Embryoid bodies (EB) were formed by TT2 embryonic stem (ES) cells in vitro. ES-like cell lines (ESLC) were established by culturing cells obtained by disaggregation of EB at 4, 8 and 20 days after culture, and designated ESLC4, ESLC8 and ESLC20, respectively. Flow cytometric analysis indicated that the cell surface expression of Le^x on ESLC was less than that of original TT2 ES cells, but the expression of L-CAM was comparable. After suspension culture, all of the ESLC cells formed cystic EB in vitro. In addition, some ESLC4- and ESLC8-derived EB showed signs of beating. Although coat color chimeras were able to be produced with ESCL4 at a lower rate than parental ES cells, the cells did not contribute to germ line cells in chimeras. These results suggest that the ESLC had less pluripotent than parental ES cells and also that EB formation is not useful in obtaining pluripotent cells.

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The effect of the karyotype and the ability to differentiate in vitro upon germ-line transmission by A3-1 embryonic stem (ES) cells in chimeric mice were examined. Germ-line transmission was confirmed in ES cells exhibiting 38% and more of the normal karyotype, but no chimeric mice and/or germ-line transmitters were observed regardless of the karyotype when the cystic embryoid body (CEB) was formed on day 8 and later in the suspension culture. Germ-line transmission of the ES cells was not significantly influenced by formation of the simple embryoid body (SEB). Germ-line transmitters were preferentially observed in chimeras when the ES cell contribution to coat color was markedly increased, but this contribution to coat color varied regardless of the karyotype or in vitro differentiation ability. These results suggest that A3-1 ES cells which exhibit CEB at 7 days after suspension culture and approximately 40% of normal karyotype are capable of germ-line transmission in chimeric mice.

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Histochemical observations of alkaline phosphatase activity of Echinococcus multilocularis during the in vivo development in golden hamster, an alternative definitive host. The present work reports on the ability of protoscoleces from a European fox strain of E. multilocularis to differentiate and develop into the adult form in the small intestine of male golden hamsters treated with prednisolone. Detection of alkaline phosphatase activity on various stages of the developing worm was performed by histochemical methods. The enzyme activity was not demonstrable in the early stages of infection but occurred with strobilization. Age-related changes in the distribution of the enzyme activity took place during strobilization. Alkaline phosphatase activity was evident in the excretory ducts of 8 to 11 day old strobila and in the tegument of mature proglottis of 16 day old worms. This in vivo procedure with rodents as definitive hosts provides interesting preliminary results on the biology of the E. multilocularis adult. Further investigations on membrane-bound enzymes involved in physiological and nutritional processes are in progress.

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Feline immunodeficiency virus (FIV) infection in cats has been reported to be a useful animal model for human AIDS studies, especially in the early stages of infection. We examined the temporal changes in provirus detection in peripheral blood mononuclear cells (PBMC) and the distribution of FIV-DNA and RNA in feline tissues by the polymerase chain reaction at 10, 35, 70 days after intravenous inoculation of FIV. Viral DNA in the PBMC was detected three to four weeks after infection and its fluctuation was demonstrated for the first time. Ten days after infection, before seroconversion, proviruses were detected only in the mesenteric lymph nodes and intestines. At 35 and 70 days after infection, after seroconversion, proviruses were detected in most lymphoid organs and the salivary glands, but the expression of FIV-RNA was limited to the thymus at 70 days after infection. These results show that FIV-RNA is transcribed from proviral DNA exclusively in the thymus at this stage. We suggest that the quantitative changes in detectable proviruses in the PBMC depend on the relation between the decrease in infected cells caused by cytolytic T lymphocytes and/or apoptosis and their increase caused by the release of a new supply of lymphocytes from the thymus.

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Forty-two of 81 dogs from a family of Japanese Shiba dogs had red blood cells with a high K and a low Na concentration (HK). Of the HK dogs, 32 were high K and low glutathione (HK/LG) and 10 were high K and high glutathione (HK/HG). These variants were found in both males and females. The phenotype of HK was inherited in a recessive mode as reported earlier. A high incidence of HK/LG dogs was found in this family, and the phenotype was also inherited in a recessive mode. Glutamate (Glu) influx, which defines the cellular glutathione concentration, was lower in HK/LG cells than in HK/HG cells (in some cases extremely low). The fact that the red blood cells of HK/LG dogs have the two varying characteristics of a remaining Na, K-pump and low Glu transport suggests that 2 or more genes may be involved. Since an extremely low Glu influx was also found in normal low K and high Na (LK) red blood cells, the characteristic of low Glu transport also exists in LK cells. The phenotype of low Glu transport may also be inherited in a recessive mode. This family therefore had a very high incidence of homozygous recessive genes which control the phenotypes for the Na, K-pump and low Glu transport.

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Changes in normal vaginal flora of African green monkeys associated with the estrous cycle were examined by the swab method. Bacteroidaceae, corynebacteria, and streptococci were predominant throughout the whole estrous cycle, although individual differences were great. It was also clear that all bacterial species tended to decrease in the menstrual phase. Lactobacilli, the most predominant bacteria in the normal vagina of humans, were detected only in very low numbers in the African green monkey, suggesting that the normal vagina of the African green monkey has a different ecosystem from that of normal human vaginal flora.

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It has shown that the human immunodeficiency virus type 1 (HIV-1) Nef protein has the high antigenicity in HIV-1 seropositive individuals. We newly obtained seven monoclonal antibodies (mAbs). To identify the antigenic determinants of HIV-1 Nef protein against murine, epitope mapping of the mAbs was performed by enzyme-linked immunosorbent assay (ELISA) by using several recombinant truncated Nef fusion proteins, that were expressed in Esherichia coli, and synthetic peptides. The results showed that mAbs A6, A7, F2, F3, F4, F8 and E5 recognized epitopes on Nef protein located at amino acid residues 18-26, 28-45, 115-137, 128-137, 115-126, 128-137, and 170-181, respectively.

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In the brains of 360-day-old Mongolian gerbils, numerous swellings immunoreactive to anti-neurofilament antibody were observed in cerebellar and vestibular nuclei. The number of these swellings was the same in two gerbil strains with different susceptibility to spontaneous motor seizures by various stimuli, but much more numerous in gerbils as compared with the 360-day-old Slc:Wistar rats. Such swellings were only occasionally found before 60 days of age in gerbils, but they increased in number about fivefold from 60 to 180 days of age and about quadruple from 180 to 360 days of age. Electron microscopic observation showed that these swellings were dystrophic axon terminals (DATs) whose cytoplasms were occupied with large bundles of neurofilaments, numerous vesicular structures containing membranous and/or granular materials, and many rod-shaped mitochondria. Additionally, other types of DATs displaying degenerative changes of cytoplasmic organelles were observed. ACPase cytochemistry showed that the vesicular structures in the DATs contained ACPase and released it into the cytoplasm.

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To investigate the effects of medetomidine on late pregnant goats, medetomidine induced changes in maternal or fetal circulation and acid-base balance, as well as changes in intrauterine pressure (IUP) and uterine blood flow (UBF), were studied. Intramuscular administration of medetomidine (40 μg/kg b.w.) decreased the heart rate (HR) and arterial blood pressure (ABP) of the mother, and the change in HR was significant statistically (p<0.05). In the fetus, HR and ABP showed a transient decrease and increase (p<0.05), respectively. A decrease in maternal arterial blood pH and oxygen partial pressure (PO₂) and an increase in carbon dioxide partial pressure (PCO₂) were recorded after the injection, but none was significant. In the fetus, arterial blood PO₂ decreased significantly (p<0.05) after 5 min of administration, and a significant metabolic acidemia supported by a decrease in base excess was observed. Within 1 to 4 min after the administration of medetomidine, IUP began to rise and remained high for 10 to 14 min. Thereafter, the rise in IUP was frequent and periodical. After the injection, UBF significantly (p<0.05) decreased, and the fall in UBF was associated with a rise in IUP. The maternal and fetal serum medetomidine concentration increased remarkably after the injection of medetomidine into the mother. These observations in late pregnant goats suggested that medetomidine induced a decrease in maternal cardiac output, a decrease in UBF arising from the induction of uterine contractions, and transplacental medetomidine can have a suppressive effect on the fetus.

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A genetic typing method for the mouse obese (ob) mutation by PCR and restriction fragment length polymorphism (RFLP) analysis was developed. Three genotypes (ob/ob, ob/+ and +/+) of the ob mouse are rapidly differentiated with this assay. Since only a small biopsy specimen (tail end) is necessary for the genotyping, the ob mouse at any age is typable and can be used in subsequent breeding or research. Obese homozygotes (ob/ob) were efficiently produced by mating heterozygotes (ob/+) only, which were selected by using the PCR-RFLP assay.

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Electron microscopic in situ hybridization (ISH-EM) was first applied to the detection of viral RNA in the germinal epithelium of mice inoculated i.p. with 10⁵ plaque-forming units/mouse of the D variant of encephalomyocarditis virus (EMC-D). Signals of viral RNA were first detected in a small number of Sertoli cells showing mild degeneration at 2 days post inoculation, and 2 days later, they were also detected in germinal cells and spermatogonia when Sertoli cells showed prominent degeneration. The results clearly demonstrated that the first site of viral attack in the germinal epithelium was Sertoli cell in the case of EMC-D-induced mouse orchitis.

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The efficacy of 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) was investigated in vivo by using a male ARC/AIDS rhesus macaque infected with simian immunodeficiency virus (SIVmac251/32H). He was administered subcutaneously 6-Cl-ddG (50 mg/kg B.W.) every 8 hr for 14 days when he showed clinical features of recurrent weight loss, severe diarrhea and neuropathy. The number of CD4⁺, CD8⁺ cells and total T cells increased rapidly after administration of 6-Cl-ddG and a high level was maintained for 2 months, but the B cell count decreased during the treatment. The antibody titer to SIV did not change significantly during or after the treatment, but the virus load in the plasma measured by RT-PCR dropped to one-third at the start of the 6-Cl-ddG treatment. Within 3 days after the start of 6-Cl-ddG administration, he began to show recovery in clinical signs including weight increase, and disappearance of diarrhea and neuropathy. These findings suggested that 6-Cl-ddG was effective at the stage of ARC/AIDS in a rhesus monkey infected with SIV.

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Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-D-glucosamine (GlcNAc)-Sepharose 6B column chromatography. It was concluded that this method is useful for clinical examination in the rat.

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"Dilute down lethal (DDL)" is a new mutation of Japanese quail (Coturnix japonica). Neonatal plumage of the DDL mutant is the same as the wild type in pattern, but its coloration is slightly lighter than that of the wild type. In addition to the down color abnormality, some DDL chicks have bent digits. All DDL chicks die within three days of age. Genetic analysis revealed that the DDL mutation is controlled by an autosomal recessive gene. The proposed gene symbol is ddl.

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