Youji Muramatsu

Wilson's disease gene is homologous to hts causing abnormal copper transport in Long-Evans cinnamon rats.

BACKGROUND/AIMS The Long-Evans Cinnamon (LEC) mutant rat shows an excess copper accumulation in the liver and low serum ceruloplasmin activity. The disorder is controlled by a single autosomal recessive gene designated as hts. Wilsons disease is an autosomal recessive disorder of copper metabolism characterized by abnormal copper accumulation in the liver and low serum ceruloplasmin activity. The gene responsible for Wilsons disease has recently been isolated. The present study was designed to examine whether the LEC rat is an ideal animal model for Wilsons disease from a genetic point of view. METHODS For chromosomal mapping of hts, genetic linkage analysis using rat microsatellite marker loci was performed. Furthermore, cosegregation between hts and a rat counterpart of the Wilsons disease gene was analyzed. RESULTS hts was finely mapped to rat chromosome 16. Complete cosegregation between hts and a rat counterpart of the Wilsons disease gene was detected. CONCLUSIONS hts is likely to correspond to a rat homologue of the Wilsons disease gene. The present results allow us to propose that the LEC rat is an ideal animal model for Wilsons disease.

Assignments of the genes for rat pituitary adenylate cyclase activating polypeptide (Adcyap1) and its receptor subtypes (Adcyap1r1, Adcyap1r2, and Adcyap1r3)

Chromosomal assignments of the genes for rat pituitary adenylate cyclase activating polypeptide (Adcyap1) and all of its receptor subtypes (Adcyap1r1, Adcyap1r2, and Adcyap1r3) were performed by PCR analysis of somatic cell hybrid DNAs. Adcyap1r1, Adcyap1r2, and Adcyap1r3 were localized to rat chromosomes 9, 4, 8, and 4, respectively.

Chromosomal mapping of the T-helper immunodeficiency (thid) locus in LEC rats.

The mechanism underlying the development of thymocytes into CD4+8± or CD4±8+ cells is not fully understood. Mutant strains have served as an invaluable tool for the study of such a mechanism. Lymphopoietic stem cells from bone marrow migrate to the thymus where they undergo gene rearrangement of the T-cell receptor (TCR), and then express TCR together with their coreceptor molecules, CD4 and CD8, on the developing thymocytes. Coengagement of TCR and coreceptor CD4 or CD8 on the maturing cells by their thymic MHC molecules will determine their cell fate. CD4+8+ thymocytes that recognize major histocompatibility complex (MHC) class I molecules on the thymic epithelial cells differentiate into CD4±8+ cells, whereas CD4+8+ thymocytes that recognize MHC class II mature as CD4+8± cells. The molecular basis of such an influence is largely unknown (von Boehmer 1994, 1996; Fink and Bevan 1995). The Long-Evans Cinnamon (LEC) rat, as far as is known, was the first naturally occurring rat mutant with a specific defect in thymocyte development. The development of CD4+8+ thymocytes into CD4+8± cells was prevented in the thymus of the LEC rat, but the development of CD4+8+ thymocytes into CD4±8+ cells was normal (Agui et al. 1990). Thymocytes from the LEC rat contained less than 1% CD4+8± cells. Unexpectedly, a small number of CD4+ T cells were present in the peripheral lymphoid organs (Agui et al. 1990). These CD4+ T cells were shown not to function as helper T cells, since LEC rats did not produce antibodies against T-cell-dependent antigen, SRBC, but did produce antibodies against T-cell-independent antigen (DNPconjugated Ficoll) (Agui et al. 1990). Moreover, CD4+ T cells in LEC rats failed to secrete IL-2 upon Concanavalin A stimulation (Agui et al. 1990; Sakai et al. 1993). Such a defect in LEC rats was brought about by a single autosomal recessive gene designated as T-helper immunodeficiency (thid) (T. Yamada et al. 1991). The inability to generate CD4+8± thymocytes together with physical and functional deficiencies in CD4+ T cells suggests that the LEC rat may serve as a model for further studies in thymic selection and T-cell functions. Here we performed linkage mapping of the mutation. The linkage map surrounding thid on rat chromosome 1 can serve as the starting point for further characterization of the mutation9s molecular basis. LEC/Tj, LEA/Tj, WKAH/Tj, and F344/Tj inbred rats were maintained in our laboratory under specific pathogenfree conditions. BN/Kyo rats were kindly provided by J. Yamada, University of Kyoto, Japan. Four backcrosses between LEC and one of the normal rats were made. Thymocytes prepared from the progenies of the backcrosses at the age of 4±6 weeks were first stained with biotinylated anti-rat CD8 mAb (OX8) (Brideau et al. 1980), and then stained with PE-conjugated streptavidin and FITC-conjugated anti-rat CD4 mAb(W3/25) (Williams et al. 1977). Stained cells were analyzed with a FACScan and Consort 30 software program (Becton Dickinson, Mountain View, Calif.). Animals with a percentage of CD4+8± thymocytes less than 1% were scored as thid/thid homozygotes, while those greater than 5% were scored as thid/+ heterozygotes (Fig. 1). DNA was also extracted from the liver of the backcross progenies using phenol and chloroform (Sambrook et al. 1989). Most of the rat microsatellite primers (Jacob et al. 1995; Gauguier et al 1996) were purchased from Research Genetics, and the remaining primers were synthesized according to published sequences (Serikawa et al. 1992). The marker was typed by polymerase chain reaction (PCR) with DNA from the backcross progenies and the above primers. PCR reaction mixture (10 ml) contained 25 ng K. Wei ? T. Yamada ? K. Matsumoto ( ) Institute for Animal Experimentation, University of Tokushima School of Medicine, Kuramoto 3, Tokushima 770, Japan

Chromosomal assignments of genes for rat glutathione S-transferase Ya (GSTA1) and Yc summits (GSTA2)

Chromosomal assignments of genes for rat glutathione S-transferase Ya (GSTA1) and Yc subunits (GSTA2) were performed by Southern blot analyses of somatic cell hybrid DNAs.GSTA1 and GSTA2 were assigned to rat chromosomes 8 and 9, respectively.

Induction of apoptosis and inhibition of DNA topoisomerase-I in K-562 cells by a marine microalgal polysaccharide

We have previously purified an extracellular polysaccharide, D-galactan sulfate associated with L(+)-lactic acid, produced from a marine microalga Dinoflagellate Gymnodinium sp. A3 (GA3). The GA3 polysaccharide, irrespective of presence or absence of lactic acid, exhibited significant cytotoxicity, which is based on an induction of apoptotic cell death, toward human myeloid leukemia K562 cells. Furthermore, we found that the GA3 polysaccharide with or without lactic acid possesses an inhibitory effect on topoisomerase-I (topo-I). The potent cytotoxic effect of GA3 polysaccharide may result from its inhibitory effect on topo-I, because the topo-I inhibition is known to trigger apoptotic cell death.

Association of a single-nucleotide polymorphism in myosin-binding protein C, slow-type (MYBPC1) gene with marbling in Japanese Black beef cattle

Bin Tong*, Seiki Sasaki*, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro*, Tatsuo Fujita and Takahisa Yamada* *Department of Agrobiology, Faculty of Agriculture, Niigata University, Nishi-ku, Niigata 950-2181, Japan; Department of Nutritional Sciences for Well-being, Faculty of Health Sciences for Welfare, Kansai University of Welfare Sciences, Kashiwara, Osaka 582-0026, Japan; Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Takatsuki, Osaka 569-1125, Japan; Department of Life Science, Division of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan; Oita Prefectural Institute of Animal Industry, Takeda, Oita 878-0201, Japan

Chromosomal assignment of the gene for rat protein phosphatase 1γ1 (Ppp1cc)

The gene for rat protein phosphatase 1γ1 (Ppp1cc) was assigned to rat chromosome 12 by analyzing somatic cell hybrid DNAs with the polymerase chain reaction, using primers specific for the rat gene.

Chromosomal assignments of genes for rat glutathione S-transferase Yb1 (GSTA3) and Yb2 (GSTA4) subunits

Chromosomal assignments of genes for rat glutathione S-transferase Yb1 (GSTA3) and Yb2 (GSTA4) subunits were performed by Southern blot analyses of somatic cell hybrid DNAs. Both GSTA3 and GSTA4 were assigned to rat chromosome 2.

Chromosomal assignments of the genes for the calcineurin Aα (Calna1) and Aβ subunits (Calna2) in the rat

Chromosomal assignments of the genes for the calcineurin Aα (Calnal) and Aβ (Cαlnα2) subunits in the rat genome were performed by polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis of somatic cell hybrid DNAs. Both genes, Cαlnαl and Cαlnαl, were assigned to rat chromosome 15.

Association of the expression levels in the longissimus muscle and a SNP in the CDC10 gene with marbling in Japanese Black beef cattle.

The septin 7 (CDC10) gene, involved in cellular proliferation, has been previously shown to be expressed at different levels in the longissimus muscle (LM) between low-marbled and high-marbled steer groups by differential-display PCR. It is located within the genomic region of a quantitative trait locus for marbling, and thus was considered as a positional functional candidate gene for marbling. In this study, we showed that the CDC10 expression levels in the LM were positively correlated with marbling in Japanese Black (JB) steers (P<0.0001). Further, an association analysis indicated that a SNP in the promoter region of the CDC10 gene was associated with marbling using 99 JB sires (P=0.03) and 542 JB paternal half-sib progeny steers from a sire homozygous for G allele at the SNP (P<0.0001). These findings suggest possible effects of the expression levels in the LM and the SNP of the CDC10 gene on marbling in JB cattle.