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Tomoko Kimura

Publications and source records attributed to Tomoko Kimura.

14 recordsLinked to original sources
PubMed2006

Biantennary glycans as well as genetic variants of alpha1-acid glycoprotein control the enantioselectivity and binding affinity of oxybutynin.

PURPOSE: The purpose of this study was to investigate the role of biantennary branching glycans of alpha1-acid glycoprotein (AGP) and its genetic variants in the enantioselective binding of oxybutynin (OXY). METHOD: Human native AGP was separated using imminodiacetate-copper (II) affinity chromatography into two fractions, the A variant and a mixture of the F1 and S variants (F1-S). These fractionated AGPs were further separated by concanavalin A affinity chromatography into two fractions, with and without biantenarry glycans. An on-line high-performance liquid chromatography (HPLC) system consisting of a high-performance frontal analysis column, an extraction column, and an analytical HPLC column was developed to determine the binding affinities of OXY enantiomers for respective AGP species. RESULTS: The total binding affinity as well as the enantiomeric selectivity of OXY in the F1-S mixed variant was significantly higher than that for the A variant, indicating that the chiral recognition ability of native AGP for the OXY enantiomers highly depends on the F1-S mixed variant. Furthermore, not only the genetic variants but also bianntenary glycans of AGP affect the binding affinity of OXY and are also responsible for the enantioselectivity. CONCLUSIONS: Both genetic variants and glycan structures significantly contribute to the enantioselectivity and the binding affinity of OXY.

Carbohydrate Conformation↗
PubMed2005

Specific determination of threonine in biological samples by gas chromatography with electron capture detection.

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.

Animals↗
PubMed2005

High-performance frontal analysis of the binding of thyroxine enantiomers to human serum albumin.

PURPOSE: The aim of this study was to characterize the binding property between thyroxine and human serum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA). METHODS: An on-line HPLC system consisting of an HPFA column, an extraction column, and an analytical HPLC column was developed to be used to determine the unbound concentrations of thyroxine enantiomers. RESULTS: Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities. The binding constant (K) and the number of binding sites on an HSA molecule (n) evaluated from Scatchard plot analysis were K = 1.01 x 10(6)m(-1) and n = 1.90 for L: -thyroxine, and K = 9.71 x 10(5) m(-1) and n = 1.97 for D: -thyroxine. The binding sites were identified using phenylbutazone and diazepam as site-specific probes for sites I and II, respectively, and each enantiomer was found to bind to both sites. Incorporation of a chiral HPLC column into the on-line system permitted the investigation of enantiomer-enantiomer interactions, which revealed that both enantiomers competitively bind to the same binding sites without significant allosteric effects.

Binding Sites↗
PubMed2005

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.

Animals↗
PubMed2005

Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection.

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq.>0.999) within 7.4-148.2 pM for T4 and 1.5-74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4+/-2.4 pM (n=15) and 7.14+/-1.04 pM (n=15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30+/-0.27 pM (n=15). The CV% values of intra-day and inter-day assays (n=15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.

Blood Proteins↗
PubMed2004

Charge-based separation of detergent-resistant membranes of mouse splenic B cells.

Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation. The low-density fraction that recovered a part of DRMs containing IgM together with those enriched in GM1a, the Src family protein tyrosine kinase Lyn, and the alpha subunit of inhibitory heterotrimeric GTP-binding protein was further resolved by FFE. FFE separated the former into more cathodally deflected fractions than the latter. In addition, FFE revealed an anodal shift of DRMs containing a transmembrane protein CD38 upon BCR-crosslinking. The results demonstrate the effectiveness of FFE for the charge-based separation of DRMs.

Animals↗
PubMed2004

Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana.

Plant metabolism is a complex set of processes that produce a wide diversity of foods, woods, and medicines. With the genome sequences of Arabidopsis and rice in hands, postgenomics studies integrating all "omics" sciences can depict precise pictures of a whole-cellular process. Here, we present, to our knowledge, the first report of investigation for gene-to-metabolite networks regulating sulfur and nitrogen nutrition and secondary metabolism in Arabidopsis, with integration of metabolomics and transcriptomics. Transcriptome and metabolome analyses were carried out, respectively, with DNA macroarray and several chemical analytical methods, including ultra high-resolution Fourier transform-ion cyclotron MS. Mathematical analyses, including principal component analysis and batch-learning self-organizing map analysis of transcriptome and metabolome data suggested the presence of general responses to sulfur and nitrogen deficiencies. In addition, specific responses to either sulfur or nitrogen deficiency were observed in several metabolic pathways: in particular, the genes and metabolites involved in glucosinolate metabolism were shown to be coordinately modulated. Understanding such gene-to-metabolite networks in primary and secondary metabolism through integration of transcriptomics and metabolomics can lead to identification of gene function and subsequent improvement of production of useful compounds in plants.

Arabidopsis↗
PubMed2003

Global expression profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-acetyl-l-serine as a general regulator of gene expression in response to sulfur nutrition.

To investigate the changes in profiles of mRNA accumulation in response to sulfur deficiency, approximately 13 000 non-redundant Arabidopsis thaliana ESTs corresponding to approximately 9000 genes were analyzed using DNA macroarray. Three-week-old Arabidopsis plants grown on an agarose-solidified control medium were transferred to a sulfate-free medium and grown for 48 h for the analyses of sulfur-related metabolites and global gene expression profiles. Concentrations of sulfate, O-acetyl-l-serine (OAS), a positive regulator of sulfur deficiency-responsive genes, cysteine and glutathione (GSH) were determined. Plants transferred to sulfate-free media had reduced concentrations of sulfate and GSH, and OAS concentrations increased. Macroarray analysis revealed a number of genes, including APR2 and Sultr1;2, whose mRNA accumulation was increased by sulfur deficiency. Profiling was also carried out with plants treated with OAS under sulfate-sufficient condition. Scatter plot analysis revealed a positive correlation between the changes of expression levels by sulfur deficiency and by OAS treatment among the clones tested, suggesting that mRNA accumulation of a number of genes under sulfur deficiency is mainly controlled by OAS concentrations in tissues. It was also revealed that the sets of genes regulated under sulfur deficiency in leaves and roots differ considerably.

Adaptation, Physiological↗
PubMed2002

Plasma protein binding study of oxybutynin by high-performance frontal analysis.

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.

Blood Proteins↗
PubMed2002

Binding study of desethyloxybutynin using high-performance frontal analysis method.

Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 microM (R)- or (S)-DEOXY was 1.19 +/- 0.001 and 2.33 +/- 0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97 x 10(7) and 1.31 x 10(7) M(-1), respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77 x 10(3) and 8.44 x 10(3) M(-1), respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53 x 10(7) M(-1)), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86 x I0(6) M(-1)). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64 x 10(4) and 2.19 x 10(4) M(-1) for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.

Chromatography, High Pressure Liquid↗
PubMed2002

[Tuberculosis control of construction workers living in Hanba].

Construction workers living in temporal quarters, HANBA, rarely have opportunity to receive the routine health screening program such as that for general inhabitants organized by the local government or that for permanent employees by the employer. Long delay in detecting TB and high drop-out rate from TB treatment among them have been reported. We carried out the following interventions to cope with the problems: In 1999 and 2000, we organized TB screening with X-ray and further examination in the same day when necessary among workers in 6 HANBA in Chiba City. A total of 382 workers were screened, and they also received structured interviews to assess their health-related behaviors and conditions. Four active pulmonary TB cases were detected, and the incidence of 1,047/1,000,000 was forty times higher than that of Chiba City. All four patients were treated and cured. According to the experiences through our intervention, we developed the following recommendations on TB control of construction workers living in HANBA: 1) A system carry out the health examination routinely in the HANBA should be provided, and its implementation be supervised by the public health center and the Labor Standards Inspection Office. 2) It is necessary to guarantee worker's minimum living conditions and medical treatment, and for this regular purpose, close cooperation should be established between clinical service providers and public health, social welfare, and work management authorities. 3) We should start "DOTS" (Directly Observed Treatment Short-Course) not only to the patients in the hospital but also to the outpatients and in the HANBA. To control TB among those workers, further effort is necessary to motivate them to receive basic regular health screening program that is provided in free of charge in Japan.

Adult↗