Biomedical subjects

Hiroaki Kawasaki

Publications and source records attributed to Hiroaki Kawasaki.

At least 19 recordsLinked to original sources

PubMed2006

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs.

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.

Amino Acid Sequence↗

PubMed2006

Genetic structure of the dopamine receptor D4 gene (DRD4) and lack of association with schizophrenia in Japanese patients.

In order to investigate the contribution of genetic variation in the human dopamine receptor D4 gene (DRD4) to the risk of developing schizophrenia, we carried out a genetic analysis of 27 polymorphisms in 216 schizophrenic patients and 243 healthy controls from the Kyushu region of Japan. Twenty-two single nucleotide polymorphisms (SNPs) and five insertion/deletion polymorphisms were analyzed in this study, including four novel SNPs and a novel mononucleotide repeat. Linkage disequilibrium (LD) and haplotype analyses reveal weak LD across the DRD4 gene. In univariate analysis female individuals with allele -521C had a higher risk for schizophrenia. However, this finding was not significant after correction for multiple hypothesis testing. No other polymorphisms or haplotypes differed between schizophrenic patients and controls. Likewise, multivariate analyses did not reveal any statistically significant associations.

Adolescent↗

PubMed2006

Sequential O- and N-acylation protocol for high-yield preparation and modification of rotaxanes: synthesis, functionalization, structure, and intercomponent interaction of rotaxanes.

A pseudorotaxane consisting of a 24-membered crown ether and secondary ammonium salt with the hydroxy group at the terminus was quantitatively acylated by bulky acid anhydride in the presence of tributylphosphane as catalyst to afford the corresponding rotaxane in high yield. Large-scale synthesis without chromatographic separation was easily achieved. The ammonium group in the resulting rotaxane was quantitatively acylated with excess electrophile in the presence of excess trialkylamine. Various N-functionalized rotaxanes were prepared by this sequential double-acylation protocol. 1H NMR spectra and X-ray crystallographic analyses of the rotaxanes showed that the crown ether component was captured on the ammonium group in ammonium-type rotaxane by strong hydrogen-bonding intercomponent interaction. The conformation around the ammonium group was fixed by the hydrogen-bonding interaction. Meanwhile, the conformation of the amide-type rotaxane was determined by the weak CH/pi interaction between the methylene group in crown ether and the benzene ring of the axle component. The N-acylation of ammonium-type rotaxane is useful for the preparation of both functionalized rotaxanes and weak intercomponent interaction-based rotaxanes.

Acylation↗

PubMed2006

Down-regulation of endogenous Wt1 expression by Sry transgene in the murine embryonic mesonephros-derived M15 cell line.

Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.

Animals↗

PubMed2006

Developmental and triiodothyronine-induced expression of genes encoding preprotemporins in the skin of Tago's brown frog Rana tagoi.

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.

Amino Acid Sequence↗

PubMed2006

Construction of microRNA-containing vectors for expression in mammalian cells.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression by single-stranded RNAs of 18 to 25 nucleotides in length. Hundreds of miRNAs have been found in animals and plants, some of which play important roles in development or differentiation. Increasing attention has thus been paid to their biogenesis and regulation mechanisms and the identification of target genes. We are constructing a comprehensive expression vector library containing predicted human miRNAs. miRNA expression vectors containing human RNA polymerase II or III promoters, and utilizing a flexible vector system, can be useful for functional analysis.

Base Sequence↗

PubMed2005

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo.

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

Animals↗

PubMed2005

Lysophosphatidic acid acyltransferase-beta is a prognostic marker and therapeutic target in gynecologic malignancies.

Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase beta (LPAAT-beta), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-beta converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-beta was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-beta using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-beta also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-beta was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-alpha. These findings identify LPAAT-beta as a potential prognostic and therapeutic target in ovarian and endometrial cancer.

Acyltransferases↗

PubMed2005

siRNA induced transcriptional gene silencing in mammalian cells.

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. It has recently become clear in human cells that RNA plays a far more profound and complex role in regulating the expression of the gene. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by small-interfering RNAs (siRNAs). Specifically, the recent observations by both our groups that siRNAs can silence target genes at the level of the chromatin in mammalian cells. We discuss here siRNA mediated transcriptional gene silencing and directed DNA methylation as well as the putative mechanism involved in human cells. Undoubtedly, the ramifications from this paradigm shift of RNA regulating the expression of the gene are immeasurable both therapeutically (i.e., directed control of a genes expression) and biologically in understanding the evolution of the cell.

Animals↗

PubMed2005

Exploration of human miRNA target genes in neuronal differentiation.

MicroRNAs (miRNAs) are endogenous non-coding RNA molecules that inhibit protein translation in a sequence specific manner. We carried out microarray analyses of 180 human pre-miRNAs in neuroblastoma cell, SH-SY5Y, following stimulation by TPA. Twelve of the pre-miRNAs were up-regulated by the TPA stimulation. We also explored miRNA target genes associated with neuronal differentiation. Some miRNAs have complementarity with 3'UTR of the Notch1 gene, a regulator of neuronal differentiation, and luciferase assay showed that overexpression of these miRNAs reduced the luciferase activity of reporter genes containing the Notch1-3'UTR sequence. Our results suggest that miRNAs can be associated with TPA induced signalling pathways and expression of Notch1 gene.

Cell Differentiation↗

PubMed2005

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Animals↗

PubMed2005

Transcriptional gene silencing by short interfering RNAs.

Double-stranded RNA (dsRNA)-induced sequence-specific gene silencing in animals and plants is known as RNA interference. In mammalian cells, although long dsRNAs induce an interferon-mediated, non-specific gene silencing, short interfering RNAs (siRNAs) that are processed from long dsRNAs by Dicer can induce sequence-specific gene silencing. Since siRNAs can be designed for genes of interest, these molecules have great potential to be used as effective gene therapies. In plants, siRNAs targeted to CpG islands within a promoter can also induce RNA-directed DNA methylation. In addition, siRNAs have a role in heterochromatic gene silencing in fission yeasts, plants and animals. Recently, siRNAs were demonstrated to induce transcriptional gene silencing (TGS) via DNA methylation in human cells. This review will focus on TGS by siRNAs, such as DNA methylation and histone methylation.

Animals↗

PubMed2004

Induction of DNA methylation and gene silencing by short interfering RNAs in human cells.

Double-stranded RNAs (dsRNAs) induce post-transcriptional gene silencing in several species of animal and plant. In plants, dsRNAs targeted to CpG islands within a promoter can also induce RNA-directed DNA methylation; however, it remains unclear whether gene silencing mediated by DNA methylation can be induced by dsRNAs in mammalian cells. Here, we demonstrate that short interfering RNAs (siRNAs; 21-25-nucleotide RNA molecules) induce DNA methylation and histone H3 methylation in human cells. Synthetic siRNAs targeted to CpG islands of an E-cadherin promoter induced significant DNA methylation and histone H3 lysine 9 methylation in both MCF-7 and normal mammary epithelial cells. As a result, these siRNAs repressed expression of the E-cadherin gene at the transcriptional level. In addition, disrupting the expression of either one of two DNA methyltransferases (DNMT1 or DNMT3B) by specific siRNAs abolished the siRNA-mediated methylation of DNA. Moreover, vector-based siRNAs targeted to the erbB2 (also known as HER2) promoter also induced DNA methylation in MCF-7 cells. Thus, siRNAs targeted to CpG islands within the promoter of a specific gene can induce transcriptional gene silencing by means of DNA-methyltransferase-dependent methylation of DNA in human cells, and might have potential as a new type of gene therapeutic agent.

Cadherins↗

PubMed2004

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.

Animals↗

PubMed2004

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.

Aged↗

PubMed2004

Non-kinase second-messenger signaling: new pathways with new promise.

Intercellular signaling by growth factors, hormones and neurotransmitters produces second messenger molecules such as cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG). Protein Kinase A and Protein Kinase C are the principal effector proteins of these prototypical second messengers in certain cell types. Recently, novel receptors for cAMP and DAG have been identified. These proteins, designated EPAC (Exchange Protein directly Activated by cAMP) or cAMP-GEF (cAMP regulated Guanine nucleotide Exchange Factor) and CalDAG-GEF (Calcium and Diacylglycerol regulated Guanine nucleotide Exchange Factor) or RasGRP (Ras Guanine nucleotide Releasing Protein) are able to mediate some of the physiologic effects of the second messengers in a protein-kinase-independent fashion. These proteins are exchange factors for Ras family GTPases that operate in pathways that run parallel to the classic kinase-dependent pathways. The rapidly emerging recognition of the functions of these "non-kinase" effectors in diverse processes such as insulin secretion, thymocyte development, asthma and malignant transformation creates new opportunities for discovery and identifies potential new therapeutic targets.

Animals↗

PubMed2004

LIM kinase-2 targeting as a possible anti-metastasis therapy.

BACKGROUND: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. METHODS: We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. RESULTS: Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. CONCLUSIONS: Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells.

Base Sequence↗

PubMed2004

Cell migration and metastasis as targets of small RNA-based molecular genetic analyses.

Metastatic tumor cells can migrate from one place to another in the body. This involves their adherence to host cell layers and subsequent transcellular movements by a complex process, molecular basis of which are yet to be clarified. Elucidation of genes functionally involved in metastasis may lead to deeper understanding of the mechanism of cell migration, and identification and designing of metastasis-modulating strategies for cancer therapeutics. We review here cell migration in tumor metastasis and the use of small RNA-based approaches to identify functional genes. We then describe our promising novel approach that uses randomized ribozyme libraries for identification of genes involved in cell migration, a consistent feature of metastatic cells.

Animals↗