Biomedical subjects

Nobuo Tanaka

Publications and source records attributed to Nobuo Tanaka.

At least 19 recordsLinked to original sources

PubMed2004

Interval immobilization technique for recognition toward a highly hydrophilic cyanobacterium toxin.

A novel adsorption medium containing selective molecular recognition site for one of the powerful cyanobacterium toxins, Cylindrospermopsin (CYN) was developed using a special technique, namely interval immobilization technique. The adsorption medium was prepared using molecular assembly derived from an alternative-template molecule coupled with functional monomers for fixing the interval between the ionic functional groups in CYN. As results of liquid chromatographic evaluations, selective molecular recognition ability for CYN was observed as expected. Further studies proved that the association constant for CYN on this medium was slightly higher than that on blank polymer.

Adsorption↗

PubMed2004

Determination of bisphenol A in environmental water at ultra-low level by high-performance liquid chromatography with an effective on-line pretreatment device.

We have developed a simple HPLC method for the microanalysis of bisphenol A (BPA), which is often contained in environmental water and is known as an endocrine disrupter. HPLC coupled with electrochemical detection requires a simpler procedure of pretreatment compared to GC-MS. In this study, we analyzed BPA using molecularly imprinted polymer as an on-line pretreatment device. This polymer has molecular recognition sites and provides specific selectivity in extraction process. Due to this effect, the detection limit obtained with this HPLC was 0.36 ng/l. This method applied to environmental water and purified water samples containing 2-70 ng/l of BPA successfully. Furthermore, UV detection was performed in some actual analyses.

Benzhydryl Compounds↗

PubMed2004

Structure of bovine carbonic anhydrase II at 1.95 A resolution.

Carbonic anhydrase (CA) is a zinc-containing enzyme that catalyzes the reversible hydration of CO2 to HCO3-. In eukaryotes, the enzyme plays a role in various physiological functions, including interconversion between CO2 and HCO3- in intermediary metabolism, facilitated diffusion of CO2, pH homeostasis and ion transport. The structure of bovine carbonic anhydrase II (BCA II) has been determined by molecular replacement and refined to 1.95 A resolution by simulated-annealing and individual B-factor refinement. The final R factor for the BCA II structure was 19.4%. BCA II has a C-terminal knot structure similar to that observed in human CA II. It contains one zinc ion in the active site coordinated to three histidines and one putative water molecule in a tetrahedral geometry. The structure of BCA II reveals a probable alternative proton-wire pathway that differs from that of HCA II.

Animals↗

PubMed2004

Novel surface-modification techniques for polymer-based separation media. Stimulus-responsive phenomena based on double polymeric selectors.

A pair of polymeric selectors potentially responding to stimulation was introduced onto monosized porous polymer particles to be evaluated as a packing material for HPLC. Possible complexes formed between polyacrylamide (PAAm) and poly(methacrylic acid) (PMAA) were utilized as stimulus responsive polymeric selectors. Uniformly sized base polymer particle was prepared by multi-step swelling and polymerization method, while the introduction of PAAm and PMAA was done by newly invented modification technique. In this technique, a solvent in which both acrylamide (AAm) and methacrylic acid (MAA) monomers are soluble, but PAAm and PMAA are insoluble, was utilized as a modification medium. The polymer particle doubly modified with PAAm and PMAA was utilized as packing material for HPLC and the stimulus responses were evaluated by changing temperature or pH to check change of the slope of a Van't Hoff plot. By using water as a mobile phase, the expected inflection point of the Van't Hoff plot was observed at upper critical solution temperature (UCST) of the polymer complexes and the temperature responsive ability was observed. Moreover, pH responsive ability was studied by using buffer of either pH 4 or 10 as mobile phase. Slope of the plot was changed in buffer of pH 4, but no change of slope was observed in the buffer of pH 10.

Chromatography, High Pressure Liquid↗

PubMed2004

Polymer-based adsorption medium prepared using a fragment imprinting technique for homologues of chlorinated bisphenol A produced in the environment.

A polymer-based adsorption medium having molecular recognition ability for homologues of chlorinated bisphenol A produced in environment was prepared using a fragment imprinting technique. 2,6-Dimethyl phenol was utilized as a pseudo-template molecule and the adsorption media prepared was evaluated by high performance liquid chromatography (HPLC) and solid-phase extraction (SPE). As results, the adsorption medium showed preferable chromatographic retention and specific adsorption ability for the chlorinated bisphenol As having chlorine substituents at 3,5-positions through fragment imprinting effect.

Adsorption↗

PubMed2004

Simple and comprehensive two-dimensional reversed-phase HPLC using monolithic silica columns.

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 cm long (4.6-mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated (C(18)) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4-0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase (C(18)) or different phases, C(18) and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of approximately 1000 in approximately 60 min in cases 1 and 2 and in approximately 30 min in case 3. The three stationary phases, FR, C(18), and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns.

Journal Article↗

PubMed2004

Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins.

Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.

Amino Acid Sequence↗

PubMed2004

Breeding severely vitamin B12-deficient mice as model animals.

Newborn mice weaned from mice fed on a B12-deficient diet during pregnancy and lactation were fed on a B12-deficient diet for 90 days after weaning, and the state of B12 deficiency was evaluated. The effect of B12 deficiency on the testicular tissue was also examined. The body weight of the mice fed on a B12-deficient diet for 90 days was slightly lower than that of the control mice administrated CN-B12, and the urinary excretion of methylmalonic acid (MMA) was increased. The B12 concentrations in the liver and testes were markedly depressed by B12 deficiency, being about 13 and 10 pmol/g, respectively, on day 90. The testes weight was clearly reduced by B12 deficiency. The testes weight/100 g body weight was also lowered. Clear morphological changes were observed in the testicular tissue of the B12-deficient mice. These results showed that mice in a severely B12-deficient state could be produced by dietary B12 deprivation. These B12-deficient mice could be useful as model animals not only for elucidating the functions of B12 in vivo, but also for biochemical studies.

Animals↗

PubMed2004

Direct observation of a stacking fault in Si(1 - x)Ge(x) semiconductors by spherical aberration-corrected TEM and conventional ADF-STEM.

Spherical aberration (C(S))-corrected transmission electron microscopy (TEM) and annular dark-field scanning TEM (ADF-STEM) are applied to high-resolution observation of stacking faults in Si(1 - x)Ge(x) alloy films prepared on a Si(100) buffer layer by the chemical vapor deposition method. Both of the images clarify the individual nature of stacking faults from their directly interpretable image contrast and also by using image simulation in the case of the C(S)-corrected TEM. Positions of the atomic columns obtained in the ADF-STEM images almost agree with a projection of the theoretical model studied by Chou et al. (Phys. Rev. B 32(1985): 7979). Comparison between the C(S)-corrected TEM and ADF-STEM images shows that their resolution is at a similar level, but directly interpretable image contrast is obtained in ultrathin samples for C(S)-corrected TEM and in slightly thicker samples for ADF-STEM.

Journal Article↗

PubMed2004

Improved detectability with a polymer-based trapping device in rapid HPLC analysis for ultra-low levels of bisphenol A (BPA) in environmental samples.

A new high-performance liquid chromatography (HPLC) method has been developed to detect ultra-low concentrations of bisphenol-A (BPA) (below 1 ng/L (ppt)) using column switching electrochemical detection (ECD). The results were superior to those obtained from manual pretreatment procedure with membrane stationary phase. BPA is inherently ubiquitous in the environment, including tools and solvents used for its analysis; to obtain meaningful results, therefore, the concentration of the overall BPA contamination must be below the detection limit for BPA using the analytical system. Therefore, purified water for preparing the standard BPA solution must be filtered with a hydrophobic membrane to suppress BPA background levels of contamination. In addition, we investigated methods for effectively preserving environmental water containing BPA. The addition of a small amount of ethylenediaminetetraacetic acid (EDTA) provided good recovery even after overnight storage. By employing these precautionary measures and procedures to reduce BPA contamination from the analytical procedure, we could accurately determine l(-10) ppt of BPA in environmental water samples using a column switching HPLC system.

Benzhydryl Compounds↗

PubMed2004

[Influence of serum homocysteine level on coronary atherosclerosis in Japanese].

OBJECTIVES: This study investigated the relationship between serum homocysteine level and coronary artery disease in Japanese. METHODS: Serum homocysteine level was measured in 200 consecutive patients who underwent coronary angiography for the assessment of ischemic heart disease. Patients with acute myocardial infarction were excluded, so 197 patients were included in this study. The patients were classified into four groups based on number of diseased vessels identified by coronary angiography: no significant stenosis group (non-vessel group), one-vessel group, two-vessel group, and three-vessel group. More than 50% stenosis was defined as diseased vessels. RESULTS: Serum homocysteine level in the three-vessel group (13.5 +/- 8.0 microM) was significantly higher than that in the non-vessel group (9.9 +/- 2.7 microM), one-vessel group (9.1 +/- 2.3 microM), and two-vessel group (10.4 +/- 3.3 microM). Patients were classified into quartile groups according to the serum homocysteine level. The number of diseased vessels and frequency of three-vessel disease tended to be higher with increasing serum homocysteine level. There was no significant relationship between serum homocysteine level and coronary risk factors (diabetes mellitus, hyperlipidemia, smoking habit) except hypertension. Multivariate analysis for the predictor of number of diseased vessels showed diabetes mellitus, hypertension, and serum homocysteine level were independent predictors. CONCLUSIONS: Elevation of plasma homocysteine level is related to the severity of coronary artery disease in Japanese.

Aged↗

PubMed2003

Crystallization and X-ray analysis of the N-terminal core domain of a tumour-associated human DEAD-box RNA helicase, rck/p54.

The RCK gene was cloned on the basis of the t(11;14)(q23;q32) chromosome translocation observed in human B-cell lymphoma cell line RC-K8. This gene was found to be overexpressed in various kinds of tumours. The gene product, rck/p54, consisting of 472 amino-acid residues with molecular weight 53.2 kDa, belongs to the family of DEAD-box RNA helicases. Its ATP-dependent RNA-unwinding activity toward c-myc RNA molecules in vitro has recently been demonstrated. In the present study, limited proteolysis experiments of rck/p54 were used to truncate the N-terminal domain (residues 1-288; 31.8 kDa) of rck/p54, leading to successful crystallization of Nc-rck/p54, i.e. the N-terminal core domain (residues 70-288; 24.5 kDa) of rck/p54. Crystals of Nc-rck/p54 were grown to a size suitable for X-ray structure analysis using polyethylene glycol 3350 as the precipitant. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 65.5, b = 73.1, c = 84.8 A, and diffracts X-rays to beyond 2.0 A resolution.

Amino Acid Sequence↗

PubMed2003

A unique dye-decolorizing peroxidase, DyP, from Thanatephorus cucumeris Dec 1: heterologous expression, crystallization and preliminary X-ray analysis.

The dye-decolorizing peroxidase DyP is a key enzyme in the decolorizing fungus Thanatephorus cucumeris Dec 1 that degrades azo and antraquinone dyes. The gene dyp from T. cucumeris Dec 1, which has low homology to other peroxidase genes, was cloned and transformed into Aspergillus oryzae and glycosylated DyP was expressed at high levels. Purified DyP was deglycosylated using GST Endo F1 and then crystallized in a strong magnetic field (10 T) at 283 K using ammonium sulfate as precipitant. X-ray diffraction data to 2.96 A resolution collected from a native crystal at the Photon Factory (Tsukuba, Japan) showed that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 136.15, c = 363.46 A. The asymmetric unit of the crystal contained four DyP molecules, with a corresponding Matthews coefficient (V(M)) of 2.50 A(3) Da(-1) and a solvent content of 51%. Heavy-atom derivatives of DyP have been obtained and electron-density maps have been calculated. The haem is visible and continuous electron density between the haem and protein clearly indicates the location of the proximal histidine ligand.

Aspergillus oryzae↗

PubMed2003

Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics.

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.

Arabidopsis↗

PubMed2003

Target-selective ion-exchange media for highly hydrophilic compounds: a possible solution by use of the "interval immobilization technique".

We prepared a crosslinked polymer as a separation and/or adsorption medium for CYN, as shown in Fig. 1. The polymers were evaluated by high-performance liquid chromatography (HPLC) and adsorption was examined under batch conditions. Results from detailed HPLC evaluation and measurement of the difference between the binding affinity for CYN and for other compounds showed the prepared polymer had specific recognition ability for CYN.

Alkaloids↗

PubMed2003

Deuterium isotope effects on hydrophobic interactions: the importance of dispersion interactions in the hydrophobic phase.

Hydrogen/deuterium isotope effects on hydrophobic binding were examined by means of reversed-phase chromatographic separation of protiated and deuterated isotopologue pairs for a set of 10 nonpolar and low-polarity compounds with 10 stationary phases having alkyl and aryl groups bonded to the silica surface. It was found that protiated compounds bind to nonpolar moieties attached to silica more strongly than deuterated ones, demonstrating that the CH/CD bonds of the solutes are weakened or have less restricted motions when bound in the stationary phase compared with the aqueous solvent (mobile phase). The interactions responsible for binding have been further characterized by studies of the effects of changes in mobile phase composition, temperature dependence of binding, and QSRR (quantitative structure-chromatographic retention relationship) analysis, demonstrating the importance of enthalpic effects in binding and differentiation between the isotopologues. To explain our results showing the active role of the hydrophobic (stationary) phase we propose a plausible model that includes specific contributions from aromatic edge-to-face attractive interactions and attractive interactions of aliphatic groups with the pi clouds of aromatic groups present as the solute or in the stationary phase.

Chromatography, High Pressure Liquid↗

PubMed2003

Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain.

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Crystallization and preliminary X-ray studies of xylanase 10B from Thermotoga maritima.

Xylanases catalyze the hydrolysis of the beta-1,4-glycosidic bonds of xylan, which is the second most abundant component of plant cell walls after cellulose. The recombinant xylanase 10B from Thermotoga maritima MSB8 was prepared and crystallized by the sitting-drop vapour-diffusion method using 40 mM zinc acetate, 20 mM MES buffer pH 6.0 and 3% ethanol. Intensity data were collected to 2.5 A resolution at beamline BL26B2 of SPring-8. Preliminary X-ray analysis showed that the crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 77.3, b = 80.6, c = 58.2 A and one molecule per asymmetric unit.

Cloning, Molecular↗