Ryoji Fukuhara

Structure, molecular evolution, and gene expression of primate superoxide dismutases.

Mn- and Cu,Zn-superoxide dismutase (SOD) cDNAs of eight primate species, Pan troglodytes, Pongo pygmaeus, Hylobates lar, Macaca fuscata, Macaca fascicularis, Macaca mulatta, Cebus apella, and Callithrix jacchus, were cloned. The whole protein-coding sequences were covered, comparing 198 and 153 (or 154) amino acids, for Mn- and Cu,Zn-SODs, respectively. Residues forming metal ligands were completely conserved in the two primate SODs and nucleotide/amino acid substitutions were more frequent in Cu,Zn-SODs than in Mn-SODs. Molecular evolutionary analyses showed Mn-SOD to have evolved at a constant rate and its phylogenetic tree well reflected primate phylogeny. Cu,Zn-SOD was shown to have evolved differently between primate lineages. The significant high ratio of a non-synonymous/synonymous rate was found in the lineage leading to great apes and humans, showing that this lineage underwent positive Darwinian selection. Southern hybridization suggested that the genes for primate Mn- and Cu,Zn-SOD exist as single copies. Northern analysis in various Japanese monkey tissues showed Mn- and Cu,Zn-SOD expression to be high in the liver, kidneys, and adrenal glands.

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR

In the last three decades, several monkeys reared in outdoor/indoor–outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)‐PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD‐PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.

Tissue Distribution, Molecular Cloning, and Gene Expression of Cytosolic Glutathione Peroxidase in Japanese Monkey

Abstract Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.

Molecular cloning and gene expression of endoplasmic reticulum stress proteins in Japanese monkey, Macaca fuscata

Background  Immunoglobulin heavy‐chain binding protein (BiP), calreticulin (Crt), and protein disulfide isomerase (PDI), are major resident endoplasmic reticulum (ER) stress proteins which are involved in diverse roles relating to successful folding, assembly, intracellular localization, and degradation of other proteins.

Tissue Distribution and Multiplicity of Enzymes That Generate and Scavenge Reactive Oxygen Species in Japanese Monkey

Abstract Five enzymes involved in the generation and scavenging of reactive oxygen species, i.e., NADH/NADPH oxidase, xanthine oxidase (XOD), superoxide dismutase (SOD), ascorbate peroxidase, and catalase were assayed in various tissues of the Japanese monkey. Their activities were largely different between tissues. Generally, small intestine, kidney, and cerebellum contained larger amounts of these enzymes than other tissues. Multiplicities of these enzymes were analyzed by staining of their enzymatic activities after electrophoresis. The number of isozymes was 2 in the case of NADPH oxidase and catalase, and 3 in the case of XOD, SOD, and ascorbate peroxidase. The expression of these isozymes differed between tissues, suggesting the occurrence of tissue-specific systems to generate and scavenge reactive oxygen species in the Japanese monkey.