Biomedical subjects

Osamu Saitoh

Publications and source records attributed to Osamu Saitoh.

At least 19 recordsLinked to original sources

PubMed2006

Association study of the vesicular monoamine transporter 1 (VMAT1) gene with schizophrenia in a Japanese population.

BACKGROUND: Vesicular monoamine transporters (VMATs) mediate accumulation of monoamines such as serotonin, dopamine, adrenaline, and noradrenaline from the cytoplasm into storage organelles. The VMAT1 (alternatively solute carrier family 18: SLC18A1) regulates such biogenic amines in neuroendocrine systems. The VMAT1 gene maps to chromosome 8p21.3, a locus with strong evidence of linkage with schizophrenia. A recent study reported that a non-synonymous single nucleotide polymorphism (SNP) of the gene (Pro4Thr) was associated with schizophrenia. METHODS: We attempted to replicate this finding in a Japanese sample of 354 schizophrenics and 365 controls. In addition, we examined 3 other non-synonymous SNPs (Thr98Ser, Thr136Ile, and Val392Leu). Genotyping was performed by the TaqMan allelic discrimination assay. RESULTS: There was no significant difference in genotype or allele distribution of the three SNPs of Pro4Thr, Thr136Ile, or Val392Leu between patients and controls. There was, however, a significant difference in genotype and allele distributions for the Thr98Ser polymorphism between the two groups (P = 0.01 for genotype and allele). When sexes were examined separately, significant differences were observed in females (P = 0.006 for genotype, P = 0.003 for allele), but not in males. The Thr98 allele was more common in female patients than in female controls (odds ratio 1.69, 95% CI 1.19-2.40, P = 0.003). Haplotype-based analyses also provided evidence for a significant association in females. CONCLUSION: We failed to replicate the previously reported association of Pro4Thr of the VMAT1 gene with schizophrenia. However, we obtained evidence for a possible role of the Thr98Ser in giving susceptibility to schizophrenia in women.

Journal Article↗

PubMed2006

Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation.

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.

Alternative Splicing↗

PubMed2006

Antipsychotic medication and cognitive function in schizophrenia.

Antipsychotic polypharmacy and excessive dosing still prevail worldwide in the treatment of schizophrenia, while their possible association with cognitive function has not well been examined. We examined whether the "non-standard" use of antipsychotics (defined as antipsychotic polypharmacy or dosage >1,000 mg/day of chlorpromazine equivalents) is associated with cognitive function. Furthermore, we compared cognitive function between patients taking only atypical antipsychotics and those taking only conventionals. Neurocognitive functions were assessed in 67 patients with chronic schizophrenia and 92 controls using the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale-Revised (WAIS-R), the Wisconsin Card Sorting Test (WCST), and the Advanced Trail Making Test (ATMT). Patients showed markedly poorer performance than controls on all these tests. Patients on non-standard antipsychotic medication demonstrated poorer performance than those on standard medication on visual memory, delayed recall, performance IQ, and executive function. Patients taking atypical antipsychotics showed better performance than those taking conventionals on visual memory, delayed recall, and executive function. Clinical characteristics such as duration of medication, number of hospitalizations, and concomitant antiparkinsonian drugs were different between the treatment groups (both dichotomies of standard/non-standard and conventional/atypical). These results provide evidence for an association between antipsychotic medication and cognitive function. This association between antipsychotic medication and cognitive function may be due to differential illness severity (e.g., non-standard treatment for severely ill patients who have severe cognitive impairment). Alternatively, poorer cognitive function may be due in part to polypharmacy or excessive dosing. Further investigations are required to draw any conclusions.

Adult↗

PubMed2006

Removal of ballistocardiogram artifacts from simultaneously recorded EEG and fMRI data using independent component analysis.

Simultaneous recording of electroencephalogram (EEG) and functional magnetic resonance imaging (fMRI) has been studied to identify areas related to EEG events. EEG data recorded in the magnetic resonance (MR) scanner with MR imaging is suffered from two specific artifacts, imaging artifact, and ballistocardiogram (BCG). In this paper, we focus on BCG. In preceding studies, average subtraction was often used for this purpose. However, average subtraction requires an assumption that BCG waveforms are precisely periodic, which seems unrealistic because BCG is a biomedical artifact. We propose the application of independent component analysis (ICA) with a postprocessing of high-pass filtering for the removal of BCG. With this approach, it is not necessary to assume that the BCG waveform is periodic. Empirically, we show that our proposed method removes BCG artifacts as well as does the average subtraction method. Power spectral density analysis of the two approaches shows that, with ICA, distortion of recovered EEG data is also as small as that associated with the average subtraction approach. We also propose a hypothesis for how head movement causes BCGs and show why ICA can remove BCG artifacts arising from this source.

Algorithms↗

PubMed2005

The association between the Val158Met polymorphism of the catechol-O-methyl transferase gene and morphological abnormalities of the brain in chronic schizophrenia.

The catechol-O-methyl transferase (COMT) gene is considered to be a promising schizophrenia susceptibility gene. A common functional polymorphism (Val158Met) in the COMT gene affects dopamine regulation in the prefrontal cortex (PFC). Recent studies suggest that this polymorphism contributes to poor prefrontal functions, particularly working memory, in both normal individuals and patients with schizophrenia. However, possible morphological changes underlying such functional impairments remain to be clarified. The aim of this study was to examine whether the Val158Met polymorphism of the COMT gene has an impact on brain morphology in normal individuals and patients with schizophrenia. The Val158Met COMT genotype was obtained for 76 healthy controls and 47 schizophrenics. The diagnostic effects, the effects of COMT genotype and the genotype-diagnosis interaction on brain morphology were evaluated by using a voxel-by-voxel statistical analysis for high-resolution MRI, a tensor-based morphometry. Patients with schizophrenia demonstrated a significant reduction of volumes in the limbic and paralimbic systems, neocortical areas and the subcortical regions. Individuals homozygous for the Val-COMT allele demonstrated significant reduction of volumes in the left anterior cingulate cortex (ACC) and the right middle temporal gyrus (MTG) compared to Met-COMT carriers. Significant genotype-diagnosis interaction effects on brain morphology were noted in the left ACC, the left parahippocampal gyrus and the left amygdala-uncus. No significant genotype effects or genotype-diagnosis interaction effects on morphology in the dorsolateral PFC (DLPFC) were found. In the control group, no significant genotype effects on brain morphology were found. Schizophrenics homozygous for the Val-COMT showed a significant reduction of volumes in the bilateral ACC, left amygdala-uncus, right MTG and left thalamus compared to Met-COMT schizophrenics. Our findings suggest that the Val158Met polymorphism of the COMT gene might contribute to morphological abnormalities in schizophrenia.

Adult↗

PubMed2005

Auditory imagery mismatch negativity elicited in musicians.

A mismatch between auditory sensation and expectant imagery of syllables elicited a possible equivalent of mismatch negativity in a previous study. The purpose of this study was to verify whether auditory imagery from musical notation could also mediate such imagery-based mismatch negativity. Neuromagnetic recording was obtained from eight musicians, who were instructed to identify unpredictably occurring pitch mismatches between a random tone sequence and a visually presented musical score. The difference between incongruent and congruent responses showed a magnetic distribution consistent with two frontal-negative current dipoles bilaterally located in the vicinity of Heschl's gyrus, peaking at approximately 150 ms in latency. This imagery-based mismatch negativity may represent an early neural process of deviance detection between the sensory input and expectant imagery.

Acoustic Stimulation↗

PubMed2005

Audiovisual phonological mismatch produces early negativity in auditory cortex.

During silent reading, visual information provided by letters is converted to auditory information in the mind. The purpose of this study was to identify the primary locus for auditory verbal imagery in the brain. Neuromagnetic recording was obtained from 10 right-handed study participants, who were instructed to identify infrequently occurring phonological mismatches between a random-ordered sequence of syllable sounds and a visually presented syllabogram sequence. The activity difference in early latency, calculated by subtracting the averaged responses to matched syllables from the averaged responses to mismatched syllables, showed a spatiotemporal profile strikingly similar to that of mismatch negativity. Auditory imagery of forthcoming verbal sounds may establish a memory trace as a template for imagery-based mismatch negativity generation in the auditory cortex.

Acoustic Stimulation↗

PubMed2005

Identification of adenosine receptor subtypes expressed in the human endothelial-like ECV304 cells.

Adenosine, acting through its receptors, is a potent endogenous regulator of endothelial cells. The cultured endothelial cells expressing adenosine receptors are thus important for elucidation of molecular mechanism of adenosine functions in these cell systems. Therefore, identification of adenosine receptors in the human ECV304 cell line derived from a human umbilical vein endothelial cell culture was performed. RT-PCR experiments revealed that ECV304 cells express mRNAs for A1 and A2B adenosine receptors. The expression of mRNA for A2A adenosine receptor was not in a significant level and that for A3 adenosine receptor was not detected. The binding study of ECV304 cell membrane fractions using various radiolabeled ligands for adenosine receptors indicated the presence of A1 adenosine receptors 245 fmol/mg of membrane proteins, but the specific binding for A2A and for A3 adenosine receptors were found to be negligible. The functional expression of A1 and A2B adenosine receptors in ECV304 cells was detected by assays for adenosine-3',5'-cyclic monophosphate and for extracellular signal-regulated kinase, but that of A2A adenosine receptors was not confirmed under the assay conditions employed. In conclusion, this study presented evidence for functional A1 and A2B adenosine receptors in human endothelial-like ECV304 cells, indicating that ECV304 cells can be a good model for the study of adenosine receptors, especially for A2B adenosine receptor, in endothelial cells.

Cell Line↗

PubMed2005

Characterization of bitter taste responses of intestinal STC-1 cells.

Cellular responses of STC-1 cells to two bitter tastants (denatonium and caffeine) were investigated using a calcium-imaging technique and compared with the response to bombesin. Caffeine is known to stimulate taste receptor cells, but the properties of its signaling have not been well studied. STC-1 cells responded to all three molecules in a dose-dependent manner, and when a reverse transcriptase-polymerase chain reaction (RT-PCR) for denatonium receptor was performed, the product of predicted size was detected in STC-1 cells. Furthermore, all three signaling pathways were blocked by a phospholipase C (PLC) inhibitor, demonstrating the essential involvement of PLC in cellular responses. To study the regulatory system of G protein signaling in STC-1 cells, we searched G protein-coupled receptor kinases (GRKs) by the degenerate-primer PCR method and found that GRK2 is expressed. We also demonstrated that three GRKs (GRK2, GRK3 and GRK5) are differentially distributed in the circumvallate papilla while only GRK2 is present in taste bud cells. Finally, we overexpressed GRK2 in SCT-1 cells and found that bombesin-induced response was strongly inhibited by GRK2 but denatonium-activated signaling was not affected. In the case of caffeine, response was decreased by expression of GRK2 only when cells were activated by 1 mM caffeine. Thus, we showed that STC-1 cells emerge as a cell model for studying the molecular mechanism of bitter taste signaling, and could indicate properties of caffeine-induced signaling in comparison with other signaling.

Animals↗

PubMed2005

Haplotype structures of EPHX1 and their effects on the metabolism of carbamazepine-10,11-epoxide in Japanese epileptic patients.

OBJECTIVE: Microsomal epoxide hydrolase (mEH) is an enzyme that detoxifies reactive epoxides and catalyzes the biotransformation of carbamazepine-10,11-epoxide (CBZ-epoxide) to carbamazepine-10,11-diol (CBZ-diol). Utilizing single nucleotide polymorphisms (SNPs) of the EPHX1 gene encoding mEH, we identified the haplotypes of EPHX1 blocks and investigated the association between the block haplotypes and CBZ-epoxide metabolism. METHODS: SNPs of EPHX1 were analyzed by means of polymerase chain reaction amplification and DNA sequencing using DNA extracted from the blood leukocytes of 96 Japanese epileptic patients, including 58 carbamazepine-administered patients. The plasma concentrations of CBZ and its four metabolites were determined using high-performance liquid chromatography. RESULTS: From sequencing all 9 exons and their surrounding introns, 29 SNPs were found in EPHX1. The SNPs were separated into three blocks on the basis of linkage disequilibrium, and the block haplotype combinations (diplotypes) were assigned. Using plasma CBZ-diol/CBZ-epoxide ratios (diol/epoxide ratios) indicative of the mEH activity, the effects of the diplotypes in each EPHX1 block were analyzed on CBZ-epoxide metabolism. In block 2, the diol/epoxide ratios increased significantly depending on the number of haplotype *2 bearing Y113H (P=0.0241). In block 3, the ratios decreased depending on the number of haplotype *2 bearing H139R (P=0.0351). Also, an increasing effect of a *1 subtype, *1c, was observed on the ratio. CONCLUSION: These results show that some EPHX1 haplotypes are associated with altered CBZ-epoxide metabolism. This is the first report on the haplotype structures of EPHX1 and their potential in vivo effects.

Adolescent↗

PubMed2005

Molecular cloning and characterization of a new RGS protein of Medaka.

We identified eight genes of putative RGS proteins in skin of Medaka fish using PCR amplification with degenerate primers for the RGS domain of known RGS proteins. Then, we cloned a full-length cDNA for a new RGS protein. This RGS protein was similar to human RGS3 within the RGS domain, but other parts were unique among known RGS proteins. RT-PCR analysis demonstrated that this Medaka RGS3-like protein (MeRGS3L) is mainly expressed in skin and heart. When coexpressed in Xenopus oocytes, MeRGS3L accelerated the turning-on and -off of Gi/o-mediated modulation of GIRK channels without apparent desensitization in the presence of ligand. MeRGS3L also decreased the response of Gq signaling upon activation of m1 muscarinic receptor. This new RGS protein may play important roles in regulation of melanophore responses in Medaka skin.

Amino Acid Sequence↗

PubMed2005

Functional expression of single-chain heterodimeric G-protein-coupled receptors for adenosine and dopamine.

The direct homo- and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A(2A)R) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A(2A)R/A(2A)R vs. A(2A)R/D2R). In order to stimulate the heteromerization of A(2A)R and D2R, we have designed a single-polypeptide-chain heterodimeric A(2A)R/D2R complex by fusing the C-terminus of the A(2A)R via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A(2A)R/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.

Adenosine↗

PubMed2005

Single nucleotide polymorphisms and haplotypes of CYP1A2 in a Japanese population.

In order to identify genetic polymorphisms and haplotype frequencies of CYP1A2 in a Japanese population, the enhancer and promoter regions, all the exons with their surrounding introns, and intron 1 were sequenced from genomic DNA from 250 Japanese subjects. Thirty-three polymorphisms were found, including 13 novel ones: 2 in the enhancer region, 5 in the exons, and 6 in the introns. The most common single nucleotide polymorphism (SNP) was -163C>A (CYP1A2*1F allele) with a 0.628 frequency. In addition to six previously reported non-synonymous SNPs, three novel ones, 125C>G (P42R, CYP1A2*15 allele, MPJ6_1A2032), 1130G>A (R377Q, *16 allele, MPJ6_1A2033), and 1367G>A (R456H, *8 allele, MPJ6_1A2019), were found with frequencies of 0.002, 0.002, and 0.004, respectively. No polymorphism was found in the known nuclear transcriptional factor-binding sites in the enhancer region. Based on linkage disequilibrium analysis, the CYP1A2 gene was analyzed as one haplotype block. Using the 33 detected polymorphisms, 14 haplotypes were unambiguously identified, and 17 haplotypes were inferred by aid of an expectation-maximization-based program. Among them, the second major haplotype CYP1A2*1L is composed of -3860G>A (*1C allele), -2467delT (*1D allele), and -163C>A (*1F allele). Network analysis suggested that relatively rare haplotypes were derived from three major haplotypes, *1A, *1M, and *1N in most cases. Our findings provide fundamental and useful information for genotyping CYP1A2 in the Japanese, and probably Asian populations.

Adrenergic beta-Antagonists↗

PubMed2004

Biochemical and electrophysiological analyses of RGS8 function.

RGS8 was identified as a regulator of G-protein signaling (RGS) protein induced in neuronally differentiated P19 cells. This article presents methods used to estimate the binding activity and selectivity between RGS8 and Galpha subunits. It describes three kinds of in vitro-binding experiments using RGS8 proteins generated by three different techniques: recombinant protein from Escherichia coli, in vitro-translated protein, and protein expressed in cDNA-transfected cultured cells. It also presents methods of the functional analysis of RGS protein using the Xenopus oocyte expression system. Electrophysiological procedures, which were used to examine the effects of RGS8 on Gi- and Gq-mediated responses, are described.

Animals↗

PubMed2004

The mechanism of membrane-translocation of regulator of G-protein signaling (RGS) 8 induced by Galpha expression.

RGS (regulators of G-protein signaling) proteins comprise a large family that modulates heterotrimeric G-protein signaling. This protein family has a common RGS domain and functions as GTPase-activating proteins for the alpha-subunits of heterotrimeric G-proteins located at the plasma membrane. RGS8 was identified as a neuron-specific RGS protein, which belongs to the B/R4 subfamily. We previously showed that RGS8 protein was translocated to the plasma membrane from the nucleus on coexpression of GTPase-deficient Galphao (GalphaoQL). Here, we first examined which subtypes of Galpha can induce the translocation of RGS8. When the Galphai family was expressed, the translocation of RGS8 did occur. To investigate the mechanism of this translocation, we generated a mutant RGS8 with reduced affinity to Galphao and an RGS-insensitive (RGS-i) mutant of GalphaoQL. Co-expression experiments with both mutants revealed that disruption of the Galpha-RGS8 interaction abolished the membrane-translocation of RGS8 despite the apparent membrane localization of RGS-i GalphaoQL. These results demonstrated that RGS8 is recruited to the plasma membrane where G-proteins are activated mainly by direct association with Galpha.

Animals↗

PubMed2003

Six novel nonsynonymous CYP1A2 gene polymorphisms: catalytic activities of the naturally occurring variant enzymes.

Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the Vmax of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower Km value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

Amino Acid Sequence↗

PubMed2003

RGS8 expression in developing cerebellar Purkinje cells.

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 belongs to B/R4 subfamily of RGS proteins and is specifically expressed in Purkinje cells of adult cerebellum. Here, to examine the expression of RGS8 mRNA in developing cerebellum, we performed in situ hybridization. Apparent signals for expression of RGS8 mRNA were first detected on day 9 after birth, then RGS8 mRNA expression in Purkinje cells increased up to day 21, and its levels decreased to some extent in adult Purkinje cells. We also studied the expression of RGS7, which is expressed in Golgi cells in the granule cell layer of adult cerebellum. The expression of RGS7 mRNA was recognized in 7 day neonatal cerebellum. When examined with anti-RGS8 antibody, the RGS8 protein was already excluded from nucleus on day 9, and was distributed in cell body and dendrites in differentiating Purkinje cells of 14 day neonates.

Animals↗

PubMed2003

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells.

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.

Cell Line↗