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T Ohkubo

Publications and source records attributed to T Ohkubo.

At least 19 recordsLinked to original sources
PubMed2001

Solution structure of the ribosome recycling factor from Aquifex aeolicus.

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.

Amino Acid Sequence↗
PubMed2001

Conformational characterization of designed minibarnase.

We have designed a minibarnase by removing one module from barnase, a bacterial RNase from Bacillus amyloliquefaciens. Barnase, consisting of 110 amino acid residues, is decomposed into six modules, M1-M6. Module is defined as a peptide segment consisting of contiguous amino acid residues that makes a small compact conformation within a globular domain. To understand the role of module in protein architecture, we analyzed NMR and CD spectra of a minibarnase, which lacked 26 amino acid residues corresponding to module M2. We demonstrated the formation of hydrophobic cores in the minibarnase similar to those of barnase. Although its conformational stability against acids and heat was reduced in comparison with barnase, the minibarnase retained cooperative folding character (two-state folding). Therefore, the folding of the minibarnase consisting of modules M1 and M3-M6 is independent to some extent of module M2. This finding may be useful for future module-based protein design.

Acids↗
PubMed2001

Stereoselective pharmacokinetics of pantoprazole, a proton pump inhibitor, in extensive and poor metabolizers of S-mephenytoin.

Pantoprazole, a proton pump inhibitor, is administered as a racemic mixture. To determine the role of cytochrome P450 (CYP) 2C19 in the stereoselective metabolism of pantoprazole, we investigated the pharmacokinetic disposition of (+)- and (-)-pantoprazole in 7 extensive metabolizers and 7 poor metabolizers of S-mephenytoin. All of the subjects received an oral 40-mg dose of racemic pantoprazole as the enteric-coated formulation. In the extensive metabolizers, the mean clearance of (-)-pantoprazole was only slightly lower than that of (+)-pantoprazole and no significant differences in the other pharmacokinetic parameters between (+)- and (-)-pantoprazole were observed. The mean (+)/(-) ratios for maximum concentration, area under the plasma concentration-time curve from 0 to infinity, and elimination half-life were 0.94, 0.82, and 0.90, respectively. In contrast, in the poor metabolizers, the clearance values of both enantiomers were significantly lower than those in the extensive metabolizers, and a significant difference in pharmacokinetics between (+)- and (-)-pantoprazole was observed. The mean elimination half-life for (+)-pantoprazole was 3.55-fold longer than that of (-)-pantoprazole, and the mean maximum concentration and area under the plasma concentration-time curve from 0 to infinity for (+)-pantoprazole were 1.31- and 3.59-fold greater, respectively, than those for (-)-pantoprazole. These results indicate that the stereoselective metabolism of pantoprazole depends on S-mephenytoin 4'-hydroxylase (CYP2C19). The metabolism of (+)-pantoprazole was impaired to a greater extent than (-)-pantoprazole in the poor metabolizers.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2001

Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization.

Chicken vasoactive intestinal polypeptide receptor (VIPR) cDNA was cloned by the reverse transcription-polymerase chain reaction method using primers designed on the basis of other species of VIPR cDNA. The cDNA obtained was sequenced by the dideoxy-mediated chain-termination method. Of the 2227 nucleotides that were sequenced, 84, 855, and 1338 bases represent the 5'-untranslated region (UTR), the 3'-UTR, and the open reading frame that predicts a peptide of 446 amino acids. The cDNA of the chicken VIPR shows 65% and 60% homologies to human cDNA of VIP1 and VIP2 receptors, respectively. The clone had the expected similarity to highly conserved features of the other G protein-coupled receptors (GPCRs) such as six cysteine residues that are functionally important in the VIPR subfamily. In addition, the seven potential membrane-spanning domains characteristic of the family B group III GPCR superfamily and highly conserved motif within the third cellular loop between transmembrane regions 5 and 6. Northern blot hybridization analysis in this study indicated mRNA expression of VIPRs in the various tissues of the chicken. Strong signal was detected in the brain and anterior pituitary gland. High levels of VIPR mRNA in the brain was consistent with VIP-binding experiments and with the function of VIP in the brain as a neuroendocrine factor or neurotransmitter. Expression of VIPR was detected in the anterior pituitary gland of chick embryos. The expression of VIPR mRNA in the chick anterior pituitary gland may indicate a regulatory function of VIP on prolactin (PRL) production or PRL cell proliferation during embryogenesis. Chicken VIPR shows high homology with mammalian type I VIPR but, in some part, possesses similarity of amino acid sequence. Expression of VIPR in various tissues supports diverse functions for VIP in the chicken.

3' Untranslated Regions↗
PubMed2001

Factors affecting the difference between screening and home blood pressure measurements: the Ohasama Study.

OBJECTIVE: To establish the factors affecting the difference between screening and home blood pressure measurements. DESIGN: A cross-sectional study. SETTING: General population in a rural Japanese community, Ohasama, Japan. PARTICIPANTS: There were 1,789 community-based subjects aged > or =40 years, for whom blood pressure was measured at screening site (screening blood pressure) and at home (home blood pressure). RESULTS: Multiple stepwise regression analysis of all subjects demonstrated that screening pulse pressure was positively associated with the difference between screening and home blood pressure measurements for systolic blood pressure. Age, the use of antihypertensive medication, and smoking status were negatively associated with the difference between measurements of both systolic and diastolic blood pressure. CONCLUSION: We have demonstrated that age, smoking, antihypertensive medication, and screening pulse pressure are independent predictors of the magnitude of the difference between screening and home blood pressure measurements, suggesting that the necessity to consider these factors, for the detection of the subjects who may be inappropriately treated or misjudged following screening blood pressure measurements.

Adult↗
PubMed2001

Usefulness of home blood pressure measurements in assessing the effect of treatment in a single-blind placebo-controlled open trial.

OBJECTIVES: Reproducibility of home blood pressure measurements (HBP), and the placebo effect on HBP, were examined to establish the number of subjects required in order to observe a significant antihypertensive effect of a drug for a comparative study between placebo and active treatment. METHODS: Reproducibility of HBP (n = 172 for systolic blood pressure and n = 137 for diastolic blood pressure) and the placebo effect on HBP (n = 35 for systolic and n = 42 for diastolic blood pressure) were examined using a semi-automatic electronic device on untreated hypertensive subjects during 21 consecutive days of measurements. From these two studies, the number of subjects required in order to observe a significant antihypertensive drug effect was assessed. In both studies, measurements from the first 3 days were excluded from the analysis. RESULTS: Reproducibility, defined as the difference between the initial 5 day average (days 4-8) and the last 5 day average (days 17-21) was -1.9 +/- 7.0/-1.4 +/- 4.8 mmHg (mean +/- SD), indicating high reproducibility with a minimal regression to the mean effect. A placebo was administered after the 8 day run-in period. The difference between the initial 5 day average (days 4-8) and the last 5 day average (days 17-21) was compared. The mean difference was 1.1 +/- 6.2/0.2 +/- 5.7 mmHg, suggesting that there was a minimal, if any, placebo effect. As determined by the power calculations, based on the standard deviations of the tests for reproducibility and the placebo effect, nine of 13 subjects are required to show that a 9/5 mmHg decrease in systolic HBP/diastolic HBP (50% of estimated effective decrease in systolic HBP/diastolic HBP according to the guidelines for clinical trials in Japan) is statistically significant CONCLUSIONS: The results suggest that the HBP measurement is highly reproducible and that there is a minimal, if any, placebo effect. These characteristics of HBP contribute to minimizing the number of subjects necessary for assessment of antihypertensive drug effects in comparative studies between an active treatment and placebo.

Adult↗
PubMed2001

The orphan nuclear receptor NOR-1 interacts with the homeobox containing protein Six3.

Neuron-derived orphan receptor 1 (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In order to identify cofactors that associate with NOR-1 in the fetal forebrain, we tested a yeast two-hybrid system with the NOR-1 cDNA fragment lacking a transactivating domain as a bait. By screening of the rat fetal brain embryonic day 17 library, a rat homologue of Six3 was identified as an associated protein. We demonstrated that NOR-1 interacted with Six3 in yeast and in vitro, and the association was required for the DNA binding and AF2 domains of NOR-1. Regarding the other members of the family (NGFI-B and RNR-1), association with Six3 was not observed in yeast. In addition, cotransfection experiments with Six3 and NOR-1 indicated that Six3 had a negative activity against the transactivation by NOR-1 through the NBRE response element in a dose-dependent manner. The overlap in expression of NOR-1 and Six3 was mainly detected in the rat fetal forebrain on embryonic day 18. Thereafter, the expression of both genes diminished rapidly. These results suggest that a dimer consisting of a homeobox containing protein Six3 and transcriptional factor NOR-1 might regulate gene expression during the late stage of the fetal forebrain development. This study provides, after the association of Ftz and Ftz-F1 in Drosophila, another example of a dimer formation of a homeobox protein and an orphan nuclear receptor.

Amino Acid Sequence↗
PubMed2001

Tyrosine phosphorylation of p120(ctn) in v-Src transfected L cells depends on its association with E-cadherin and reduces adhesion activity.

Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell-cell adhesion. Using L cells expressing one of three functional E-cadherin constructs, the wild-type, a chimeric molecule with alpha-catenin (EalphaC), and a tail-less one, we determined the effect of v-Src expression on E-cadherin-mediated adhesion. The aggregation of L cells expressing the wild-type or EalphaC chimeric protein, which both interact with p120(ctn), was reduced by v-Src expression, whereas that of L cells expressing the tail-less E-cadherin was not affected by the expression. Tyrosine phosphorylation of p120(ctn) was observed in v-Src-transformed L cells expressing the wild-type or EalphaC chimeric protein, but not in ones expressing the tail-less E-cadherin. Thus, tyrosine phosphorylation of p120(ctn) depends on the complex formation with E-cadherin and the resulting membrane localization. Constitutive phosphorylation of p120(ctn) on serine and threonine residues also depends on the complex formation and membrane localization. Coexpression of the p120(ctn) protein with an N-terminal deletion, which eliminates some potential tyrosine phosphorylation sites, or the protein with a single amino acid substitution (tyrosine at 217 to phenylalanine) resulted in an increase in the aggregation of v-Src-transformed EL and EalphaCL cells. These results indicate that tyrosine phosphorylation of p120(ctn) is involved in the v-Src modulation of E-cadherin-mediated cell adhesion.

Amino Acid Substitution↗
PubMed2001

Risk factors and predictors of coronary arterial lesions in Japanese hypertensive patients.

The morbidity rate of coronary artery disease has recently increased in Japan. This is attributable to changes from traditional to more westernized lifestyles. In this study, we therefore examined the risk factors and predictors of coronary arterial lesions in Japanese patients with essential hypertension. Coronary angiography was performed in 109 consecutive essential hypertension patients (57 men and 52 women; 66 +/- 8.0 years of age) with either angina pectoris or atypical chest pain, who were chosen from 485 consecutive hypertensive patients in a hypertension clinic in Sendai, Japan. Coronary arterial stenosis of greater than 50% was defined as significant and used as a dependent variable for the multiple regression analysis. Risk factors were defined as factors confirmed to have a causal relationship with coronary arterial lesions, whereas arteriosclerotic complications and hypertensive target organ damage were defined as predictors. Multiple logistic regression analysis was performed using these parameters as independent variables. Of 109 patients, 25 had a coronary arterial stenosis greater than 50%. A smoking habit (odds ratio (OR): 4.48; 95% confidence interval (CI): 1.13-17.82; p<0.05), hypercholesterolemia (OR: 5.34; 95% CI: 1.52-18.73; p<0.05), and 24-h diastolic blood pressure (OR: 2.33; 95% CI: 1.06-5.16; p<0.05) were significant risk factors, whereas carotid intima-media thickness (OR: 5.85; 95% CI: 1.48-23.2; p<0.05) was a significant predictor of coronary arterial lesion. When two of the major risk factors (a smoking habit, hypercholesterolemia, or impaired glucose tolerance including diabetes mellitus) were clustered in addition to the hypertension, the risk of coronary arterial lesions increased by 6.7 to 10.1 times. These findings indicate that the major risk factors established in Caucasians, i.e., a smoking habit, hypercholesterolemia and blood pressure level, are also risk factors for coronary arterial lesions in Japanese with essential hypertension. The presence of two or more risk factors increases the risk of coronary arterial lesions synergistically in the presence of hypertension.

Aged↗
PubMed2001

[Surgical analysis for N 2 factor in non-small cell lung cancer].

In this study we analyzed 33 cases which underwent complete surgical resection to assess the role of surgery in the treatment of patients with N 2 NSCLC. The 3 year survival rate was 33.3% and the median survival time was 26.1 months. The survival curve for patients with T 3 factor was statistically worse than those with T 1 or T 2 factor. Further, the survival curve for patients with p 2 or p 3 factor was significantly lower than than that for those classified as p 0. Patients classified with p 2 or p 3 had a survival rate under 2 years. Only one patient diagnosed as T 3 achieved 3 year survival. This patient had a pm1 tumor with p 0 factor. We thereby recommend that surgery should only be performed for those N 2 NSCLC patients diagnosed as T 1 or T 2 with a classification of p1 or less.

Adult↗
PubMed2000

Prognosis of isolated systolic and isolated diastolic hypertension as assessed by self-measurement of blood pressure at home: the Ohasama study.

BACKGROUND: Although the clinical significance of systolic-diastolic hypertension and isolated systolic hypertension has been established, the significance of isolated diastolic hypertension has not been fully investigated. OBJECTIVE: To clarify the prognostic significance of isolated systolic and isolated diastolic hypertension as assessed by self-measurement of blood pressure (BP) at home (home BP measurements), which has a better reproducibility and prognostic value than casual BP measurements in the general population. SUBJECTS AND METHODS: We obtained home BP measurements for 1913 subjects aged 40 years or older, then followed up their survival status (mean, 8.6 years). We classified the subjects into the following 4 groups according to their home BP levels: systolic-diastolic hypertension, isolated systolic hypertension, isolated diastolic hypertension, and normotension. The prognostic significance of each type of hypertension for the risk of cardiovascular mortality risk was investigated using a Cox proportional hazards regression model adjusted for possible confounding factors. RESULTS: The risk for isolated systolic hypertension and systolic-diastolic hypertension were significantly higher than the relative hazard for normotension, while isolated diastolic hypertension was associated with no significant increase in risk. Home pulse pressure measurement was also independently associated with an increase in the risk of cardiovascular mortality. CONCLUSIONS: Isolated diastolic hypertension, as assessed by home BP measurements, carried a low risk of cardiovascular mortality, similar to that found in subjects with normotension, suggesting that the prognosis of hypertension would be improved by treatment focused on systolic rather than on diastolic home BP measurements. To our knowledge, this study is the first to demonstrate the clinical significance of pulse pressure as assessed by home BP measurement. Arch Intern Med. 2000;160:3301-3306.

Adult↗
PubMed2000

Structure of lithocholic acid binding to the N-terminal 8-kDa domain of DNA polymerase beta.

The purpose of this study was to investigate the molecular action of lithocholic acid (LCA), known as a selective inhibitor of DNA polymerase beta (pol beta). The 39-kDa pol beta was separated proteolytically into two fragments of the template-primer binding domain (8 kDa) and the catalytic domain (31 kDa). LCA bound tightly to the 8-kDa fragment but not to the 31-kDa fragment. We examined the structural interaction with the 8-kDa domain using LCA. On (1)H-(15)N HMQC NMR analysis of pol beta with LCA, the 8-kDa domain bound to LCA as a 1:1 complex with a dissociation constant (K(D)) of 1.56 mM. The chemical shifts were observed only in residues mainly in helix-3, helix-4, and the 79-87 turn of the same face. No significant shifts were observed for helix-1, helix-2, and other loops of the 8-kDa domain. This region was composed mainly of three amino acid residues (Lys60, Leu77, and Thr79) of pol beta on the LCA interaction interface. The inhibition mechanism and the structure-function relationship between pol beta and LCA is discussed.

Animals↗
PubMed2000

Two novel first exons in the prolactin receptor gene are transcribed in a tissue-specific and sexual maturation-dependent manner to encode multiple 5'-truncated transcripts in the testis of the chicken.

Cloning and sequencing of the chicken prolactin receptor (PRLR) gene segment from the transmembrane domain to the box 2 motif revealed the presence of the two testis-specific first exons, TSE-1 and TSE-2, encoding the unique 5'-end sequences of the reported and newly identified multiple 5'-truncated PRLR transcripts containing only the cytoplasmic domain in the testis. TSE-1 was located downstream of the exon encoding the transmembrane domain and TSE-2 presented downstream of the exon encoding the box 1 motif. These findings indicate that the box 1-containing 5'-truncated transcripts are generated by the utilization of TSE-1 as the first exon with distinct splicing donor sites to the box 1-containing exon, and that the utilization of TSE-2 as the first exon and its splicing to the box 2-containing exon results in the generation of the box 1-lacking transcript. Three transcription initiation sites for the box 1-containing 5'-truncated transcripts and two transcription initiation sites for the box 1-lacking transcript were detected by the RNase protection assays. Reverse transcription-polymerase chain reaction analysis showed that the expression levels of all these 5'-truncated PRLR transcripts are simultaneously increased during sexual maturation, accompanying the decrease of the amount of the canonical full-length transcript for PRLR.

Animals↗
PubMed2000

Structure and tissue distribution of chicken leptin receptor (cOb-R) mRNA.

Chicken leptin receptor (cOb-R) cDNA has been cloned, sequenced and characterized. The predicted cOb-R preprotein was composed of 1148 amino acids showing approximately 60% sequence identity with the long isoform of mammalian leptin receptor, and contained a putative signal peptide, a single transmembrane domain and the conserved box 1, 2 and 3 motifs in the cytoplasmic region. High levels of cOb-R mRNA expression were observed in ovary and brain, and less abundant expression of the mRNA was detected in liver, kidney and intestine in juvenile females and sexually matured hens. The expression levels of cOb-R mRNA did not change during sexual maturation in most tissues, but the mRNA level in the intestine was higher in matured hens than in juveniles. Estrogen treatment was found to enhance the Ob-R mRNA expression in the intestine, but not in other tissues.

Amino Acid Sequence↗
PubMed2000

Early induction of the orphan nuclear receptor NOR-1 during cell death of the human breast cancer cell line MCF-7.

The neuron-derived orphan receptor (NOR-1) is a member of the NGFI-B subfamily within the nuclear receptor superfamily. In T-cell apoptosis, where NGFI-B plays an essential role, a functional redundancy between NGFI-B and NOR-1 has been demonstrated. Here, we examined the regulation and expression of the NOR-1 gene during cell death induced by a calcium ionophore A23187 in the human breast cancer cell line MCF-7. A23187 caused a transient increase in NOR-1 mRNA levels within 6 h after treatment. To delineate the sequences required for the transitional response to A23187, a series of promoter deletion mutants were constructed. From the transient transfection experiments, the element responsive to A23187 was identified between -94 and -42 base pairs upstream from the transcription initiation site. This 53-base pairs region contains three copies of the cAMP response element (CRE). Furthermore, phosphorylation of the CRE-binding protein (CREB), which affects the transcription of the CRE dependent-genes, was detected 30 min after A23187 stimulation. Our findings are consistent with NOR-1 involvement in A23187-induced cell death via the CRE-CREB signaling pathway.

Apoptosis↗
PubMed2000

Missense variations of the gene responsible for Wolfram syndrome (WFS1/wolframin) in Japanese: possible contribution of the Arg456His mutation to type 1 diabetes as a nonautoimmune genetic basis.

Recently, a novel gene for a putative transmembrane protein (WFS1/wolframin) was found to be mutated in patients with Wolfram syndrome or DI-DM-OA-D (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness) syndrome. It is suggested that the WFS1 protein is important in the survival of islet beta-cells. We studied the WFS1 gene in a Japanese population to assess its possible role in common type 1 diabetes. Mutation screening revealed four missense mutations; R456H, G576S, H611R, and I720V. By genetic association studies of 185 type 1 diabetes patients and 380 control subjects, we found that R456H was significantly increased in the type 1 diabetes group compared to the control group (P = 0.0005); H611R and I720V were also significantly increased with weaker significance. Furthermore, in patients with the R456H mutation, type 1 diabetes-resistant HLA-DRB1 alleles (DRB1*0406, 1501, and 1502) were significantly increased compared to mutation-negative patients while susceptible DRB1*0901 was significantly decreased. Frequencies of autoimmunity characteristics (ICA or GAD-Ab positiveness and combination of autoimmune thyroid disease) were decreased in the R456H-positive patients compared to the R456H-negative patients. These data suggest that the WFS1 gene may have a role in the development of common type 1 diabetes as a nonautoimmune genetic basis.

Adult↗
PubMed2000

Molecular cloning of the chicken prolactin gene and activation by Pit-1 and cAMP-induced factor in GH3 cells.

Transcription of the prolactin (PRL) gene has been reported to be activated by a nuclear factor, Pit-1. However, the precise molecular mechanisms of the Pit-1-mediated PRL gene activation are still unclear. We have cloned the chicken PRL (cPRL) gene and its 5'-flanking region to analyze their structure and transcription-initiating mechanism. In luciferase assay, forskolin activated the proximal promoter region between -248 and -76 to transcribe the cPRL gene in GH3 cells, although there is no canonical cyclic AMP-responsive element in the promoter region. In gel mobility shift assay, a DNA fragment between -104 and -76 containing a putative Pit-1 binding site was bound by nuclear factors from the GH3 cells. Furthermore, it was observed that Pit-1 protein specifically bound to the DNA fragment in the supershift assay. These results indicate that both Pit-1 and cAMP-induced factor(s) associated with the cis element on the proximal promoter region to activate cPRL gene expression in GH3 cells.

Animals↗