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T Arakawa

Publications and source records attributed to T Arakawa.

At least 19 recordsLinked to original sources
PubMed1992

Structure and chromosomal localization of human arachidonate 12-lipoxygenase gene.

Arachidonate 12-lipoxygenase introduces a molecular oxygen into the C-12 position of arachidonic acid to produce 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid. With the aid of cDNA probes for the enzyme, we isolated overlapping lambda clones encompassing the human 12-lipoxygenase gene and flanking regions from a human genomic library. The gene consists of 14 exons with 13 introns and spans approximately 15 kilobases of DNA. All the exon-intron junctions conform to the GT/AG rule. Neither a typical TATA box nor a CAAT box was found in approximately 1-kb sequence of 5'-upstream region of the translation initiation site. However, this region contains several regulatory elements including four GC boxes, two CACCC boxes, three AP-2 binding sequences, and a glucocorticoid-responsive element. The major transcription initiation site was determined by primer-extension analysis as an adenosine residue at 306 bases upstream from the translation initiation codon. The chromosomal localization of the human 12-lipoxygenase gene was examined by fluorescence in situ hybridization, and the gene was assigned to the sub-band p13.1 of chromosome 17.

Arachidonate 12-Lipoxygenase↗
PubMed1992

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor.

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended ("other") conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.

Circular Dichroism↗
PubMed1992

Molecular structure and function of the porcine arachidonate 12-lipoxygenase gene.

The gene encoding arachidonate 12-lipoxygenase was cloned from a porcine EMBL3 genomic library using a cDNA probe of the enzyme, and its nucleotide sequence was determined. The gene consists of 14 exons with 13 introns, and spans approximately 8 kilobases. Analysis of splice junctions indicated that all of the splice donor and acceptor sites conformed to the GT/AG rule. An approximately 1-kilobase region upstream of the coding sequence contains nine GC-boxes for potential Sp1-binding sites at positions -77, -135, -145, -165, -214, -636, -643, -684, and -813. There are two sets of AP-2 binding sequence at positions -234 and -402. Neither typical TATA box nor CCAAT box is found in this region. The transcriptional start site was determined by primer extension analysis, and was tentatively identified as a cytidine residue located 19 bases upstream from initiation codon. Southern blot analysis revealed the presence of one copy of 12-lipoxygenase gene per haploid genome. We found striking similarities in genomic organization as well as the promoter sequences between the porcine 12-lipoxygenase and the rabbit 15-lipoxygenase genes, suggesting that these genes are evolutionarily related.

Amino Acid Sequence↗
PubMed1992

Molecular weights of glycosylated and nonglycosylated forms of recombinant human stem cell factor determined by low-angle laser light scattering.

The molecular weight of recombinant human stem cell factor (SCF) was determined using a low-angle laser light scattering combined with a differential refractometer and a uv detector. The protein samples were applied to these detectors through a gel filtration column by a high-performance liquid chromatographic pump. The Chinese hamster ovary (CHO) cell-derived SCF gave a molecular weight of 53,000 for the entire molecule and 35,000 for the protein moiety only at pH 7.0, indicating that the CHO cell-derived protein is glycosylated by 34%. Since the molecular weight of the polypeptide is 18,600, the results demonstrate that the CHO cell-derived SCF forms a dimer. The molecular weight of Escherichia coli-derived SCF was determined to be 39,000, similar to the above value (35,000). Essentially identical molecular weights were obtained at pH 3.0, indicating no dissociation of the dimer.

Animals↗
PubMed1992

Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.

Circular Dichroism↗
PubMed1992

Stability of fungal alpha-amylase in sodium dodecylsulfate.

Unfolding of a fungal alpha-amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the alpha-amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90 degrees C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49 degrees C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the alpha-amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50 degrees C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the alpha-amylase is a slow process.

Chromatography, Liquid↗
PubMed1992

Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor.

The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35 degrees C. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41 degrees C, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.

Fibroblast Growth Factor 2↗
PubMed1992

Effect of sofalcone on localization of 15-hydroxyprostaglandin dehydrogenase, an enzyme that metabolizes prostaglandin E2, in rat gastric mucosa: an immunohistochemical study.

We identified the cells containing 15-hydroxyprostaglandin dehydrogenase (15-HPGD) in rat gastric mucosa and examined the effects of sofalcone on the localization of the enzyme by use of an immunohistochemical technique. Also, we investigated the effects of sofalcone on the localization of prostaglandin E2 (PGE2). Specific stainings for 15-HPGD and PGE2 were similarly observed in a granular pattern mainly in the cytoplasm of parietal and surface epithelial cells. The number of the stained cells for 15-HPGD, especially surface epithelial cells, decreased when rats were given sofalcone, with a concomitant increase in PGE2 staining. These results suggest that parietal and surface epithelial cells are responsible for the degeneration of PGE2 in the rat gastric mucosa, and that sofalcone increased the PGE2 level in the mucosa through inactivation of 15-HPGD in these cells, especially surface epithelial cells.

Animals↗
PubMed1992

Early ultrastructural changes of surface epithelial cells isolated from rat gastric mucosa after exposure to ethanol with or without 16,16-dimethylprostaglandin E2.

Early ultrastructural changes of surface epithelial cells isolated from rat gastric mucosa caused by 15% ethanol were studied. The effect of 16,16-dimethylprostaglandin E2 (16,16-dimethyl-PGE2) on these changes was also examined. Findings by transmission electron microscopy showed ballooning and decreased density of mitochondria in addition to loss of microvilli and partial disruption of the surface cell membrane in cells exposed to 15% ethanol, although the nucleus looked normal. These ultrastructures were well preserved even after exposure to 15% ethanol when the cells were treated with 10(-6) M 16,16-dimethyl-PGE2 beforehand. These results may indicate that the surface cell membrane and mitochondria are the major targets for early injury by ethanol and protection by 16,16-dimethyl-PGE2 in surface epithelial cells. The mechanism by which 16,16-dimethyl-PGE2 prevents the damage is not known.

Animals↗
PubMed1992

Immunohistochemical localization of cells that synthesize leukotriene B4 in human gastric mucosa.

We evaluated the cell location of leukotriene B4 (LTB4) in human gastric mucosa by immunohistochemistry using a novel fixation procedure with 0.5% glutaraldehyde-8% paraformaldehyde followed by periodate-lysine-2% paraformaldehyde. The location of LTA4 hydrolase, which synthesizes LTB4, was also investigated in human gastric mucosa. Staining for LTB4 was mostly in a granular pattern in the cytoplasm of parietal and surface epithelial cells. LTB4 was also stained in some vascular endothelial cells and a few polymorphonuclear cells in the lamina propria. LTA4 hydrolase was similarly stained in these cells. These results suggest that parietal, surface epithelial, and vascular endothelial cells are the cells that synthesize LTB4 in the human gastric mucosa. Polymorphonuclear cells might also synthesize a smaller amount of LTB4.

Biopsy↗
PubMed1992

Circular dichroism of reduced and oxidized recombinant human epidermal growth factor.

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mM sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of alpha-helix, beta-sheet, and unordered structure. The addition of the alpha-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of alpha-helix, at the apparent expense of beta-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.

Circular Dichroism↗
PubMed1992

[Extracorporeal shock wave lithotripsy of staghorn stones in solitary kidney].

Extracorporeal shock wave lithotripsy (ESWL) has been established as noninvasive treatment of choice for the great majority of upper urinary tract stones. However the management of staghorn stones in solitary kidney still deserves special consideration. We reviewed retrospectively 11 patients with staghorn stones in solitary kidney treated mainly with the Dornier HM-3 lithotripter during the period between December 1984 and December 1989 at the Sagamidai Hospital. The contralateral kidneys of the 8 patients were nephrectomized or nonfunctioning due to stone disease and those of 3 patients were nephrectomized due to tuberculosis. They were consisted of 6 males and 5 females with average ages of 60.0 years and 48.6 years respectively. The size of the stones ranged from 30 x 30 mm to 85 x 40 mm in KUB. ESWL was the first treatment for all the cases except for one patient a cystine stone. In that patient, ESWL was preceded by percutaneous nephrolithotripsy (PNL). In three patients the treatment was successfully by ESWL alone without any obvious complication. In 6 patients percutaneous nephrostomy was required and in two patients PNL was performed as an auxiliary procedure. Seven patients developed high fever (over 38.5 degrees C) and two of them became septic during the course of treatment. In five patients serum creatinine elevated over 2.0 mg/dl, but returned to within normal limits postoperatively. Extracorporeal shock wave lithotripsy can play a major role in the patient of the staghorn stones in solitary kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Effects of active oxygen species on damage to and prostaglandin synthesis in cultured rat gastric cells.

Active oxygen species cause gastric mucosal damage in vivo. However, it is not known if these species are directly cytotoxic toward gastric cells. Prostaglandins have important physiological roles in the gastric mucosa, including direct cell protection against damaging factors. So, to find if active oxygen species affect prostaglandin synthesis in gastric mucosal cells is important, but this also is not known. This study was done to investigate the effects of such species on damage to and prostaglandin synthesis in cultured mucus-producing cells from rat gastric mucosa. Active oxygen species were produced by the addition of xanthine and xanthine oxidase to the culture medium. Cytotoxicity was assayed by 51Cr release. Xanthine (1 mM) and xanthine oxidase (100 mU/ml) increased specific 51Cr release as the thiobarbituric acid reactants increased. This increase in 51Cr release was inhibited by catalase, a scavenger of hydrogen peroxide, or dimethyl sulfoxide, a scavenger of hydroxyl radicals, but not by superoxide dismutase, a scavenger of superoxide, nor deferoxamine, an inhibitor of hydroxyl radical generation. Catalase, dimethyl sulfoxide, and superoxide dismutase each had no effect on prostaglandin E2 synthesis when xanthine and xanthine oxidase were not added. In the presence of xanthine and xanthine oxidase, catalase and dimethyl sulfoxide stimulated the synthesis of prostaglandin E2 and superoxide dismutase inhibited it. Indomethacin, a prostaglandin synthetase inhibitor, did not affect the decrease in 51Cr release caused by catalase in the presence of xanthine and xanthine oxidase, but it abolished the decrease caused by dimethyl sulfoxide. These results suggest that hydrogen peroxide, but not superoxide nor hydroxyl radicals, is involved in damage to cultured rat gastric cells, and that superoxide stimulates prostaglandin E2 synthesis, but that hydrogen peroxide inhibits it. Protection of the cells by dimethyl sulfoxide may be related to stimulation of prostaglandin E2 synthesis in the cells, but not via scavenging hydroxyl radicals.

Animals↗
PubMed1992

[H2-receptor antagonist-refractory ulcer--its pathophysiology and role of proton-pump inhibitors].

Among H2-receptor antagonist (H2RA)-refractory ulcers, non-responders that did not heal after 5 months therapy had high intraluminal pH in the basal condition and high sensitivity to inhibition of acid secretion by H2RA but possessed gastric mucosa to generate less prostaglandins. Combination therapy of PGE1-analogue with H2RA healed these ulcers by 60%. Proton-pump inhibitor (PPI) exerted a complete inhibition of acid secretion in these patients and the rate of healing was 88%. Helicobacter pylori was present in the mucosa of all 4 ulcer patients refractory to treatment with PPI. The ulcers healed in 3 out of 4 patients after eradication of H. pylori. It is suggested that PG supplement or complete inhibition of acid secretion is effective for ulcers in H2RA-non-responders. PPI-refractory ulcers may relate to H. pylori infection.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1992

Refractory peptic ulcers.

Ulcers that do not heal after 8 weeks of treatment with standard-dose regimens of antiulcer drugs are considered refractory. Incidences of duodenal and gastric ulcers refractory to H2-receptor antagonists are 9% and 20%, respectively. When treated with a single daily dose of omeprazole 20 mg, duodenal ulcers have a refractory incidence of 3% and gastric ulcers, 11%. Omeprazole's benefit may result from its potent gastric antisecretory action. Acid hypersecretion is an important pathophysiologic factor associated with refractoriness to H2 antagonists. Some patients with refractory ulcer, however, have a normal pharmacologic response of decreasing intragastric acidity following administration of H2 antagonists. Two possible mechanisms may explain refractoriness in such cases: the relative preservation of daytime acidity, which is the usual pharmacologic response to standard doses of H2 antagonists, and excessive impairment of mucosal defense. The first possibility is consistent with the results that increased doses of H2 antagonists or more potent antisecretory drugs such as omeprazole do heal a subgroup of ulcers refractory to H2 antagonists. The second possibility is supported by reports that drugs that mainly enhance mucosal defense, such as misoprostol, sucralfate, and bismuth compounds, also effectively heal refractory ulcers, and that gastrointestinal prostaglandin levels are extremely low in the mucosa of such patients. Other reasons for refractoriness to omeprazole treatment include gastric hyperacidity and impaired gastric emptying that may disturb drug absorption. Infection with Helicobacter pylori might, to some extent, be involved in refractoriness to potent antisecretory drugs. Single daily doses of omeprazole 40 mg seem superior to omeprazole 20 mg or increased doses of H2 antagonists for maintenance therapy of H2 antagonist-refractory ulcers.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Ulcer Agents↗