Biomedical subjects

Hirohisa Hirai

Publications and source records attributed to Hirohisa Hirai.

At least 19 recordsLinked to original sources

PubMed2006

Identification and characterization of a nuclear receptor subfamily I member in the Platyhelminth Schistosoma mansoni (SmNR1).

A cDNA encoding a nuclear receptor subfamily I member in the platyhelminth Schistosoma mansoni (SmNR1) was identified and characterized. SmNR1 cDNA is 2406 bp long and contains an open reading frame encoding a 715 residue protein. Phylogenetic analysis demonstrates that SmNR1 is a divergent member of nuclear receptor subfamily I with no known orthologue. SmNR1 was localized to S. mansoni chromosome 1 by fluorescent in situ hybridization. Gene structure of SmNR1 was determined showing it to consist of eight exons spanning more than 14 kb. Quantitative real-time RT-PCR showed that SmNR1 was expressed throughout schistosome development with a higher expression in eggs, sporocysts and 21-day worms. SmNR1 contains an autonomous transactivation function (AF1) in the A/B domain as demonstrated in a yeast one-hybrid assay; it interacts with SmRXR1 in a yeast two-hybrid assay and in a glutathione S-transferase pull-down assay. Electrophoretic mobility shift assay showed that SmNR1 could form a heterodimer with SmRXR1 to bind to DNA elements containing the half-site AGGTCA, a direct repeat of the half-site separated by 0-5 nucleotides (DR1-DR5) and a palindrome repeat of the half-site not separated by nucleic acids (Pal0). Transient transfection in mammalian COS-7 cells showed that SmNR1/SmRXR1 could enhance the transcriptional activation of a DR2-dependent reporter gene. Our results demonstrate that SmNR1 is a partner of SmRXR1.

Amino Acid Sequence↗

PubMed2006

Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases.

Egg production is responsible for life cycle progression and host immunopathology during schistosomiasis, with the associated parasite molecules being investigated as potential novel chemotherapeutic targets. Here, we characterize two Schistosoma mansoni products, tyrosinase 1 and tyrosinase 2 (SmTYR1/SmTYR2) and show that their diphenol oxidase enzyme activities are critical for eggshell formation and production. The genes encoding these bifunctional enzymes (monophenol and diphenol oxidases) result from a duplication event that likely occurred before speciation and exist in the parasite's genome as multiple copies, which are linked and localized to chromosomes 4 and W. SmTYR1/SmTYR2 transcription and diphenol oxidase action are developmentally regulated with most enzyme activity localized to the eggshell-producing cells contained within the vitellaria of adult female worms. Importantly, kojic-acid mediated inhibition (IC50=0.5 microM) of SmTYR1/SmTYR2's diphenol oxidase activity during in vitro culture of sexually mature adult worms resulted in a significant decrease in the production of phenotypically normal eggs. Therefore our data suggest that SmTYR1/2 inhibition represents a novel and potentially effective strategy for combating schistosomiasis and furthermore, it may point to new methods for combinatorial control of immunopathology and egg transmission during platyhelminth infection.

Animals↗

PubMed2006

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells.

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.

Actins↗

PubMed2006

Human Endogenous Retrovirus (HERV)-R family in primates: Chromosomal location, gene expression, and evolution.

Hitherto, full-length endogenous retrovirus (HERV)-R has been located at human chromosome 7q11.2, and mRNA and envelope proteins have been detected in placenta and a variety of other cell types. In the present study, using a probe derived from the gorilla fosmid library, we detected the paralogous locus (7q31.3) of the HERV-R env gene in human chromosome 7q11.2, and also determined the chromosomal location in apes and Old World monkeys. The HERV-R gene was not detected in New World monkeys or prosimians with FISH and PCR analyses. We determined the sequences of the HERV-R env genes obtained from the genomic DNA of primates using PCR and sequencing tools. Except for a HERV-R env sequence derived from gorilla DNA, the functional domains of putative envelope proteins are conserved, suggesting that those domains could have a functional capacity in the primate genome. In addition, we investigated the env gene expression of HERV-R in various human tissues and cancer cells. An RT-PCR approach indicated that the env gene was expressed in several human tissues (brain, prostate, testis, kidney, placenta, thymus, and uterus) and cancer cells (RT4, BT-474, MCF7, OVCAR-3, LOX-IMVI, and AZ521). Taken together, our data could be of great use for understanding the evolutionary dynamics of HERV-R through primate radiation as well as the implications of its functional role in human tissues and cancers cells.

Animals↗

PubMed2005

Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes, the genera Hylobates and Symphalangus.

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.

AT Rich Sequence↗

PubMed2005

Evidence for a dispersed Hox gene cluster in the platyhelminth parasite Schistosoma mansoni.

In most bilaterian organisms so far studied, Hox genes are organized in genomic clusters and determine development along the anteroposterior axis. It has been suggested that this clustering, together with spatial and temporal colinearity of gene expression, represents the ancestral condition. However, in organisms with derived modes of embryogenesis and lineage-dependent mechanisms for the determination of cell fate, temporal colinearity of expression can be lost and Hox cluster organization disrupted, as is the case for the ecdysozoans Drosophila melanogaster and Caenorhabditis elegans and the urochordates Ciona intestinalis and Oikopleura dioica. We sought to determine whether a lophotrochozoan, the platyhelminth parasite Schistosoma mansoni, possesses a conserved or disrupted Hox cluster. Using a polymerase chain reaction (PCR)-based strategy, we have cloned and characterized three novel S. mansoni genes encoding orthologues of Drosophila labial (SmHox1), deformed (SmHox4), and abdominal A (SmHox8), as well as the full-length coding sequence of the previously described Smox1, which we identify as an orthologue of fushi tarazu. Quantitative reverse transcriptase-PCR showed that the four genes were expressed at all life-cycle stages but that levels of expression were differentially regulated. Phylogenetic analysis and the conservation of "parapeptide" sequences C-terminal to the homeodomains of SmHox8 and Smox1 support the grouping of platyhelminths within the lophotrochozoan clade. However, Bacterial Artificial Chromosome (BAC) library screening followed by genome walking failed to reconstitute a cluster. The BAC clones containing Hox genes were sequenced, and in no case were other Hox genes found on the same clone. Moreover, the SmHox4 and SmHox8 genes contained single very large introns (>40 kbp) further indicating that the schistosome Hox cluster is highly extended. Localization of the Hox genes to chromosomes using fluorescence in situ hybridization showed that SmHox4 and SmHox8 are on the long arm of chromosome 4, whereas SmHox1 and Smox1 are on chromosome 3. In silico screening of the available genome sequences corroborated results of Southern blotting and BAC library screening that indicate that there are no paralogues of SmHox1, SmHox4, or SmHox8. The schistosome Hox cluster is therefore not duplicated, but is both dispersed and disintegrated in the genome.

Amino Acid Sequence↗

PubMed2005

A truncated tropomyosin-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Amino Acid Motifs↗

PubMed2005

A whole-arm translocation (WAT8/9) separating Sumatran and Bornean agile gibbons, and its evolutionary features.

Gibbons, like orangutans, are a group of threatened Asian apes, so that genetic monitoring of each species or subspecies is a pressing need for conservation programmes. We conducted a project to take, as far as possible, samples of known origin from wild-born animals from Sumatra and Borneo (Central Kalimantan) for genetic monitoring of agile gibbons. As a result, we found a whole arm translocation between chromosomes 8 and 9 (WAT8/9) specific to Sumatran agile gibbons. Furthermore, population surveys suggested that the form with the WAT8/9 seems to be incompatible with an ancestral form, suggesting that the former might have extinguished the latter from Sumatran populations by competition. In any case, this translocation is a useful chromosomal marker for identifying Sumatran agile gibbons. Population genetic analyses with DNA showed that the molecular genetic distance between Sumatran and Bornean agile gibbons is the smallest, although the chromosomal difference is the largest. Thus, it is postulated that WAT8/9 occurred and fixed in a small population of Sumatra after migration and geographical isolation at the last glacial period, and afterwards dispersed rapidly to other populations in Sumatra as a result of the bottleneck effect and a chromosomal isolating mechanism.

Animals↗

PubMed2004

Characterization and evolution of major histocompatibility complex class II genes in the aye-aye, Daubentonia madagascariensis.

Major histocompatibility complex genes (Mhc-DQB and Mhc-DRB) were sequenced in seven aye-ayes (Daubentonia madagascariecsis), which is an endemic and endangered species in Madagascar. An aye-aye from a north-eastern population showed genetic relatedness to individuals of a north-western population and had a somewhat different repertoire from another north-eastern individual. These observations suggest that the extent of genetic variation in Mhc genes is not excessively small in the aye-aye in spite of recent rapid destruction of their habitat by human activities. In light of Mhc gene evolution, trans-species and allelic polymorphisms can be estimated to have been retained for more than 50 Ma (million years) based on the time scale of lemur evolution.

Animals↗

PubMed2004

Schistosoma mansoni genome project: an update.

A schistosome genome project was initiated by the World Health Organization in 1994 with the notion that the best prospects for identifying new targets for drugs, vaccines, and diagnostic development lie in schistosome gene discovery, development of chromosome maps, whole genome sequencing and genome analysis. Schistosoma mansoni has a haploid genome of 270 Mb contained on 8 pairs of chromosomes. It is estimated that the S. mansoni genome contains between 15000 and 25000 genes. There are approximately 16689 ESTs obtained from diverse libraries representing different developmental stages of S. mansoni, deposited in the NCBI EST database. More than half of the deposited sequences correspond to genes of unknown function. Approximately 40-50% of the sequences form unique clusters, suggesting that approximately 20-25% of the total schistosome genes have been discovered. Efforts to develop low resolution chromosome maps are in progress. There is a genome sequencing program underway that will provide 3X sequence coverage of the S. mansoni genome that will result in approximately 95% gene discovery. The genomics era has provided the resources to usher in the era of functional genomics that will involve microarrays to focus on specific metabolic pathways, proteomics to identify relevant proteins and protein-protein interactions to understand critical parasite pathways. Functional genomics is expected to accelerate the development of control and treatment strategies for schistosomiasis.

Animals↗

PubMed2004

Transcriptional repression mechanisms of nucleolus organizer regions (NORs) in humans and chimpanzees.

Polymorphisms related to transcriptional inactivation of nucleolus organizer regions (NORs) have long been described in many animals, particularly humans. However, the precise aetiology of such variations is not always clear. We conducted analyses to investigate the repression mechanisms in humans and chimpanzees using FISH (fluorescence in situ hybridisation) with 18S rDNA, Ag-NOR (silver nitrate) staining, C-banding, and the in situ nick translation technique with the HpaII restriction enzyme. Examination of 48 humans and 46 chimpanzees suggested that there are at least three different mechanisms that produce inactivation of NORs. These include: (1) elimination of rDNA; (2) DNA methylation: (3) gene silencing due to position effects induced by heterochromatin (C-bands) and/or telomeres.

Animals↗

PubMed2004

FISH mapping for helminth genome.

Basic techniques for fluorescence in situ hybridization (FISH) mapping that have been used in genome projects on schistosomes and filariae are introduced. The chapter shows techniques specific for bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones and includes experiences of chromosome preparation, DNA labeling, hybridization, microscopy, and localization of BAC clones.

Animals↗

PubMed2003

Frequent segmental sequence exchanges and rapid gene duplication characterize the MHC class I genes in lemurs.

Major histocompatibility complex (MHC) class I genes have complicated and profound evolutionary histories. To reconstruct and better understand their histories, partial class I genes (exon 2-intron 2-exon 3) were sequenced in a sampling of prosimians (Strepsirhini, Primates). In total, we detected 117 different sequences from 36 Malagasy prosimians (lemurs) and 1 non-Malagasy prosimian (galago) representing 4 families, 7 genera, and 13 species. Unlike the MHC class II genes ( MHC-DRB), MHC class I genes show a generally genus-specific mode of evolution in lemurs. Additionally, no prosimian class I loci were found to be orthologous to HLA genes, even at highly conserved loci (such as HLA-E, HLA-F). Phylogenetic analysis indicates that nucleotide diversity among loci was very small and the persistence time of the polymorphisms was short, suggesting that the origin of the lemur MHC class I genes detected in this study was relatively recent. The evolutionary mode of these genes is similar to that of classic HLA genes, HLA-A, HLA-B, and HLA-C, in terms of their recent origin and rarity of pseudogenes, and differs from them with respect to the degree of gene duplications. From the viewpoint of MHC genes evolution, some interlocus sequence exchanges were apparently observed in the lemur lineage upon phylogenetic and amino acid motif analyses. This is also in contrast to the evolutionary mode of HLA genes, where intralocus exchanges have certainly occurred but few interlocus exchanges have taken place. Consequently, the gene conversion model for explaining the generation of the MHC diversity among different loci can be thought to play more important roles in the evolution of lemur MHC class I genes than in that of HLA genes.

Animals↗

PubMed2003

The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species.

The mammalian amelogenin (AMEL) genes are found on both the X and Y chromosomes (gametologous). Comparison of the genomic AMEL sequences in five primates and three other mammals reveals that the 5' portion of the gametologous AMEL loci began to differentiate in the common ancestor of extant mammals, whereas the 3' portion differentiated independently within species of different mammals. The boundary is marked by a transposon insertion in intron 2 and is shared by all species examined. In addition, 540-kb DNA sequences from the short arm of the human X chromosome are aligned with their Y gametologous sequences. The pattern and extent of sequence differences in the 5' portion of the AMEL loci extend to a proximal region that contains the ZFX locus, and those in the 3' portion extend all the way down to the pseudoautosomal boundary (PAB)1. We concluded that the AMEL locus spans an ancient PAB, and that both the ancient and present PABs were determined by chance events during the evolution of mammals and primates. Sex chromosome differentiation likely took place in a region that contains the male-determining loci by suppressing homologous recombination.

Amelogenin↗

PubMed2003

Genetic mechanism and property of a whole-arm translocation (WAT) between chromosomes 8 and 9 of agile gibbons (Hylobates agilis).

C-banding analysis with 47 gibbons of the subgenus Hylobates (Hylobates) (44-chromosome gibbons) uncovered that the gibbons had a characteristic complicated C-banding pattern. The C-band pattern also revealed that a whole-arm translocation (WAT) between chromosomes 8 and 9 existed only in the species H. agilis (agile gibbon). Comprehensive consideration allows postulation that the translocation seemed to be restricted to two subspecies: H. agilis agilis (mountain agile gibbon) and H. agilis unko (lowland agile gibbon), found in Sumatra and part of the Malay Peninsula. Moreover, combined intensive analyses of C-banding and chromosome painting provided strong evidence for a plausible evolutionary pathway of chromosome differentiation of chromosomes 8 and 9. The C-banded morph 8M(t/c) seemed to be the primary type of chromosome 8 in the subgenus and to have altered into the three morphs through three pericentric inversions. The newest morph (8A(M/ci)) produced by the third inversion exchanged the long arm for chromosome 9, and subsequently constructed the WAT morphs of 8/9A(Mc/ct) and 9/8M(i/ci).

Animals↗

PubMed2003

A probe generated by chromosome microdissection, useful for analyzing Y chromosome evolution in Old World monkeys.

We isolated a DNA probe, designated MMDYZ1, using a chromosome microdissection technique from the Y chromosome of the Rhesus monkey. The probe obtained from eight whole Y chromosomes shows higher specificity for the Y short arm of the Rhesus monkey, which consists totally of constitutive heterochromatin. Two microclones (MMY#3 and MMY#4) were constructed from the Y-specific primary PCR products. Sequence analysis of these two microclones revealed that both were essentially identical to each other and the sizes were 870 and 686 bp, respectively. From alignment analysis using the Genbank database of primates, the alphoid DNA has the highest affinity with the probe. However, the total composition of this probe has extremely high homology with the Y short arm of the Rhesus monkey, as demonstrated by fluorescence in-situ hybridization (FISH). Comparative FISH-mapping disclosed that this DNA-sequence cluster was located at extremely different sites on the Y chromosome in several species of the Old World monkey. Accordingly, this probe seems to be a high-quality tool, now established for the first time, for investigating Y chromosome evolution of the Old World monkey.

Animals↗

PubMed2002

Mhc-DRB genes evolution in lemurs.

Partial exon 2 sequences (202 bp) of the lemur Mhc-DRB genes were sequenced. A total of 137 novel sequences were detected in 66 lemurs, representing four out of the five extant families. Trans-species polymorphisms and even identical sequences were observed not only among genera but also among families. Based on the time-scale of lemur evolution, these findings suggest that some identical sequences have been maintained for more than 40 million years. This is in contrast to the evolutionary mode of simian DRB genes, where such identical sequences have been retained for at most several million years. To explore the reasons behind these unexpected findings, the degree of recombination and the synonymous substitution rate in lemurs and simians were examined. We found that (1) little difference existed in the extent of recombination, (2) frequent recombination occurred within the alpha-helix as well as between the beta-pleated sheet and the alpha-helix, and (3) the synonymous substitution rate was significantly reduced in lemur lineages. Upon phylogenetic analysis, lemur DRB genes were clustered by themselves and separated from the other primate DRB genes (simians and non-Malagasy prosimians). This result suggests that the DRB variations in extant lemur populations have been generated after the divergence of the lemurs from the remaining primates. This mode of substitution accumulation is also supported by a pattern of mismatch distribution among lemur DRB genes. These observations correspond with the postulation that a severe bottleneck occurred when the ancestors of lemurs settled into Madagascar from the African continent.

Amino Acid Motifs↗

PubMed2002

Adenylosuccinate lyase of Schistosoma mansoni: gene structure, mRNA expression, and analysis of the predicted peptide structure of a potential chemotherapeutic target.

Adenylosuccinate lyase is an enzyme used in parasite nucleotide salvage pathways that cleaves adenylosuccinate into adenosine 5'-monophosphate and fumarate. A cDNA encoding adenylosuccinate lyase from the trematode parasite Schistosoma mansoni has been cloned for analysis. Sequencing of the cDNA revealed an open reading frame of 1454 nucleotides that codes for a protein with a predicted mass of about 54.5 kDa. Comparative analysis of the predicted protein sequence shows that S. mansoni adenylosuccinate lyase has a lot of similarity with human adenylosuccinate lyase. Genomic analysis using S. mansoni adenylosuccinate lyase-containing bacterial artificial chromosome (BAC) clones revealed a gene of approximately 19.4 kb consisting of eight exons and seven introns. Intron 6 was found to contain a novel 2.9 kb long terminal repeat retrotransposon with direct terminal repeats of 500 nucleotides. Fluorescence in situ hybridisation mapping localised S. mansoni adenylosuccinate lyase to the Z and W chromosomes. Analysis of S. mansoni adenylosuccinate lyase mRNA expression levels using real time reverse transcriptase (RT)-PCR showed that S. mansoni adenylosuccinate lyase is expressed at higher levels in the female worms than in the male worms and is expressed at different levels than other purine nucleotide salvage enzymes. Male homogenate showed a specific activity of 10.3 units/mg protein while the female showed a specific activity of 24.2 units/mg protein. These data indicate that S. mansoni adenylosuccinate lyase is an important parasite enzyme and should be examined as a potential chemotherapeutic target.

Adenylosuccinate Lyase↗