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Biomedical subjects

H Kumagai

Publications and source records attributed to H Kumagai.

At least 19 recordsLinked to original sources
PubMed2000

The effect of 6-methylthiohexyl isothiocyanate isolated from Wasabia japonica (wasabi) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-buatnone-induced lung tumorigenesis in mice.

The present study was undertaken to estimate the effect of 6-methylthiohexyl isothiocyanate (6MHITC) isolated from Wasabia japonica (wasabi) pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice. Pretreatment with 6MHITC for 4 consecutive days at a daily dose of 5 micromol significantly inhibited NNK-induced O(6)-methylguanine formation in lungs at 4 h after the injection. In conjugation with this inhibitory effect, 6MHITC suppressed the increase in proliferating nuclear cell antigen level as well as ornithine decarboxylase activity at a promotion stage of NNK-induced lung tumorigenesis. Finally, this treatment of 6MHITC suppressed the NNK-induced lung tumorigenesis in mice. These results suggest that 6MHITC inhibits the development of lung tumors in mice treated with NNK, due to the suppression of initiation stage.

Animals↗
PubMed2000

Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile.

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.

Aminobutyrates↗
PubMed2000

Identification of novel membrane and secreted proteins upregulated during adipocyte differentiation.

Adipose tissue is the largest organ in the body that secretes soluble proteins such as cytokines. A preadipocyte cell line 3T3-L1 has been widely used for investigations of mechanisms of adipocyte differentiation. 3T3-L1 cells convert to adipocytes in the presence of 1-methyl-3-isobutylxanthine, dexamethasone, and insulin. We screened a cDNA library derived from differentiated 3T3-L1 cells, using the SST-REX method (signal sequence trap by retrovirus-mediated expression screening method). Screening of 4 x 10(5) clones gave rise to 63 known and 8 novel clones. The known clones represented 28 independent proteins, 21 of which were secreted proteins and 7 were membrane proteins. The novel clones represented 7 independent proteins, 5 of which had no similarity to known proteins. Interestingly, most of these novel genes showed differentiation- and tissue-specific expression. The present results indicate that adipocytes specific genes or adipocyte differentiation-related genes encoding membrane and secreted proteins can be readily identified if signal sequence trap screening of differentiated adipocyte-derived cDNAs is done.

3T3 Cells↗
PubMed2000

Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase.

The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein. A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M. luteus genomic DNA library. The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa. The enzyme was found to be a homodimer with a molecular weight of 49,000. It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors. Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases. A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling.

Amino Acid Sequence↗
PubMed2000

Characterization of endo-alpha-N-acetylgalactosaminidase from Bacillus sp. and syntheses of neo-oligosaccharides using its transglycosylation activity.

Endo-alpha-N-acetylgalactosaminidase was purified to homogeneity from the culture fluid of Bacillus sp. isolated from soil and characterized. The molecular mass of the enzyme was estimated as 110 kDa. The enzyme was stable at pH 4.0-10.0, up to 55 degrees C, and was most active at pH 5.0. The substrate specificity of the enzyme was strict for the disaccharide, galactosyl beta1, 3 N-acetyl-d-galactosamine, bound to aglycone in alpha configuration. On the other hand, the specificity of the enzyme for the aglycone structure was fairly relaxed. The enzyme could transfer the disaccharide from para-nitrophenyl substrate to various acceptors, such as monosaccharides, disaccharides, and sugar alcohols. Using this transglycosylation activity of the endoglycosidase, it may be possible to synthesize neo-oligosaccharides.

Asialoglycoproteins↗
PubMed2000

GPR1 regulates filamentous growth through FLO11 in yeast Saccharomyces cerevisiae.

Cell growth and differentiation are regulated by nutrient availability in the yeast Saccharomyces cerevisiae. Under conditions of nitrogen limitation, diploid cells of S. cerevisiae differentiate to a filamentous growth known as a pseudohyphal growth, while haploid cells produce invasive filaments which penetrate the agar in nutrient-rich medium. We have found that GPR1, which encodes a putative G-protein-coupled receptor, is required for both pseudohyphal and invasive growth. Pseudohyphal growth was defective in Deltagpr1/Deltagpr1 mutant strain and this defect was reversed by addition of cAMP. Also, haploid Deltagpr1 mutant strain was defective in invasive growth. Northern blot analysis revealed that the transcriptional level of FLO11, which encodes a recently identified cell surface flocculin required for pseudohyphal growth, was reduced in Deltagpr1 mutant strain. These results indicate that GPR1 regulates both pseudohyphal and invasive growth by a cAMP-dependent mechanism.

Blotting, Northern↗
PubMed2000

Time-resolved two-dimensional thick-slice magnetic resonance digital subtraction angiography in assessing brain tumors.

The aim of this study was to evaluate clinical applicability of two-dimensional (2D) thick-slice, contrast-enhanced magnetic resonance digital subtraction angiography (MRDSA) with high temporal resolution in diagnosis of brain tumors. Forty-four patients with brain tumors including, 15 meningiomas, 8 gliomas, 6 metastatic tumors, 4 neuromas, and 2 hemangioblastomas, were studied with 2D MRDSA with frame rate approximately 1 s. Images were continuously obtained following the initiation of bolus injection of gadolinium chelates for 40 s and subtraction images were generated in a workstation. We evaluated visualization of normal cranial vessels on MRDSA and compared MRDSA and intra-arterial digital subtraction angiography (IADSA) with regard to hemodynamic information. Large cerebral arteries, all venous sinuses, and most tributaries were clearly visualized. A stain was present in hypervascular tumors including all 15 meningiomas and 2 hemangioblastomas on MRDSA. Presence of a stain demonstrated on MRDSA and that on IADSA coincided in 16 of 20 cases (Spearman rank correlation value was 0.85). The location, shape, and phase of the stain on MRDSA were similar to those on IADSA. Two-dimensional MRDSA with high temporal resolution has a unique ability to demonstrate cerebral hemodynamics, such as IADSA, and can play an important role in assessing brain tumors.

Adolescent↗
PubMed2000

Effect of water content on dielectric relaxation of gelatin in a glassy state.

The dielectric properties of gelatin in the glassy state were measured from 100 Hz to 1 MHz over a temperature range of -20 to 60 degrees C. Samples with different water contents were prepared by varying the drying time for desalted gelatin solution; they were confirmed to be in the glassy state from DSC measurements. The dielectric relaxation (the decrease in dielectric constant, epsilon', and the maximum of the dielectric loss, epsilon' ') was observed for each sample; the relaxation time tau was evaluated from the peak of epsilon' '. The activation energy E obtained from an Arrhenius plot of tau decreased with increasing water content. On the basis of the order of magnitude of E, the dielectric relaxation observed was considered to be beta-relaxation reflecting the local motion of molecules. E and tau seem to describe the enhancement effect of water on the mobility of gelatin molecules in the glassy state; tau and E are considered to be suitable parameters for the characterization of the plasticizing effect of water on a glassy material.

Calorimetry, Differential Scanning↗
PubMed2000

Interactions between thermal cycled resilient denture lining materials, salivary and serum pellicles and Candida albicans in vitro. Part II. Effects on fungal colonization.

In the present study, the growth of a single isolate of Candida albicans on saliva-, serum-coated or protein free (uncoated), thermocycled (4-70 degrees C for 1 min, respectively; 0, 1000 and 10 000 times) seven commercial soft lining materials were investigated, by adenosine triphosphate (ATP) analysis. In the case of control resilient liners (not thermocycled and uncoated), the fungal colonization appeared to depend upon the type of commercial resilient liner used. Thus, the lowest colonization was observed with fluoric and heat-cured silicone materials, cold-cured silicone materials, except for one product, and heat-cured acrylic resin exhibited the highest colonization capacity, and cold-cured acrylic resilient liners exhibited the intermediate. However, the fungal colonization on the materials was significantly promoted both by thermal cycling (ANOVA, P<0.01) and a layer of protein coating (saliva, P<0.01; serum, P<0.01). These results, taken together, suggest that the ageing of the materials and the biological fluids of the host promote yeast colonization on resilient lining materials.

Acrylic Resins↗
PubMed2000

Standardized thigh muscle area measured by computed axial tomography as an alternate muscle mass index for nutritional assessment of hemodialysis patients.

BACKGROUND: Quantification of muscle mass, which represents the largest protein pool in the body, is important for nutritional assessment but is difficult to achieve with conventional methods in hemodialysis patients. OBJECTIVE: We measured the cross-sectional area of the thigh occupied by muscle by using computed tomography and compared this with other muscle mass indicators. DESIGN: Thigh muscle area (TMA) was examined and correlated with creatinine production and various nutritional indexes in 163 patients undergoing hemodialysis. Where appropriate, TMA was expressed relative to bone area in the thigh (TBA) to avoid the influence of body size. RESULTS: TMA was highly correlated with creatinine production as measured in the spent dialysate (r = 0.85, P < 0.001), indicating that TMA substantially reflects total-body muscle mass. TMA standardized for TBA was negatively correlated with age and positively correlated with other nutritional indicators including body weight, body mass index, serum albumin, serum transthyretin, and protein catabolic rate. Multiple regression analysis revealed that of these variables, age, serum albumin, and protein catabolic rate independently predicted TMA standardized for TBA. By using correlations with various nutritional indicators, we concluded that patients with a value <10.0 for TMA standardized for TBA were likely to be malnourished whereas those with a value >13.0 were likely to be well nourished. CONCLUSIONS: These results indicate that TMA standardized for TBA, measured by computed tomography, is a reliable indicator of muscle mass that could be used for nutritional assessment of hemodialysis patients.

Aged↗
PubMed2000

Dynamic contrast-enhanced MR angiography and MR imaging of the carotid artery: high-resolution sequences in different acquisition planes.

BACKGROUND AND PURPOSE: First-pass contrast-enhanced MR angiography has become the technique of choice for studying the carotid bifurcation, but this method has some limitations. We evaluated the clinical utility of performing 3D contrast-enhanced MR angiography in the axial plane immediately after performing angiography in the coronal plane. METHODS: Cervical carotid arteries of 80 consecutive patients were studied on a 1.5-T MR imager with phased-array coils. Coronal 3D MR angiography was performed after administering a bolus injection of contrast material (20 mL) with automatic triggering. This was immediately followed by an axial acquisition. We measured carotid diameters on the contrast-enhanced MR angiograms as well as on intra-arterial digital subtraction angiograms according to established criteria. We also evaluated original source MR angiograms. RESULTS: Angiograms obtained in the axial plane correlated better with the intra-arterial digital subtraction angiograms than did the coronal angiograms. When first-pass contrast-enhanced MR angiography was incomplete because of a failure of triggering, the second-phase acquisition provided sufficient image quality. Original source images suffered from ring artifacts, low axial resolution, and a low level of soft-tissue visualization. Axial-based source images showed flow-independent contrast filling to the patent lumen with sufficient visualization of plaque morphology, thickened arterial wall, and surrounding disease processes, such as tumors. CONCLUSION: With the addition of a 1-minute second-phase 3D acquisition in a different plane immediately after first-pass contrast-enhanced MR angiography, one can obtain a more accurate depiction of the carotid bifurcation, insurance against failure of triggering, and diagnostic source images.

Adolescent↗
PubMed2000

[A case of saccular aneurysm of the coronary artery post PTCA].

We experienced a 48-year-old man who was diagnosed as having a saccular aneurym of the coronary artery by CAG after an otherwise uneventful percutaneous transluminal coronary angioplasty (PTCA). At the time of operation, the aneurysm, measured 15 mm, and arose from the 3rd segment of the right coronary artery. Aneurysmal formation after successful PTCA is not rare and is related to the overinflating balloon. It is necessary to evaluate the coronary artery after PTCA by IVUS. There were no symptoms after the operation and he was discharged at 21th postoperative day.

Angioplasty, Balloon, Coronary↗
PubMed2000

Minimally invasive direct coronary artery bypass grafting using the saphenous vein in redo CABG.

We describe a patient who underwent minimally invasive direct coronary artery bypass (MIDCAB), who had previously undergone coronary artery bypass grafting (CABG) through a median sternotomy with a left internal mammary artery (LIMA) graft to the left anterior descending artery (LAD) and a right gastroepiploic artery (GEA) graft to the posterior descending artery. MIDCAB was less invasive and was an effective alternative procedure for the second operation. Because the patient had no LIMA or GEA available for a graft because of prior use, we used a saphenous vein graft (SVG) for bypassing from the left subclavian artery to the coronary artery by MIDCAB via a left minithoracotomy. The left subclavian artery was selected as the proximal anastomotic site because this artery was less diseased and was easier to reach. The SVG-to-coronary artery anastomosis was facilitated by firm adhesion between the epicardium and the pericardium, which reduced the motion of the epicardium itself. These results suggest that the procedure is safe and promising in selected cases of redo CABG.

Cardiopulmonary Bypass↗
PubMed1999

Conductance and relaxations of gelatin films in glassy and rubbery states.

The dielectric constant, epsilon', and the dielectric loss, epsilon'', for gelatin films were measured in the glassy and rubbery states over a frequency range from 20 Hz to 10 MHz; epsilon' and epsilon'' were transformed into M* formalism (M* = 1/(epsilon' - i epsilon'') = M' + iM''; i, the imaginary unit). The peak of epsilon'' was masked probably due to dc conduction, but the peak of M'', e.g. the conductivity relaxation, for the gelatin used was observed. By fitting the M'' data to the Havriliak-Negami type equation, the relaxation time, tauHN, was evaluated. The value of the activation energy, Etau, evaluated from an Arrhenius plot of 1/tauHN, agreed well with that of Esigma evaluated from the DC conductivity sigma0 both in the glassy and rubbery states, indicating that the conductivity relaxation observed for the gelatin films was ascribed to ionic conduction. The value of the activation energy in the glassy state was larger than that in the rubbery state.

Electric Conductivity↗
PubMed1999

Differential inhibition of HMG-CoA synthase and pancreatic lipase by the specific chiral isomers of beta-lactone DU-6622.

A synthetic beta-lactone trans-DU-6622 (3-hydroxy-2-(hydroxymethyl)-5-[7-(methylcarbonyl)-naphthalen++ +-1-yl]pentanoic acid 1,3-lactone, a mixture of (2R, 3R)- and (2S, 3S)-beta-lactones) was found to inhibit HMG-CoA synthase (IC(50): 0. 15 microM) and pancreatic lipase (IC(50): 120 microM). The effects of the optically pure DU-6622 isomers on the two enzymes were compared. The (2R, 3R)-isomer was shown to be a highly specific inhibitor of HMG-CoA synthase (IC(50): 0.098 microM vs 270 microM for pancreatic lipase), while the (2S, 3S)-isomer markedly increased the specificity of lipase inhibition (IC(50): 27 microM vs 31 microM for HMG-CoA synthase). Furthermore, the (2R, 3R)-isomer strongly inhibited the binding of [(14)C]hymeglusin to HMG-CoA synthase, indicating that the isomer was bound to the same site of the synthase as hymeglusin. The (2R, 3R)-beta-lactone is responsible for the specific inhibition of HMG-CoA synthase, while the (2S, 3S)-beta-lactone is responsible for the inhibition of pancreatic lipase.

Animals↗
PubMed1999

Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.

The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990) J. Biol. Chem. 265, 22004-22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.

Dimerization↗
PubMed1999

Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p.

Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.

Amino Acid Sequence↗