Yosuke Kameoka

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

BackgroundThe genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome.ResultsWe identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks.ConclusionsThis list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.

Molecular Characterization of Cryptosporidium Isolates Obtained from Human and Bovine Infections in Japan

Abstract. Cryptosporidium oocysts, morphologically identified as Cryptosporidium parvum, were isolated from 22 human and 14 bovine cases in Japan, and were genotyped by means of a PCR/RFLP analysis of the poly-threonine gene. DNA profiles of human isolates gave three distinct genotypes, namely an anthroponotic genotype 1, zoonotic genotype 2 and a new genotype. Isolates from bovine samples gave zoonotic genotype 2. The unusual genotype of Cryptosporidium was isolated from the feces of three immunologically healthy adults, and was further characterized by the sequence analysis of the 18S rRNA gene. The third genotype was identified as Cryptosporidium meleagridis, demonstrating that C. meleagridis, which occurs worldwide, has the potential to infect humans regardless of their immunological condition.

In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimers disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimers disease.

Ancient genome‐wide admixture extends beyond the current hybrid zone between Macaca fascicularis and M. mulatta

Macaca fascicularis and Macaca mulatta are two of the most commonly used laboratory macaques, yet their genetic differences at a genome‐wide level remain unclear. We analysed the multilocus DNA sequence data of 54 autosomal loci obtained from M. fascicularis samples from three different geographic origins and M. mulatta samples of Burmese origin. M. fascicularis shows high nucleotide diversity, four to five times higher than humans, and a strong geographic population structure between Indonesian‐Malaysian and Philippine macaques. The pattern of divergence and polymorphism between M. fascicularis and M. mulatta shows a footprint of genetic exchange not only within their current hybrid zone but also across a wider range for more than 1 million years. However, genetic admixture may not be a random event in the genome. Whereas randomly selected genic and intergenic regions have the same evolutionary dynamics between the species, some cytochrome oxidase P450 (CYP) genes (major chemical metabolizing genes and potential target genes for local adaptation) have a significantly larger species divergence than other genes. By surveying CYP3A5 gene sequences of more than a hundred macaques, we identified three nonsynonymous single nucleotide polymorphisms that were highly differentiated between the macaques. The mosaic pattern of species divergence in the genomes may be a consequence of genetic differentiation under ecological adaptation and may be a salient feature in the genomes of nascent species under parapatry.

Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta

BackgroundCynomolgus macaques (Macaca fascicularis) are widely used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as rhesus macaques (M. mulatta). We isolated 85,721 clones and determined 9407 full-insert sequences from cynomolgus monkey brain, testis, and liver. These sequences were annotated based on homology to human genes and stored in a database, QFbase http://genebank.nibio.go.jp/qfbase/.ResultsWe found that 1024 transcripts did not represent any public human cDNA sequence and examined their expression using M. fascicularis oligonucleotide microarrays. Significant expression was detected for 544 (51%) of the unidentified transcripts. Moreover, we identified 226 genes containing exon alterations in the untranslated regions of the macaque transcripts, despite the highly conserved structure of the coding regions. Considering the polymorphism in the common ancestor of cynomolgus and rhesus macaques and the rate of PCR errors, the divergence time between the two species was estimated to be around 0.9 million years ago.ConclusionTranscript data from Old World monkeys provide a means not only to determine the evolutionary difference between human and non-human primates but also to unveil hidden transcripts in the human genome. Increasing the genomic resources and information of macaque monkeys will greatly contribute to the development of evolutionary biology and biomedical sciences.

Susceptibility Loci to Coronary Arteritis in Animal Model of Kawasaki Disease Induced with Candida albicans-Derived Substances

We have established an animal model of coronary arteritis which is histopathologically similar to that observed in cases of Kawasaki disease (KD), is a well‐known childhood vasculitis syndrome. Coronary arteritis in this mouse model has been induced by intraperitoneal injection of Candida albicans‐derived substances (CADS). Arteritis varied by mouse strain with the highest incidence by 71.1% (27/38) found in C3H/HeN mice, but absent in CBA/JN mice (0%, 0/27), suggesting association of genomic background to develop the disease. The present study aims to elucidate the susceptibility loci associated with coronary arteritis by using this animal model. The association of the onset of arteritis with polymorphic microsatellite markers between the two strains was examined using one hundred and fifteen of N1 backcross progeny [(CBA×C3H)F1×C3H]. Based on our analysis, arteritis‐susceptibility loci with suggestive linkage were mapped on D1Mit171 and D1Mit245 (map position 20.2 cM) on chromosome 1 (P=0.0019). These loci include several kinds of inflammatory cytokine receptors, such as interleukin 1 receptor and tumor necrosis factor receptor. We also found the cytokine response against CADS, levels of inflammatory cytokines interleukin‐1β, tumor necrosis factor‐α, and interleukin‐6 in sera increased within 24 hr after CADS injection. Our results may indicate based on genomics that ligand‐receptor interaction between these inflammatory cytokines and the receptors of these cytokines may affect the onset of arteritis.

MacroH2A1.2 binds the nuclear protein Spop

X-chromosome inactivation is a phenomenon by which one of the two X chromosomes in somatic cells of female mammals is inactivated for life. The inactivated X chromosomes are covered with Xist (X-inactive specific transcript) RNA, and also enriched with the histone H2A variant, macroH2A1.2. The N-terminal one-third of macroH2A1.2 is homologous to core histone H2A, but the function of the C-terminal two-thirds, which contains a basic, putative leucine zipper domain, remains unknown. In this study, we tried analyzing protein-protein interaction with a yeast two-hybrid system to interact with the nonhistone region of mouse macroH2A1.2. The results showed that macroH2A1.2 interacts with mouse nuclear speckled type protein Spop. The Spop protein has a unique composition: an N-terminal MATH, and a C-terminal BTB/POZ domain. Further binding domain mapping in a glutathione-S-transferase (GST) pull-down experiment revealed that macroH2A1.2 binds the MATH domain of Spop, which in turn binds to the putative leucine zipper domain of macroH2A1.2.

Myeloperoxidase has directly-opposed effects on nitration reaction--study on myeloperoxidase-deficient patient and myeloperoxidase-knockout mice.

Myeloperoxidase (MPO) catalyzes a nitration reaction to form nitrotyrosine in the presence of high nitrite, the metabolite of NO. Human leukocyte was shown to cause phenolic nitration using released MPO as a catalyst in the presence of nitrite. It opposes our previous finding that inhibition of MPO was essential for phenol nitration in human leukocyte study. To clarify the role of MPO, we utilized MPO-deficient human leukocytes and MPO-knockout mice. Even in the absence of exogenously added nitrite, high nitration product was observed in MPO-deficient leukocytes. In liver subjected to ischemia/reperfusion injury, a significantly higher amount of nitrotyrosine was produced in MPO-knockout mice than in normal mice. These results clearly demonstrate that MPO inhibits the accumulation of nitration products in vivo . Further experiments showed that MPO could degrade nitrotyrosine in the presence of glutathione. Thus, MPO-induced degradation of nitration products may cause the underestimation of the nitration product generated in vivo . We conclude that MPO may act predominantly to scavenge nitrotyrosine under physiological nitrite condition, and protect against injurious effect of nitrotyrosine.

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

The heme protein myeloperoxidase (MPO) contributes critically to O2-dependent neutrophil antimicrobial activity. Two Japanese adults were identified with inherited MPO deficiency because of mutations at Arg-499 or Gly-501, conserved residues near the proximal histidine in the heme pocket. Because of the proximity of these residues to a critical histidine in the heme pocket, we examined the biosynthesis, function, and spectral properties of the peroxidase stably expressed in human embryonic kidney cells. Biosynthesis of normal MPO by human embryonic kidney cells faithfully mirrored events previously identified in cells expressing endogenous MPO. Mutant apopro-MPO was 90 kDa and interacted normally with the molecular chaperones ERp57, calreticulin, and calnexin in the endoplasmic reticulum. However, mutant precursors were not proteolytically processed into subunits of MPO, although secretion of the unprocessed precursors occurred normally. Although δ-[14C]aminolevulinic acid incorporation demonstrated formation of pro-MPO in both mutants, neither protein was enzymatically active. The Soret band for each mutant was shifted from the normal 430 to ∼412 nm, confirming that heme was incorporated but suggesting that the number of covalent bonds or other structural aspects of the heme pocket were disrupted by the mutations. These studies demonstrate that despite heme incorporation, mutations in the heme environs compromised the oxidizing potential of MPO.

IL-10 Is a Negative Regulatory Factor of CAWS-Vasculitis in CBA/J Mice as Assessed by Comparison with Bruton’s Tyrosine Kinase-Deficient CBA/N Mice

Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, exhibits vasculitis-inducing activity (CAWS-vasculitis) in mice. The sensitivity to CAWS-vasculitis varies greatly among mouse strains. This study examined the factors contributing to or inhibiting CAWS-vasculitis using CAWS-vasculitis-resistant CBA/J mice and Bruton’s tyrosine kinase-deficient CBA/N mice, which is a CAWS-vasculitis-sensitive strain that has the same origin as CBA/J mice. After stimulation with various kinds of pathogen-associated molecular patterns, the production of inflammatory cytokines IL-6 and IFN-γ was induced in CBA/N mice, whereas that of immunosuppressive IL-10 was induced in CAWS-vasculitis-resistant CBA/J mice. Furthermore, the production of tissue inhibitor of metalloproteinase 1, an endogenous matrix metalloproteinase inhibitor, was observed in CBA/J mice. The results strongly suggest that the difference in the production of these cytokines is closely linked to the development of CAWS-vasculitis.