Biomedical subjects

T Tsuchiya

Publications and source records attributed to T Tsuchiya.

At least 181 records · Page 10Linked to original sources

PubMed1999

Geranylgeranylacetone suppresses spontaneous apoptotic DNA fragmentation in cultured guinea pig gastric pit cells.

We recently found that guinea pig gastric pit cells in culture undergo spontaneous and rapid apoptotic DNA fragmentation, which may represent the rapid death of gastric pit cells in vivo. In this study, we observed that pretreatment of cells with geranylgeranylacetone, an antiulcer drug with heat-shock protein-inducing activity, suppressed the spontaneous apoptotic DNA fragmentation in a dose-dependent manner. Pretreatment of cells with low concentrations of ethanol or heat-shock also prevented the spontaneous apoptotic DNA fragmentation. These observations indicate that the suppression of the apoptotic DNA fragmentation by geranylgeranylacetone involve the induction of heat-shock proteins.

Animals↗

PubMed1999

Identification of a high-copy-number plasmid suppressor of a lethal phenotype caused by mutant DnaA protein which has decreased intrinsic ATPase activity.

The induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells causes a lethal phenotype. Based on our results that the decreased ATPase activity of DnaA E204Q is activated to the level of a wild-type protein in the presence of partially purified stimulating factors for DnaA ATPase, we tested the hypothesis that genes encoding the stimulating factors can be cloned as high-copy-number plasmid suppressors for the lethality caused by DnaA E204Q. We isolated a number of high copy-number plasmids that suppress the lethal phenotype. The genes responsible for the suppression of the lethal phenotype were revealed to be dnaN, relA, pcnB and cyaA by subcloning, and a site-directed mutational analysis. The mechanisms by which these genes suppressed the lethal phenotype were examined in vivo and in vitro.

Adenosine Triphosphatases↗

PubMed1999

Phenolic constituents of Cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant Staphylococcus aureus.

Thirteen phenolic glycosides including six new compounds were isolated from seeds of Cassia tora (Leguminosae). The structures of the new compounds, rubrofusarin triglucoside (7), nor-rubrofusarin gentiobioside (9), demethylflavasperone gentiobioside (10), torachrysone gentiobioside (11), torachrysone tetraglucoside (12) and torachrysone apioglucoside (13), were elucidated on the basis of spectroscopic and chemical evidence. The effects of the phenolic glycosides, their aglycones and several other compounds structurally related to them on Escherichia coli K12, Pseudomonas aeruginosa PAO1 and some strains of Staphylococcus aureus were then examined. Among them, torachrysone (15), toralactone (16), aloe-emodin (18), rhein (19) and emodin (20) showed noticeable antibacterial effects on four strains of methicillin-resistant Staphylococcus aureus with a minimum inhibitory concentration of 2-64 micrograms/ml. On the other hand, the phenolic compounds tested did not show strong antibacterial effects on E. coli and P. aeruginosa.

Anthraquinones↗

PubMed1999

Non-suture end-to-end anastomoses between polytetrafluoroethylene graft and vessels for blood access.

BACKGROUND: Non-suture end-to-end anastomoses between polytetrafluoroethylene grafts and blood vessels were achieved using absorbable cuff material in experimental and clinical studies. The cuff was made of a synthetic biodegradable material, a lactic-glycolic acid copolymer, similar in composition to conventional absorbable surgical sutures. METHODS: In the experimental study, vascular anastomoses for prosthetic interposition of the infrarenal aorta in rabbits were created using the cuff method. Six months after surgery, the cuff anastomoses sites were examined angiographically and microscopically and found to be patent and smooth without neointimal hyperplasia. RESULTS: The cuff layer had been completely absorbed. The clinical application used a similar technique and involved the creation of forearm bridge graft fistula in twelve patients for hemodialysis. In eight patients, Doppler fistula flow rate ranged from 167 ml/min to 392 ml/min. Ten of the twelve patients continued dialysis uneventfully. The one-year patency rate was 78% (7/9). The longest patency period was 920 days and the graft access continued to maintain sufficient blood flow for hemodialysis. CONCLUSIONS: This absorbable cuff material is therefore well suited for the construction of prosthetic vascular end-to-end anastomoses.

Aged↗

PubMed1999

[A case of pulmonary alveolar proteinosis presenting with peripheral infiltrates].

We report a case of pulmonary alveolar proteinosis (PAP). A 39-year-old asymptomatic woman was admitted to our hospital because of abnormal shadows on chest X-ray films. Chest X-ray films revealed peripheral infiltrates in both lungs. Computed tomographic examination showed patchy peripheral ground-glass attenuation, concentrated subpleurally. Bronchoalveolar lavage fluid was clear. Because transbronchial lung biopsy findings were inconclusive, a VATS-biopsy was performed. The specimens demonstrated accumulation of proteinaceous materials within alveolar spaces. The patient was given a diagnosis of PAP. Although the distribution of radiographic shadows varies in patients with PAP, perihilar or centralized shadows usually predominate. In our patient, subpleural areas of the lung were affected almost exclusively.

Adult↗

PubMed1999

[PIVKA-II].

Explore the source record for details and available documents.

Biomarkers↗

PubMed1998

Frequent aberration of FHIT gene expression in acute leukemias.

We analyzed the mRNA expression of the FHIT gene by reverse transcription-PCR (RT-PCR) in 54 cases of acute lymphoblastic leukemia (ALL; 11 cases of T-cell ALL [T-ALL] and 43 cases of non-T-ALL) and 40 cases of acute myeloid leukemia (AML). In 46% of the ALL cases and 55% of the AML cases, FHIT expression was absent or markedly decreased. Only abnormal short bands were detected in 30% of the ALL cases and 5% of the AML cases. Eighteen of 19 abnormal transcripts had the same fusion of exons 2-7, and all lacked the starting codon in exon 5. No obvious normal-sized PCR products were detected in cases exhibiting abnormal transcripts. These findings suggest that the expression of functional FHIT protein was lost in the majority of ALL (76%) and AML (60%) cases. Differential quantitative PCR of exons 3-9 of the FHIT gene and RT-PCR of the PTPRG gene, which is centromeric to the FHIT gene, showed the presence of the target sequences. Fluorescence in situ hybridization analysis using probes covering exons 5 and 8 revealed no difference in the signal patterns between leukemia and normal cells, showing one or two signal doublets in more than 90% of nuclei, and indicated that gross segments of the FHIT gene were not homozygously deleted in these cases. A small number of transcripts with an aberrant fusion between exons 2 and 7 were detected by RT-PCR in the bone marrow cells from four healthy individuals. Granulocytes, lymphocytes, and monocytes in the bone marrow cells of a healthy individual contained transcripts with the same fusion. This unique fusion of exons 2 and 7 might be preferentially seen in either neoplastic or normal hematopoietic cells, regardless of their lineage. The finding that FHIT expression was abolished in the majority of leukemia cases might support the hypothesis that the FHIT gene acts as a tumor suppressor, at least in leukemia.

Acid Anhydride Hydrolases↗

PubMed1998

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.

Adenosine Diphosphate↗

PubMed1998

Effects of pilsicainide and propafenone on vagally induced atrial fibrillation: role of suppressant effect in conductivity.

The effects of pilsicainide on vagally induced atrial fibrillation and on electrophysiological parameters were compared with those of propafenone in alpha-chloralose-anesthetized dogs. Conduction velocity, effective refractory period, wavelength, averaged atrial fibrillation cycle length and activation sequence in the right atrial free wall were determined before and after drug administration. Pilsicainide (2 mg/kg/5 min and 3 mg/kg/h)(n=10) or propafenone (2 mg/kg/15 min and 4 mg/kg/h)(n=10) was intravenously infused during stable atrial fibrillation sustaining > 30 min. Pilsicainide terminated atrial fibrillation in nine dogs, while propafenone did so in three (p < 0.01). After the drug, conduction velocity was suppressed more in the pilsicainide than in the propafenone group(p < 0.01). There was no difference in effective refractory period after drug between the two groups. Mean wavelength was prolonged from 46.0 to 70.4 mm in the pilsicainide group and from 45.0 to 110.8 mm in the propafenone (p < 0.01 vs. pilsicainide). Activation mapping during atrial fibrillation showed Type II or III atrial fibrillation as previously defined [Konings, K.T.S., Kirchhof, C.J.H.J., Smeets, J.R.L.M., Wellens, H.J.J., Penn, O.C., Allessie, M.A., 1994. High-density mapping of electrically induced atrial fibrillation in humans. Circulation. Vol. 89, pp. 511-521.] before the drug, and changed to Type I before atrial fibrillation termination. Thus, pilsicainide was more effective to terminate vagally induced atrial fibrillation than was propafenone despite a greater effect of propafenone than of pilsicainide on wavelength. In this canine atrial fibrillation model, the suppression of conduction velocity may play an important role in changing the activation pattern of atrial fibrillation and thus, terminating atrial fibrillation.

Animals↗

PubMed1998

The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Animals↗

PubMed1998

Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.

Adenosine Triphosphate↗

PubMed1998

Identification of two essential arginine residues in UhpT, the sugar phosphate antiporter of Escherichia coli.

Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (>/=30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.

Amino Acid Sequence↗

PubMed1998

Cloning and sequencing of a novel Na+/H+ antiporter gene from Pseudomonas aeruginosa.

We cloned a gene for Na+/H+ antiporter from chromosomal DNA of Pseudomonas aeruginosa. Introduction of the gene into host Escherichia coli mutant cells lacking all of the major Na+/H+ antiporters enabled the cells to grow in the presence of 0.2 M NaCl, although the original host cells could not. Membrane vesicles prepared from cells of the transformant possessing the cloned gene showed Na+/H+ antiport activity. As a result of DNA sequencing, we found one open reading frame (nhaP). The deduced amino acid sequence suggests that the Na+/H+ antiporter (NhaP) of P. aeruginosa consists of 424 amino acid residues with molecular mass of 45486 Da, and hydropathy analysis suggested the presence of 12 putative transmembrane domains. We found no bacterial Na+/H+ antiporter which showed significant sequence similarity with the NhaP in the protein sequence database. The NhaP showed partial sequence similarity with animal Na+/H+ exchangers. Thus, the NhaP of P. aeruginosa is unique among bacterial antiporters.

Amino Acid Sequence↗

PubMed1998

Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the melibiose operon in Escherichia coli.

The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 degreesC. The reason for this temperature sensitivity was thought to be that the melibiose transporter (MelB) of W3133 cells was temperature-sensitive. A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize melibiose between 37 and 42 degreesC. However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the melibiose transporter at 37 degreesC (or 40 degreesC) in both E. coli W3133 and W3133-2 were exactly the same. Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the melibiose operon) of W3133-2 was exactly the same as that of W3133. Activity of alpha-galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 degreesC was very low, although that of W3133-2 cells grown at 40 degreesC was high. These observations suggested that expression of the melibiose operon in W3133 is also temperature-sensitive. In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method. Furthermore, we identified mutations in the promoter region of the melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the -35 region. This may stabilize a possible stem structure due to the inverted repeat at 37-42 degreesC.

Base Sequence↗

PubMed1998

Patch clamp studies on ion pumps of the cytoplasmic membrane of Escherichia coli. Formation, preparation, and utilization of giant vacuole-like structures consisting of everted cytoplasmic membrane.

Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H+ because of respiration and to ATP hydrolysis by the FoF1-ATPase. Such current was inhibited by inhibitors of the respiratory chain or FoF1-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.

Cytoplasm↗

PubMed1998

Identification of the hydrophobic amino acid residues required for heme assembly in the rhizobial oxygen sensor protein FixL.

Rhizobial FixL is a novel heme protein, which senses environmental oxygen tension and directs signal transduction via protein phosphotransfer. To identify the essential residues for heme assembly in Rhizobium meliloti FixL, we individually replaced the 18 invariant hydrophobic amino acid residues (F, I, L, and V) in the heme-containing domain with alanine and histidine. Spectroscopic measurements of the soluble fractions from fixL recombinant Escherichia coli revealed that V152, F162, F170, I172, L185, F226, L230, and F243 as well as the proximal ligand H194 were indispensable for heme assembly. Autoxidation rates of purified I209H, I210A, and I210H were 65-fold, 15-fold, and 15-fold, respectively, faster than that of the wild type, although they retained heme in the protein. The absorption peak in the Soret region of the ferric I209H or I210H was red-shifted, suggesting that the ferric heme is a hexa-coordinate form in these mutants.

Amino Acid Sequence↗

PubMed1998

A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus.

A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this antiporter. Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters.

Amino Acid Sequence↗

PubMed1998

Embryonal carcinoma P19 cells produce erythropoietin constitutively but express lactate dehydrogenase in an oxygen-dependent manner.

Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3' enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Base Sequence↗