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Biomedical subjects

T Ishibashi

Publications and source records attributed to T Ishibashi.

At least 19 recordsLinked to original sources
PubMed1992

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.

Anilino Naphthalenesulfonates↗
PubMed1992

Expression cloning of a human dual-specificity phosphatase.

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

Amino Acid Sequence↗
PubMed1992

Identification of the fatty acid binding site on glutathione S-transferase P.

Glutathione S-transferase P (GST-P) bound a series of endogenous fatty acids (C12-C18). To clarify the function and the binding site of the fatty acids, interaction between fatty acids and GST-P was investigated by using 12-(9-anthroyloxy) stearic acid conjugated with Woodward's reagent K. The fluorescence-conjugated fatty acid noncompetitively inhibited GST activity. After GST-P was covalently labeled with the fatty acid, the enzyme was digested with Lysyl Endopeptidase. From the peptide mapping, a single fluorescence-labeled peptide was obtained. By the sequence analysis, the peptide binding fatty acid was determined as the residues of 141-188 from the amino terminus.

Affinity Labels↗
PubMed1992

Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.

Amino Acid Sequence↗
PubMed1992

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.

Amino Acid Sequence↗
PubMed1992

Analysis of the defective signal transduction mechanism through the platelet thromboxane A2 receptor in a patient with polycythemia vera.

We previously reported a patient with polycythemia vera whose platelets showed subnormal responses to thromboxane A2 (TXA2) (Thromb Haemostas 1987; 57: 158-64). The patient's platelets showed normal binding activity to TXA2 analogues but the generation of second messengers was defective. We further studied the mechanism of this dysfunction in this work. The coupling of TXA2 receptor with its relevant GTP binding protein was examined using STA2(a synthetic TXA2 mimetic)-induced augmentation of GTPase activity as its measure. Only subnormal increase in the GTPase activity of the patient's platelet membrane was found after STA2 stimulation. Down regulation of TXA2 receptor of the patient's platelet also showed abnormal behavior. These results suggested that the defective coupling of TXA2 receptor with GTP binding protein could be included as a cause of defective signal transduction in this patient's platelets. This defect might be due to the abnormality of TXA2 receptor itself if the receptor was down regulated by its phosphorylation through agonist-induced conformational change.

Blood Platelets↗
PubMed1992

Maturation of newly-formed subretinal vessels.

The maturation process of newly-formed subretinal vessels was studied ultrastructurally. Primitive new vessels have thick endothelial cells with cytoplasm that contains many ribosomes and rough endoplasmic reticulum. Some immature endothelial cells show budding, and many of the intercellular junctions have small gaps. As the newly-formed vessels mature, the endothelial cells become attenuated and fenestrated, and their junctions become tight. Mature subretinal new vessels closely resemble those of the normal choriocapillaris.

Animals↗
PubMed1992

Effects of sodium nitroprusside (MR7S1) and nitroglycerin on the systemic, renal, cerebral, and coronary circulation of dogs anesthetized with enflurane.

In beagle dogs anesthetized with enflurane-nitrous oxide, effects of sodium nitroprusside (SNP; MR7S1) and nitroglycerin (NTG) on hemodynamics and main organ circulation were studied to evaluate their effectiveness and safety as hypotensive agents during anesthesia. SNP (MR7S1) infusion (1-10 micrograms/kg/min) decreased arterial blood pressure in a dose-dependent manner. The hypotension was stable during the infusion. After discontinuation of infusion, the blood pressure rapidly returned to the initial level. The hypotension was associated with decreases in cardiac output and total peripheral resistance. NTG infusion (3-10 micrograms/kg/min) decreased arterial blood pressure, too, but the hypotension was less marked and not dose dependent, and the recovery was slower. Neither drug changed the heart rate. Infusion of SNP (MR7S1) and NTG did not change the hypotension induced by the injection of adenosine, SNP, and NTG. Furthermore, cerebral blood flow, cerebral oxygen consumption, and renal blood flow were unchanged during the hypotension produced by either drug. Coronary blood flow was decreased, but this was due to decreases in cardiac oxygen consumption. In conclusion, SNP (MR7S1) is superior to NTG as a hypotensive agent during anesthesia in efficacy, clear dose dependency, and rapid recovery. The hypotension induced by NTG as well as SNP (MR7S1) seems to have no undesirable effects on the circulation of important organs.

Anesthesia↗
PubMed1992

Vasodilating properties of KRN2391: structural basis of a new pyridine-type potassium channel opener with a nitrate moiety.

The vasodilating mechanism of a new compound, cyanoimino-3-pyridylmethylaminoethyl nitrate methanesulfonate (KRN2391), a derivative of nicorandil, was examined in the isolated rabbit aorta. To elucidate the structure activity relationship, a comparison was made with the two denitrated derivatives: cyanoimino-3-pyridylmethylaminoethyl acetate methanesulfonate (Ki4032) and cyanoimino-3-pyridylmethylaminoethyl alcohol (Ki3315). In preparations precontracted with phenylephrine (10(-7) mol/l), KRN2391, Ki4032 and Ki3315 caused concentration-dependent relaxation. pD2 values (-log [EC50]) were 6.74 +/- 0.03, 5.67 +/- 0.05 and 3.63 +/- 0.03, respectively. Both methylene blue and glibenclamide produced a shift to the right of the concentration-response curves for KRN2391. The shift by glibenclamide became greater in the presence of methylene blue. An elevation of the cGMP content was not detected until the concentration of KRN2391 was increased to a level enough to produce a full relaxation (3 x 10(-6) mol/l). In contrast, in the case of Ki3315 a parallel shift to the right of the concentration-response curve was observed after glibenclamide (10(-5) mol/l). Methylene blue (10(-5) mol/l) had no effect on the concentration-response curve, and there was no increase in cyclic GMP (cGMP) with 10(-3) mol/l of the compound. The concentration-response curve of Ki4032 was also attenuated by glibenclamide. Though this compound lacks the nitrate moiety, it (10(-4) mol/l) showed a slight tendency to increase the cGMP content, and methylene blue slightly but significantly modified the concentration-response curve of this compound. However, the co-administration of glibenclamide and methylene blue resulted in no further modification of the concentration-relaxation curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Immunohistochemical detection of extravasated fibrinogen (fibrin) in human diabetic retina.

In diabetic retinopathy, breakdown of the blood-retinal barrier is an early functional disorder that can cause retinal edema, which in turn results in visual disturbance. Hard exudates, composed mainly of lipid and proteinaceous material, are one sign of chronic retinal edema caused by long-standing leakage from the vessels due to breakdown of the blood-retinal barrier. Utilizing diabetic retinas in which hard exudates were present, we performed immunohistochemical staining for fibrinogen. Because fibrinogen is a serum protein, its extravascular localization implies the existence of blood-retinal barrier breakdown. Our studies showed that the extravasated fibrinogen from blood-retinal barrier breakdown accumulated in the hard exudates and in areas of hemorrhage found primarily in the outer plexiform layer and was then phagocytosed by macrophages.

Aged↗
PubMed1992

An in vitro reversible interconversion of rat liver fatty acid binding protein having different isoelectric points by virtue of the fatty acid content.

Rat liver fatty acid binding protein (FABP) was purified to homogeneity by procedures including Sephadex G-100 and DEAE-cellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these two fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions has revealed that they are virtually identical or closely resemble each other. However, their fatty acid content was significantly different and heterogeneity was clearly demonstrated in the patterns of isoelectric focusing. In this communication, a single isoform (pI 5.0) from peak B FABP was further purified by successive DEAE-cellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining fatty acids. These in vitro manipulations of bound fatty acid content made clear its possible cause of the microheterogeneity of FABP.

Amino Acids↗
PubMed1992

Effusions after anterior cruciate ligament reconstruction using the ligament augmentation device.

Persistent effusions occurred in 4.3% (seven patients) of 164 patients who underwent anterior cruciate ligament (ACL) reconstruction using an autograft augmented with the ligament augmentation device (LAD) between 1986 and 1989. No bacteria were cultured from the joint fluid. The radiograph in each case showed a tibial drill hole located anteriorly or anterolaterally. Arthroscopy confirmed an abraded LAD in six cases and breakage of the LAD in one case and impingement of the graft on the femoral condyle during extension. Under polarized light microscopy, the histology of the synovia showed foreign body giant cells in one case and particles among the proliferative cells in four cases. The effusions subsided in two cases after notchplasty and in five cases after removal of the LAD. Impingement of the graft on the femoral condyle due to incorrect positioning of the tibial drill hole presumably produced particles that induced synovitis and effusions.

Adolescent↗
PubMed1992

Localization of proteins immunologically related to erythrocyte protein 4.1, spectrin and ankyrin in thyroid gland.

Analogues of erythrocyte protein 4.1, spectrin and ankyrin were examined in the thyroid gland of pig and rat by immunohistochemical techniques. Analysis with immunofluorescence microscopy revealed that the peripheral cytoplasm and apical-lateral plasma membrane of follicle epithelial cells of thyroid glands were stained with antibodies against erythrocyte protein 4.1, spectrin, or ankyrin. The results indicate that membrane skeletal protein lattice might exist in thyroid follicle epithelial cells.

Animals↗
PubMed1992

A case of amniotic band syndrome with bilateral epibulbar choristoma.

An autopsy case of amniotic band syndrome with bilateral epibulbar choristoma is described. The left eye reveals a complex choristoma and the right eye a dermis-like choristoma. Both choristomatous lesions included lenticular tissue suggesting that rupture of the amnion, which is the initial event of amniotic band syndrome, might have occurred at about the fourth week of gestation. Since the other systemic manifestations of amniotic band syndrome are considered to be compression deformities of the fetus caused by oligohydramnios or amniotic band, the occurrence of epibulbar choristomas in both eyes in this case suggests that a compression mechanism may play a role in the pathogenesis of epibulbar choristoma.

Adult↗
PubMed1992

Ibudilast may improve retinal circulation in patients with diabetes mellitus.

Ibudilast selectively vasodilates cerebral vessels without reducing blood pressure. We investigated the effect of the drug on retinal circulation in 8 patients with diabetes mellitus, using the video-densitometric image analysis of fluorescein angiography. We compared the build up time, the time constant of washout rate and the mean circulation time (MCT) before and after oral therapy with ibudilast. After 2 weeks of daily administration (30 mg), MCT was shortened significantly (4.2 +/- 2.8 vs. 3.0 +/- 1.6 s, p = 0.0215). Since retinal circulation in patients with diabetes mellitus was improved by ibudilast, the drug may be useful to treat disorders such as diabetic retinopathy.

Administration, Oral↗
PubMed1992

Morphological changes of epiphyseal plate in the long bone of chondrodysplastic dwarfism in Japanese brown cattle.

A total of 20 cases of disproportionate calves on Japanese Brown cattle were examined morphologically. Investigation of 5 affected calves revealed that the insufficiency of endochondral ossification was confined only to the long bones of the limbs and was not accompanied by other associated changes. On the basis of the histological changes of epiphyseal plate and the affected site, this condition may be called chondrodysplasia of the rhizomelic type. Therefore, this disorder was defined as a bovine dwarfism. From three dimensional image analysis and histological changes of the tibial proximal portion in the other 15 cases, deformity and shortness of the tibia were related to the state of distribution and the degree of damages of the epiphyseal plate. In the affected cartilage matrix, cystoid degeneration, fibrous striation, bone spicule, necrotic foci and rent were found. Inadequate metachromasia suggests the abnormal staining of sulfated glycosaminoglycans and alcian blue stainability may be attributable to the abnormal interactions between proteoglycan and other matrix components. At the interface of cartilage and bone, irregular calcification, fibrotic scar and sealing by osseous tissue with relation to vascularity were found. These changes in this study indicated the failure of modeling on the epiphyseal plate, showing disturbance of chondrocytic differentiation and abnormal formation of the matrix.

Animals↗