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T Ohta

Publications and source records attributed to T Ohta.

At least 145 records · Page 8Linked to original sources
PubMed2000

Proteasome-dependent degradation of cytosolic chaperonin CCT.

The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We show here that degradation of CCT in mammalian cells is inhibited by a proteasome-specific inhibitor, lactacystin. When CCT synthesis was inhibited by growth arrest of cells, the decrease in CCT levels was much slower in the presence of lactacystin than in its absence. Pulse-chase experiments indicated that degradation of CCT is inhibited 2- to 2.5-fold by addition of lactacystin. In addition, CCT degradation rate in ts85 cells that produce thermolabile ubiquitin-activating enzyme E1 was reduced 3-fold at the nonpermissive temperature compared to the degradation at the permissive temperature. These results indicate that the ubiquitin-proteasome system is involved in CCT degradation.

Acetylcysteine↗
PubMed2000

Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.

Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Mechanisms of molecular evolution.

Both drift and selection are important for nucleotide substitutions in evolution. The nearly neutral theory was developed to clarify the effects of these processes. In this article, the nearly neutral theory is presented with special reference to the nature of weak selection. The mean selection coefficient is negative, and the variance is dependent on the environmental diversity. Some facts relating to the theory are reviewed. As well as nucleotide substitutions, illegitimate recombination events such as duplications, deletions and gene conversions leave indelible marks on molecular evolution. Gene duplication and conversion are sources of the evolution of new gene functions. Positive selection is necessary for the evolution of novel functions. However, many examples of current gene families suggest that both drift and selection are at work on their evolution.

Animals↗
PubMed2000

Excision repair of uracil-containing DNA in a mitochondrial extract of Xenopus oocytes.

A mitochondrial extract of Xenopus oocytes was prepared to characterize DNA repair reactions operating in mitochondria. We asked whether and how uracils, spontaneously formed in DNA as a result of cytosine deamination, would be repaired in the extract. When a 40-mer oligonucleotide duplex with a single deoxyuridine at position 21 was incubated in the extract, incision took place at the 5' side of the lesion, and the uracil-containing strand was mostly cleaved within 15 min. Subsequent repair DNA synthesis produced DNA fragments of various sizes. Analysis of these repair intermediates indicated that the 3' border of the repair patches was at position 22. Completion of the repair reaction however was rare in this system, due in part to insufficient ligation activity of the extract.

Animals↗
PubMed2000

Exploring the structural basis of neurotoxicity in C(17)-polyacetylenes isolated from water hemlock.

Water hemlock, Cicuta virosa, belonging to the Umbelliferae, is well-known as a toxic plant responsible for lethal poisonings in humans as well as animals, causing tonic and clonic convulsions and respiratory paralysis. Cicutoxin (1), being a major violent toxin of the plant, is a chemical in the class of C(17)-polyacetylenes bearing a long pi-bond conjugation system, a terminal hydroxyl, and an allylic hydroxyl in its structure, and a variety of its analogues have been isolated from the plant. In the present study, various derivatives of these toxins were synthesized through acetylation, methylation, and oxidation of cicutoxin (1) and virol A (3) and B (4). 1-Dehydroxyvirol A (28) was prepared through the coupling of (7S)-dodeca-3,5-dien-1-yn-7-ol and 1-iodopentyne under Sonogashira's conditions. A monoacetylenic compound (29) was also prepared through the coupling of (5S)-1-chlorodeca-1,3-dien-5-ol and 1-iodopentyn-5-ol. The structure-activity relationships involved in the acute toxicity of cicutoxin derivatives in mice were investigated, and the length and geometry of pi-bond conjugation and the O-functional groups were found to be important for activity. The potency in inhibition of the specific binding of the noncompetitive GABA antagonist, [(3)H]EBOB, to GABA-gated Cl(-) channels of GABA receptors in rat brain cortex was found to be correlated with acute toxicity, indicating that the ability to bind to these channels plays an important role in the acute toxicity of these compounds.

Acetylene↗
PubMed2000

cDNA cloning and characterization of vascular apoptosis-inducing protein 1.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.

Amino Acid Sequence↗
PubMed2000

Vacuolar H(+)-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts in cultured human saphenous vein.

BACKGROUND: The molecular mechanism of neointimal hyperplasia after vein graft surgery remains elusive. Vacuolar H(+)-ATPase (V-ATPase) is involved in intracellular trafficking and may play a crucial role in neointimal cell growth. METHODS AND RESULTS: Cultured human saphenous vein segments developed neointimal formation within 10 days. Neointimal cells were positive for vimentin and alpha-smooth muscle actin but negative for desmin, which is indicative of myofibroblasts. Those myofibroblasts were found to have originated from periadventitial fibroblasts, which upregulated the expression of 16-kDa proteolipid of V-ATPase before proliferation and phenotypic modulation. Neointimal myofibroblast growth and survival were highly sensitive to inhibition of V-ATPase by bafilomycin A(1) (BA(1)), because the incorporation of [(3)H]thymidine into the myofibroblasts was significantly inhibited by nanomolar concentrations of BA(1) and apoptotic cell death was induced by a similar concentration range of BA(1). In contrast, endothelial cells and differentiated smooth muscle cells were resistant to apoptosis by BA(1). CONCLUSIONS: These results suggest that V-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts that contributes to neointimal formation in cultured human saphenous vein.

Adult↗
PubMed2000

Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes.

A disulfide-bridged dicopper(I) complex, [Cu2(Py2SSPy2)](ClO4)2 (1) (Py2SSPy2 = bis(2-[N,N-bis(2-pyridylethyl)-amino]-1,1- dimethylethyl)disulfide), a thioether-copper(I) complex, [Cu(iPrSPy2)](ClO4) (2) (iPrSPy2 = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine, and a thioether-copper(II) complex, [Cu-(PheSPy2)(H2O)](ClO4)2 (3) (PheSPy2 = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2- pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)2.6H2O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)-ethyl]-1,1-dimethyl-2- mercaptoethylamine) and thioether ligands of iPrSPy2 and PheSPy2, respectively. For complexes 1 and 2, X-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P1, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a = 15.165 (3) A, b = 22.185 (4) A, c = 14.989 (3) A, alpha = 105.76 (1) degrees, beta = 90.82 (2) degrees, gamma = 75.23 (1) degrees, and Z = 2; for 2, a = 17.78 (2) A, b = 17.70 (1) A, c = 15.75 (1) A, and Z = 8. Complex 1 is the first structurally characterized example obtained by the redox reaction Cu(II) + RSH-->Cu(I) + RSSR and has two independent structures (1a, 1b) which mainly differ in S-S bond distances, Cu(I)...Cu(I) separations, and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) A in 1a and 2.070(7) A in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment molecular orbital (FMO) analyses and molecular orbital overlap population (MOOP) analyses based on the extended Hückel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-copper(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were investigated with peroxides (H2O2 and tBuOOH) as oxidants. Complex 1 catalyzed the selective oxidation of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidation in the presence of H2O2. A transient dark green intermediate observed in the reaction of 1 with H2O2 is characterized by UV-vis, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the nu(O-O) bands at 822 and 836 cm-1, which are red-shifted to 781 and 791 cm-1, respectively, upon introduction of H2(18)O2, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for one-electron oxidation, which has been reported so far unfavorable for other vibrationally characterized Cu-OOH species.

Journal Article↗
PubMed2000

TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor.

Amino Acid Sequence↗
PubMed2000

Inhibitory effect of a prostaglandin E receptor subtype EP(1) selective antagonist, ONO-8713, on development of azoxymethane-induced aberrant crypt foci in mice.

We previously reported that prostaglandin E(2) contributes to colon carcinogenesis through its binding to the prostaglandin E receptor subtype EP(1) using a genetic approach in EP(1)-knockout mice and a pharmacological approach with the EP(1) selective antagonist, ONO-8711. In the present study, we examined the effects of another more selective EP(1) receptor antagonist, ONO-8713, on development of azoxymethane (AOM)-induced aberrant crypt foci (ACFs) in male C57BL/6J mice treated i.p. with 10mg/kg body weight AOM once a week for 3weeks. Administration of ONO-8713 at doses of 250, 500 and 1000ppm in diet during and post-AOM treatment for 5weeks resulted in a dose-dependent reduction of ACF formation, being 15, 30 and 36% inhibition of the control value, respectively. The level of inhibition was similar to that with ONO-8711. Moreover, ONO-8713 suppressed the development of ACF when administered at post-AOM, as in the case of ONO-8711. The data confirm EP(1) receptor involvement in colon carcinogenesis.

Animals↗
PubMed2000

Reinterpretation of the molecular O2 chemisorbate in the initial oxidation of the si(111)7 x 7 surface.

The initial oxygen adsorption on the Si(111)7 x 7 surface was investigated by high-resolution x-ray absorption spectroscopy. Below 220 K, a molecular adsorption species is identified by distinctive absorption resonances due to the 1 pi(g) molecular orbitals. The molecular species is metastabilized to have a lifetime of 15-35 min at 135 K only with the presence of atomic adsorbates of more than 0. 1 ML (monolayer). It is thus clearly evidenced that the very initial adsorption is dissociative even at 100 K and the molecular species is not a precursor state. The molecular adsorption structures with the coadsorbed oxygen atoms are suggested.

Journal Article↗
PubMed2000

Inhibition selectivity of grapefruit juice components on human cytochromes P450.

Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19 (K(i) = 0.8 and 0.5 microM, respectively).

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135.

A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled-coil protein with an extensive alpha-helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA- or GFP-tagged full (amino acids 1-1144) as well as truncated (#10, 29-1144; Delta3, 29-812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive approximately 7 nm filaments and dense particles organized in a whorl-like arrangement in which parallel-oriented dense lines appear with a regular approximately 7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher-ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Delta3-containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers ( approximately 120 nm), the dense stripe of Delta3 polymers ( approximately 40 nm) is wider than #10 ( approximately 30 nm). The light band of Delta3 ( approximately 40 nm) is thus narrower than #10 ( approximately 60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled-coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain-specific manner.

Animals↗
PubMed2000

Allopurinol induces renal toxicity by impairing pyrimidine metabolism in mice.

We investigated the relationship between the toxic effect of allopurinol and pyrimidine metabolism in mice. Allopurinol-induced increases in plasma transaminase levels in dinitrofluorobenzene (DNFB)-sensitized mice were not affected by uridine. In contrast, plasma creatinine and BUN tended to decrease 18 hr after the last injection of uridine. Both plasma and urinary orotidine (OD) were detected in DNFB-sensitized mice after administration of a single dose of allopurinol. In contrast, TEI-6720, a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, caused neither pyrimidine metabolism abnormality nor renal impairment in DNFB-sensitized mice. Also, normal mice administered high doses of allopurinol showed abnormal pyrimidine metabolism together with renal toxicity which could be ameliorated by uridine, indicating that allopurinol essentially causes pyrimidine metabolism abnormality leading to renal impairment. In DNFB-sensitized mice, allopurinol increased urinary OD excretion to an extent similar to that in normal mice administered the same dose of allopurinol. However, renal impairment by allopurinol was more striking in DNFB-sensitized mice than in normal mice. Histopathological observations showed that allopurinol induced calculus formation in the collecting tubules and papillary duct. Calculus formation was increased by DNFB and decreased by uridine. These observations indicate that the enhancement of the renal toxicity of allopurinol by DNFB-sensitization may be due to some biological interactions between DNFB and allopurinol. In humans, it is possible that there are some biological interactions which serve to enhance the toxicity of allopurinol, resulting in the development of allopurinol hypersensitivity syndrome (AHS). In contrast, TEI-6720, had no effect on pyrimidine metabolism and showed no toxic effect.

Allopurinol↗
PubMed2000

Synthesis and antifungal activity of rhodopeptin analogues. 1. Modification of the east and south amino acid moieties.

Structure-activity relationships of the east and south amino acid modified analogues of rhodopeptins, novel antifungal cyclic tetrapeptides isolated from Rhodococcus species Mer-N1033, have been investigated. It was observed that a basic amino acid moiety (lysine or ornithine) as the east amino acid and a hydrophobic and bulky neutral amino acid (i.e., gamma-methylleucine) as the south amino acid were indispensable structure motifs for antifungal activity of rhodopeptin analogues.

Amino Acids↗
PubMed2000

Alteration of beta-catenin expression in esophageal squamous-cell carcinoma.

beta-catenin regulates cadherin-mediated cell-cell adhesion and also functions as a signaling molecule. In this study, we examined the expression pattern of E-cadherin, alpha-catenin and beta-catenin in 22 cases of esophageal squamous-cell carcinoma by Western-blot analysis. Expression of E-cadherin, alpha-catenin and beta-catenin was lower in carcinomas than in normal esophageal mucosa in 4 cases (18.2%) for E-cadherin, 6 cases (27.3%) for alpha-catenin and 9 cases (40.9%) for beta-catenin. Expression of beta-catenin was not always correlated with that of E-cadherin. Over-expression of beta-catenin was observed in 3 cases (13.6%). Of 3 cases that presented with over-expression of beta-catenin, 2 showed cytoplasmic staining by immunohistochemistry. Nuclear localization of beta-catenin was observed in one case that had higher beta-catenin level in tumor tissue (1.4-fold higher than normal mucosa). The genomic DNA sequences of the beta-catenin and the APC gene were analyzed. No mutation of the beta-catenin gene was observed in any cases. Silent mutation of the APC gene was found in all the cases that showed over-expression or nuclear localization of the beta-catenin protein. These results indicate that alterations of the cadherin-catenin complex may play an important role in a sub-set of esophageal carcinogenesis. Furthermore, it is suggested that beta-catenin over-expression is not caused by genetic alteration of either the beta-catenin or the APC gene.

Blotting, Western↗