectivity assay. The reason for this was considered to be that RVs included spikeless and damaged particles which were produced under conditions of low or high pH. However, in the time course of virus yield, titers of N-ELISA correlated well with those of the infectivity assay.

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