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Biomedical subjects

Shinya Watanabe

Publications and source records attributed to Shinya Watanabe.

At least 19 recordsLinked to original sources
PubMed2006

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

Arteries↗
PubMed2006

Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions.

Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and PsiTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.

Anti-Bacterial Agents↗
PubMed2006

Comparison of the growth performances of offspring produced by a pair of cloned cattle and their nuclear donor animals.

In the present study, the growth performance of a calf produced by mating a somatic cell cloned dam and sire was compared with that of its full siblings produced by mating the cattle used as nuclear donors for the cloned animals. The somatic cell cloned dam and sire were derived from cultured cumulus cells and ear cells, respectively. The cloned dam was artificially inseminated with semen from the cloned sire. A female calf was produced that was reared under general group feeding conditions. The calf was subjected to a clinical examination and to hematology, serum biochemistry, and telomere length analyses; all of these tests indicated that the calf was normal. The growth characteristics (body weight and shoulder height) of the calf fell within the range of the full siblings of the same sex produced by mating the animals used as the nuclear donors of clones. These findings suggest that the same breeding performance is expected from mating a cloned dam and sire as from mating the animals used as nuclear donors for the clones.

Animals↗
PubMed2006

Chemoenzymatic synthesis and application of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination.

Sialoglycopeptide (SGP) is referred as the glycopeptide in hen's egg yolk, which has an N-linked, complex-type, disialyl biantennary oligosaccharide with an alpha-(2-->6)-sialyl N-acetyllactosamine residue. The residue is known as a binding ligand of type-A human influenza virus hemagglutinin. We describe herein a simple synthesis of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination that makes use of the natural source ingredient, SGP, and the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). Its inhibitiory activity for influenza virus hemagglutination is 40 times higher than that of SGP, and its competitive inhibition is determined to be over 300 times higher than that of fetuin. These results indicate that a sialoglycopolymer having a multivalent sialo-oligosaccharide could potentially be used for the prevention of influenza virus infection.

Acetylglucosamine↗
PubMed2006

KSHV RTA induces a transcriptional repressor, HEY1 that represses rta promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus that is frequently associated with the formation of tumors like Kaposi's sarcoma and primary effusion lymphomas. KSHV RTA is required for lytic replication/reactivation in KSHV-infected cells. We developed an inducible RTA expression cell line to search for RTA-inducible genes. Using DNA chip technology, we showed that hey1, a basic helix-loop-helix-containing transcription factor, increased three- to fourfold in this line. In a luciferase reporter system, RTA activated the hey1 promoter in several cell lines; conversely, HEY1 repressed the rta promoter. Chromatin immunoprecipitation assays indicated that HEY did not bind the rta promoter directly, although it may repress rta expression as part of a complex. Thus, hey1 may help to downregulate RTA after its burst of expression during lytic replication/reactivation. Similar repression mechanisms may downregulate immediate-early gene expression in other herpesviruses, although different transcription factors are probably involved.

Animals↗
PubMed2006

Mechanoenzymatic characterization of human myosin Vb.

There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.

Actins↗
PubMed2006

Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism.

Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. We analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s(-1)) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s(-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s(-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from our kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure.

Actins↗
PubMed2005

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs.

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.

Animals↗
PubMed2005

Influence of inhalation anesthesia assessed by comprehensive gene expression profiling.

Although general anesthesia is routinely used as an essential surgical procedure and its harmlessness has been evaluated and endorsed by clinical outcomes, little is known about its comprehensive influence that is not reflected in mortality and morbidity. In this paper, we have shown that inhalation anesthesia affected the expression of <1.5% of >10,000 genes, by analyzing the expression profiles for multiple organs of rats anesthetized with sevoflurane. The small number of transcripts affected by the inhalation anesthesia comprised those specific to single and common in multiple organs. The former included genes mainly associated with drug metabolism in the liver and influenced by agents such as amphetamine in the brain. The latter contained multiple circadian genes. In the brain, we failed to detect the alteration of the clock gene expression with the exception of Per2, assuming that anesthesia perturbs circadian rhythms. Our findings provide the first assessment for the influence of inhalation anesthesia by approaches of experimental biology and genome science.

Anesthetics, Inhalation↗
PubMed2005

Growth and fertility of bulls cloned from the somatic cells of an aged and infertile bull.

In the present study, somatic cell cloning technology was used to produce eight newborn calves from an aged, infertile bull. Average birth weight of these calves was significantly higher than that of calves produced using AI. Four of the cloned calves died during the peripartum period; the remaining four (Clones A-D) survived and were used in this study. Two of the surviving calves (Clones C and D) were castrated; growth rates of the intact and castrated clones were similar to those of intact and castrated bulls, respectively, that had been derived by AI. Both uncastrated bulls (Clones A and B) began to produce normal semen at approximately 12 months of age. Semen produced by these clones, and their nuclear donor, was subsequently used for IVF; the proportion of IVM-IVF oocytes developing to the blastocyst stage was 23.4% (50/214), 28.4% (52/183) and 30.9% (63/204), respectively. Conception rates for AI were 54.5% (12/22) and 62.7% (64/102) for semen derived from Clone A and from the nuclear donor, respectively. The length of pregnancy and birth weight of the calves derived from semen collected from clones were similar to those of calves obtained by conventional AI using semen from their nuclear donor. Therefore, sires cloned from the somatic cells of an aged and infertile bull had normal fertility.

Aging↗
PubMed2005

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.

Database Management Systems↗
PubMed2005

Whole-genome sequencing of staphylococcus haemolyticus uncovers the extreme plasticity of its genome and the evolution of human-colonizing staphylococcal species.

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S.haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S.haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S.haemolyticus, S.aureus, and S.epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S.haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.

Biological Evolution↗
PubMed2004

Genetic characterization of methicillin-resistant Staphylococcus aureus.

The genome structure of Staphylococcus aureus is analyzed. The genome is composed of two domains. The first domain, descendent from an ancestral bacterial species, contains house-keeping genes that showed highest homology to those of Bacillus species. The second domain contained the genes responsible for virulence and drug-resistance in human infection that seems to have been acquired from other bacterial species via lateral gene transfer. The latter domain constitutes the genetic information that makes S. aureus a notorious hospital pathogen.

Biological Evolution↗
PubMed2004

The myosin cardiac loop participates functionally in the actomyosin interaction.

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Actomyosin↗
PubMed2004

New macrocyclic musk compounds.

The syntheses and olfactory evaluations of eight new macrocyclic musks with a 1,6-dioxa structure (1a-d and 1'a-d) as well as of twelve optically active 3-methyl macrolides (5a-c and 5'a-c) are reported. These macrocycles were synthesized via intramolecular metathesis mediated by the Grubbs catalyst. Despite the absence of a C=O function, the 1,6-dioxa compounds, both unsaturated (1) and saturated (1'), possess musky odors similar to those of macrocyclic ketones and lactones. Especially 16-membered rings were found to display an intense and pleasant musk character. However, in the case of optically active 3-methyl macrolides (5, 5'), only the (R)-configured 15- and 16-membered rings had intense and pleasant musk notes.

Fatty Acids, Monounsaturated↗
PubMed2004

Dynamic regulation of gene expression by the Flt-1 kinase and Matrigel in endothelial tubulogenesis.

A nontubulogenic endothelial cell line, NP31, can be transformed by the active form of the Flt-1 kinase (BCR-FLTm1) into Tb3 cells, which show a tubulogenic property only when cultured in Matrigel. By utilizing this strict dependence of NP31 on BCR-FLTm1 and Matrigel for experimental angiogenesis, we performed microarray analyses under several conditions and found 97 genes whose dynamically regulated profiles of gene expression are divided into nine groups, in two major clusters. In one major cluster, gene expression is interdependently regulated by BCR-FLTm1 or Matrigel. The second major cluster contains genes whose expression patterns under BCR-FLTm1 influence are reversed by Matrigel. Based on these gene expression patterns in NP31 driven by BCR-FLTm1 and/or Matrigel, we propose a model in which sequential and alternate stimulation by BCR-FLTm1 and Matrigel induces cooperative regulation of subsets of genes. Microarray analyses of Tb3 under 11 different conditions revealed 5 candidate genes whose gene expression regulation is most closely associated with tubulogenesis.

Animals↗