Syuichi Oka

βKlotho Is Required for Fibroblast Growth Factor (FGF) 21 Signaling through FGF Receptor (FGFR) 1c and FGFR3c

Fibroblast growth factor (FGF) 21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that betaKlotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of betaKlotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Conversely, coexpression of FGFR1c and Klotho, a protein related to betaKlotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of betaKlotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF21 was associated with expression of betaKlotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that betaKlotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion.

A-type proanthocyanidins from peanut skins

Abstract Six A-type proanthocyanidins were isolated from the water-soluble fraction of peanut skins. On the basis of spectral data, reductive cleavage with sodium cyanoborohydride, and chiral HPLC analysis, three new compounds, epicatechin-(2β→O→7, 4β→6)-catechin, epicatechin-(2β→O→7, 4β→6)-ent-catechin and epicatechin-(2β→O→7, 4β→6)-ent-epicatechin were unambiguously identified, together with the three known compounds, proanthocyanidin A-1, proanthocyanidin A-2 and epicatechin-(2β→O→7, 4β→8)-ent-epicatechin. 13 C NMR chemical shift rules to distinguish between [2→O→7, 4→8] and [2→O→7,4→6] double-linked heptamethyl ethers of A-type proanthocyanidins are proposed. Bioassay experiments showed that these six compounds possess substantial activity against hyaluronidase.

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 μg) 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 μg 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.

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.

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 μg, 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.

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.

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.

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.

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 ≤ −1; −1 < Ratio < 1; 1 ≤ 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. Copyright

RAPID DETECTION OF MYCOPLASMA CONTAMINATION IN CELL CULTURES USING SYBR GREEN-BASED REAL-TIME POLYMERASE CHAIN REACTION

SummaryWe 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 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 evaluated 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, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.