Biomedical subjects

Syuichi Oka

Publications and source records attributed to Syuichi Oka.

At least 19 recordsLinked to original sources

PubMed2005

Alteration of gene expression of G protein-coupled receptors in endocrine disruptors-caused hyperactive rats.

We examined the effects of endocrine disruptors on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A, p-octylphenol, nonylphenol, dibutylphthalate (DBP), dicyclohexylphthalate (DCHP), or diethylhexylphthalate (DEHP) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.3- to 1.6-fold more active in the nocturnal phase than control rats. Based on DNA macroarray analyses of the midbrain at 8 weeks of age, the endocrine disruptors altered the levels of gene expression of G protein-coupled receptors that were involved in not only dopaminergic neurotransduction but also many peptidergic neurotransduction. The gene expression of dopamine receptor D1A was decreased by nonylphenol, DBP, or DEHP by 0.23- to 0.4-fold, whereas that of dopamine D2 was increased by nonylphenol or DBP by 2- to 2.8-fold. It was notable that four of six endocrine disruptors tested, i.e. nonylphenol, DBP, DCHP, and DEHP largely downregulated the levels of gene expression of galanin receptor 2 by 0.11- to 0.28-fold. Bisphenol A, DBP or DCHP significantly decreased the levels of gene expression of dopamine transporter at 8 weeks more than 0.5-fold. Immunohistochemical analyses revealed that p-octylphenol impaired the immunoreactivity for tyrosine hydroxylase in substantia nigra pars compacta. Thus, endocrine disruptors caused hyperactivity in the rat, probably regulating the levels not only of gene expression but also of proteins of both G-protein-coupled receptors systems and dopaminergic neurotransduction system.

Animals↗

PubMed2005

A short peptide GPIGS promotes proliferation of hair bulb keratinocytes and accelerates hair regrowth in mice.

The aim of this study was to discover a novel agent that promotes hair growth. We carried out a screening test in 298 types of conditioned medium (CM) from cultures of bacteria by using a hair bulb keratinocyte (HBK) growth assay. As a result, we found a HBK growth factor in the CM of Bacillus sp. M18. This HBK growth factor was purified by collecting biologically active fractions in three steps, including HP-20 batch processing, LH-20 chromatography and C18 reverse-phase high-pressure liquid chromatography, and identified as a short peptide GPIGS. GPIGS increased Akt phosphorylation in HBKs. Moreover, the GPIGS-stimulated HBK growth was inhibited by the treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI-3K). These results suggest that GPIGS promotes HBK growth via the PI-3K/Akt pathway. In addition to in vitro tests, GPIGS was found to accelerate hair regrowth in telogen mice. Our results indicate that GPIGS is a potential agent to promote hair growth.

Animals↗

PubMed2004

Motor hyperactivity caused by a deficit in dopaminergic neurons and the effects of endocrine disruptors: a study inspired by the physiological roles of PACAP in the brain.

Recent studies have revealed that the pituitary adenylate cyclase-activating polypeptide (PACAP) might act as a psychostimulant. Here we investigated the mechanisms underlying motor hyperactivity in patients with pervasive developmental disorders, such as autism, and attention-deficit hyperactivity disorder (ADHD). We studied the effects of intracisternal administration of 6-hydroxydopamine (6-OHDA) or endocrine disruptors (EDs) on spontaneous motor activity (SMA) and multiple gene expression in neonatal rats. Treatment with 6-OHDA caused significant hyperactivity during the dark phase in rats aged 4-5 weeks. Motor hyperactivities also were observed after treatment with endocrine disruptors, such as bisphenol A, nonylphenol, diethylhexyl phthalate and dibutyl phthalate, during both dark and light phases. Gene-expression profiles produced using cDNA macroarrays of 8-week-old rats with 6-OHDA lesions revealed the altered expression of several classes of gene, including the N-methyl-D-aspartate (NMDA) receptor 1, glutamate/aspartate transporter, gamma-aminobutyric-acid transporter, dopamine transporter 1, D4 receptor, and peptidergic elements such as the galanin receptor, arginine vasopressin receptor, neuropeptide Y and tachykinin 2. The changes in gene expression caused by treatment with endocrine disruptors differed from those induced by 6-OHDA. These results suggest that the mechanisms underlying the induction of motor hyperactivity and/or compensatory changes in young adult rats might differ between 6-OHDA and endocrine disruptors.

Animals↗

PubMed2004

p-Nitrotoluene causes hyperactivity in the rat.

It has not been known which endocrine disruptors exert their effects on neuronal functions, particularly leading to behavioral alterations. To address this, we examined the effects of p-nitrotoluene, an endocrine disruptor, on rat behavior and gene expression. Single intracisternal administration of p-nitrotoluene (ca. 10 microg) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. They were about 1.4-fold more active in the nocturnal phase after administration of p-nitrotoluene than control rats. Based on DNA array analyses, p-nitrotoluene decreased more than two-fold the levels of gene expression of the mesencephalic dopamine transporter at 8 weeks old. Thus, it was demonstrated for the first time that p-nitrotoluene definitely affected the developing brain, resulting in hyperactivity in the rat.

Animals↗

PubMed2004

Bisphenol A causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity.

We examined the effects of bisphenol A, an endocrine disruptor, on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A (0.2-20 microg) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. Rats were about 1.6-fold more active in the nocturnal phase after administration of both 2 and 20 microg of bisphenol A than were control rats. The response was dose-dependent. Based on DNA macroarray analyses of the midbrain, bisphenol A decreased by more than twofold gene expression levels of the dopamine D4 receptor at 4 weeks of age and the dopamine transporter at 8 weeks of age. Furthermore, bisphenol A decreased by more than twofold gene expression levels of the dopamine D4 receptor at 4 weeks of age and the dopamine transporter at 8 weeks of age. We conclude that bisphenol A affected central dopaminergic system activity, resulting in hyperactivity due most likely to a large reduction of tyrosine hydroxylase activity in the midbrain.

Age Factors↗

PubMed2004

Constitutively active Src facilitates NGF-induced phosphorylation of TrkA and causes enhancement of the MAPK signaling in SK-N-MC cells.

Here we investigated a biological association of constitutively active Src with TrkA in SK-N-MC human neuroblastoma cells. Activation of TrkA and extracellular signal-regulated kinase (ERK) by nerve growth factor (NGF) was inhibited by pretreatment with PP2, an inhibitor of Src family kinases. Moreover, NGF-induced phosphorylation of TrkA and ERK was also attenuated by the transfection with a dominant-negative src construct. On the other hand, the transfection with a constitutively active src construct enhanced these phosphorylations. In addition, we showed that active Src phosphorylates TrkA directly in vitro, and that Src associates with TrkA through Grb2 after NGF stimulation. These results suggest that constitutively active Src that associates with TrkA through Grb2 after NGF stimulation participates in TrkA phosphorylation and in turn enhances the mitogen-activated protein kinase signaling in SK-N-MC cells.

Blotting, Western↗

PubMed2004

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes.

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.

Cluster Analysis↗

PubMed2004

Polyphenols from peanut skins and their free radical-scavenging effects.

Separation of the water-soluble fraction of peanut skins led to the isolation of five proanthocyanidins. Based on the spectroscopic investigation and partial acid catalyzed degradation, their structures were determined to be epicatechin-(2beta-->O -->7, 4beta -->6)-[epicatechin-(4beta-->8)]-catechin (1), epicatechin-(2beta-->O -->7, 4beta-->8) epicatechin-(4beta-->8)-catechin-(4alpha-->8)-epicatechin (2), and procyanidins B2 (3), B3 (4) and B4 (5). The absolute configuration of the new compounds was determined from their circular dichroism curves and the (1)H NMR spectra of analysis of flavan-3-ols formed by thiolytic degradation of 1 and 2 in the presence of a chiral dirhodium complex (dirhodium tetra-(R)-(trifluoromethyl) phenyl acetate).

Antioxidants↗

PubMed2004

Motor activity and gene expression in rats with neonatal 6-hydroxydopamine lesions.

A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders.

Animals↗

PubMed2004

Dicyclohexylphthalate causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity.

Endocrine disruptors possibly exert effects on neuronal functions leading, in particular, to behavioural alterations. In this study, we examined the effects of dicyclohexylphthalate (DCHP), an endocrine disruptor, on rat behavioural and cellular responses. Single intracisternal administration of DCHP (0.87-87 nmol) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.4-fold more active in the nocturnal phase after administration of 87 nmol of DCHP than control rats (p < 0.001). The response had a tendency to be dose-dependent. Based on DNA macoarray analyses, DCHP down-regulated the levels of gene expression of the dopamine D4 receptor at 4 weeks old in both the midbrain and the striatum, and the dopamine transporter in the midbrain at 8 weeks old 1.7- to 2-fold. The gene expression of several subtypes of glutamate receptors was facilitated in the striatum at 4 weeks old and in the midbrain at 8 weeks old. Some normalization and/or compensatory changes seemed to occur in gene expression of GABA or glycine transmission. Furthermore, DCHP abolished immunoreactivity of tyrosine hydroxylase in the substantia nigra at 8 weeks of age, where TUNEL-positive cells were seen. We conclude that DCHP affected the developing rat brain, resulting in hyperactivity, probably as a result of degeneration of mesencephalic tyrosine hydroxylase rather than alteration of the level of gene expression.

Animals↗

PubMed2004

Effects of neonatal treatment with 6-hydroxydopamine and endocrine disruptors on motor activity and gene expression in rats.

To investigate the mechanisms underlying motor hyperactivity, we performed intracisternal injection of 6-hydroxydopamine or endocrine disruptors in rats on postnatal day 5. 6-Hydroxydopamine (100 microg, 488 nmol) caused a significant increase in spontaneous motor activities at 4 weeks of age. Gene-expression profiling using a cDNA membrane array revealed alterations in several classes of gene at 8 weeks of age. In the midbrain, gene expression was enhanced in dopamine transporter 1; a platelet-derived growth factor receptor; dopamine receptor D4; galanin receptor 2; arginine vasopressin receptor 2; neuropeptide Y; tachykinin 2; and fibroblast growth factor 10. Expression was also enhanced in the glutamate/aspartate transporter gene in the striatum. Rats received an endocrine disruptor (87 nmol), such as bisphenol A, nonylphenol, p-octylphenol, or diethylhexylphthalate, which also caused motor hyperactivity at 4 weeks. The effects of bisphenol A on motor activity were dose-dependent from 0.87 to 87 nmol. The phenols caused a deficit in dopamine neurons, similarly to the deficit caused by 6-hydroxydopamine. Gene-expression profiles after treatment with endocrine disruptors showed variation and differed from those of 6-hydroxydopamine. The results suggest that neonatal treatment with environmental chemicals can generate an animal model of attention-deficit hyperactivity disorder, in which clinical symptoms are pervasive.

Animals↗

PubMed2003

Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5.

Earlier studies demonstrated that knock-out of fibroblast growth factor-5 gene (Fgf-5) prolonged anagen VI phase of hair cycle, resulting long hairs in the mice. We showed the activities on hair growth of the two Fgf-5 gene products, one of which, FGF-5 suppressed hair growth by inhibiting anagen proceeding and inducing the transition from anagen to catagen, and FGF-5S, a shorter polypeptide with FGF-5-antagonizing activity translated from alternatively spliced mRNA, suppressed this activity of FGF-5. As the results suggested that FGF-5 antagonist would increase hair growth, we synthesized various peptides having partial sequences of human FGF-5 and FGF-5S and determined their FGF-5 antagonist activity. Among them, a decapeptide designated P3 (95-VGIGFHLQIY-104) that aligns with receptor binding sites of FGF-1 and FGF-2 suppressed FGF-5-induced proliferation of BALB/3T3 A31 and NIH/3T3 murine fibroblasts, and FGF receptor-1c (FGFR-1c)-transfected Ba/F3 cell line (FR-Ba/F3 cells). IC50s of this peptide on these cell proliferations were 64, 28, 146 microM, respectively. On the other hand, IC50 of this peptide on binding of FGF-5 to the FGFR-1(IIIc)/Fc chimera was 483 microM. Examination in dorsal depilated mice revealed that the P3 peptide reduced the activity of FGF-5 to recover hair pigmentation and hair follicle lengths. The classification of histologically observed skin sections showed FGF-5-induced delations of anagen procedure had reduced by the P3 peptide. The anti-Ki67 antibody staining of hair follicles was inhibited by administration of FGF-5, and this inhibition by FGF-5 was recovered by administration of the P3 peptide. The P3 peptide alone did not affect hair follicle length and hair cell proliferation. These results indicate that the decapeptide antagonized FGF-5 activity in vivo, and reduced the inhibition of FGF-5 in hair growth, confirming that FGF-5 inhibitors are promising substances against hair loss and/or for promoting hair growth.

3T3 Cells↗

PubMed2003

Screening of genes that respond to cryopreservation stress using yeast DNA microarray.

We studied the response of yeast cells after cryopreservation treatment using DNA microarray technology. Genes that contribute to "Cell rescue, defense and virulence," "energy," and "metabolism," were significantly induced. These genes were classified as encoding heat shock proteins, oxidative stress scavenger, and enzymes involved in glucose metabolism. The expression profile of mRNA after cryopreservation treatment was calculated to be closer to that following treatment with detergent or plant oils rather than by other stress factors such as heavy metals and agricultural chemicals. These results suggest that the cryopreservation treatment caused damage to the structure of the cell wall and cellular organelles. This was supported by the localization of the products of the induced genes at the cell wall and within cellular organelles.

Cell Wall↗

PubMed2003

Differential effects of vitamin E and three hydrophilic antioxidants on the actinomycin D-induced and colcemid-accelerated apoptosis in human leukemia CMK-7 cell line.

The actinomycin D (AD)-induced apoptosis in human leukemia CMK-7 cell line is greatly accelerated by microtubule disruption with colcemid (CL). We studied the effect of antioxidants on this apoptosis in order to learn how the universal signal mediators, reactive oxygen species (ROS), are involved. Caspase-3 activation and DNA fragmentation were both suppressed by vitamin E (VE), t-butylhydroxyanisole, and luteolin. The ROS formation in the AD treatment was evidenced by flow cytometry, and further supported by suppression of caspase-3 activation by superoxide radical-forming enzyme inhibitors (TTFA, rotenone, and DPI). The inhibition of apoptosis by VE was completed during the initial 1-h treatment with AD, but it did not appear when VE was added with CL to washed cells after AD treatment. Luteolin, an iron chelator PDTC, and a water-soluble VE analogue, trolox, inhibited the apoptosis when added with CL after the AD treatment. Western blot analysis showed that the proteolytic cleavage of procaspase-9 and procaspase-3 were both inhibited when VE was added with AD or when luteolin was added with CL, and that the cytochrome c liberation was suppressed by both antioxidants. This result implies that the ROS are initially formed in lipophilic environments (e.g. mitochondrial membrane) and then they diffuse into an aqueous environment (i.e. cytoplasm) where they promote the apoptotic process in combination with the cytoskeletal disruption. Thus, the different antioxidants are effective to scavenge ROS for preventing the apoptosis in its different phases.

Antioxidants↗

PubMed2003

Elemental mapping of DNA chains by energy-filtering TEM.

DNA chains were detected by phosphorus mapping based on energy-filtering transmission electron microscopy (EF-TEM). The three-window method and the image-merging system were used to make phosphorus-mapping images. It was impossible to observe DNA chains without the image-merging system, because of the low signal levels of single images. It became easy to assign DNA molecules when the typical superstructure of plasmids was preserved. To preserve the structure during specimen preparation, rapid-freezing and freeze-drying methods were used. Mapping images can be obtained only when carbon-supporting films are extremely thin, as thick supporting films weaken the signals. The thickness of the supporting film, which was estimated to be < 2 nm, was the most important factor in this study. In the three-window method, the calculation to remove the background absolutely includes a few errors, because the precise spectrum was not observed. To obtain higher quality mapping images, several improvements in the hardware are suggested.

DNA↗

PubMed2002

Effects of neonatal 6-hydroxydopamine lesion on the gene expression profile in young adult rats.

Patients with pervasive developmental disorders, including autism, and attention-deficit hyperactivity disorder show behavioral hyperactivity during childhood. We investigated the effects of a neonatal 6-hydroxydopamine lesion on multiple gene expression in the rat striatum and midbrain. Spontaneous motor activity was significantly increased at 4-5 weeks of age. The animals were sacrificed, and the striatum and midbrain were subjected to gene expression profiling using a membrane array with 1176 kinds of cDNAs. Alterations were found in several classes of gene expression, depending on the brain region. Enhanced expression of the glutamate transporter gene was found in the striatum. Expression of the dopamine receptor D4 gene and dopamine transporter gene was also increased in the midbrain. These results suggest that 6-hydroxydopamine-treated rats may partly mimic human hyperkinesia not only in behavior but also in gene expression.

Animals↗

PubMedDate not supplied

Rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Animals↗

PubMedDate not supplied

Effects of polyphenolic anthrone derivatives, resistomycin and hypercin, on apoptosis in human megakaryoblastic leukemia CMK-7 cell line.

A tetrahydroxyanthrone derivative, resistomycin, was isolated from the culture broth of Streptomyces sulphureus and a similar polyphenolic dianthraquinone, hypericin, was isolated from an extract of Hypericum perforatum L. as modulators for apoptosis. Resistomycin inhibited apoptosis induced by actinomycin D (AD) with or without acceleration by colcemid (CL) in human megakaryoblastic leukemia CMK-7 cells, IC50 for inhibition against AD-induced apoptosis was about 0.5 microM and IC50 for inhibition against AD plus CL-induced apoptosis was about 1 microM. CL alone induced weak apoptosis in cells, which was enhanced by resistomycin. Hypericin did not inhibit AD-induced apoptosis and slightly enhanced CL-induced apoptosis. Emodin, corresponding to 1 of 2 anthraquinone units in hypericin, did not show any effect on this apoptotic system. AD-induced apoptosis was inhibited by the antioxidative flavonoid, luteolin (IC50 45 microM), and a protein kinase C (PKC) inhibitor, staurosporine (IC50 1.5 microM), but these compounds did not affect the CL-induced apoptosis. Hypericin and resistomycin scavenged superoxide anion radicals at the same rate as luteolin. PKC in CMK-7 cells was inhibited by hypericin and luteolin, but not significantly inhibited by resistomycin. This result suggests that the inhibition of AD-induced apoptosis by resistomycin is at least partly correlated with its antioxidative activity, and that the enhancement of CL-induced apoptosis by this compound depends upon the lack of PKC inhibitory activity. Though the mechanism is not clear, the enhancement of the CL-induced apoptosis might be hindered by PKC inhibition in the case of hypericin and luteolin.

Anthracenes↗