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T Ohta

Publications and source records attributed to T Ohta.

At least 793 records · Page 44Linked to original sources
PubMed1993

Amino acid substitution at the Adh locus of Drosophila is facilitated by small population size.

The number of amino acid replacement substitutions and that of synonymous substitutions are examined by using DNA sequences of the Adh locus of Drosophila. The ratio of replacement to synonymous substitutions is higher in sequence comparisons between species than in polymorphisms within species. The ratio for the between-species comparisons is highest in the Hawaiian group and lowest in the obscura group. These observations suggest that amino acid substitutions are facilitated by small population size. The result is in accord with the nearly neutral theory of molecular evolution.

Alcohol Dehydrogenase↗
PubMed1993

Selective on-line enrichment and separation of peptides having aromatic amino acids at their C-termini by column-switching high-performance liquid chromatography using an anhydrochymotrypsin-immobilized precolumn.

A column switching high-performance liquid chromatographic system in which peptides retained on an anhydrochymotrypsin (AHC)-immobilized diol-silica precolumn were selectively transferred to and separated on a reversed-phase analytical column was developed. An investigation of the affinity characteristics of 40 peptides to the AHC precolumn showed that among eleven peptides having Tyr or Phe at their C-termini and more than five amino acid residues, ten were retained almost quantitatively on the precolumn, and two peptides having Trp at their C-termini were less retained. Two peptides having C-terminal PheNH2 were also retained, but the peptide having C-terminal D-PheNH2 was not retained. Among eighteen peptides having no aromatic amino acids at their C-termini, two were retained, one slightly and the other moderately. Calibration graphs for rat atrial natriuretic peptide constructed at various sample sizes were nearly identical, indicating that the peptide could be enriched by this system. The AHC precolumn showed no loss of analytical performance over 1 year, during which about 450 samples were analysed.

Amino Acid Sequence↗
PubMed1993

Purification, crystallization and preliminary X-ray investigation of aqualysin I, a heat-stable serine protease.

Aqualysin I, a thermostable protease found in the culture medium of Thermus aquaticus YT-1, has been purified to homogeneity using a combination of ion-exchange and affinity chromatography. It is a polypeptide with a molecular weight of 28 350 [Kwon, Terada, Matsuzawa & Ohta (1988). Eur. J. Biochem. 173, 491-497] and is most active at 343-353 K and pH about 10.0 [Matsuzawa, Tokugawa, Hamaoki, Mizoguchi, Taguchi, Terada, Kwon & Ohta (1988). Eur. J. Biochem. 171, 441-447]. Crystals of the enzyme are monoclinic, space group P2(1), with cell dimensions a = 40.80 (5), b = 64.39 (6), c = 45.51 (6) A and beta = 109.1 (1) degrees. The asymmetric unit consists of a single molecule (V(m) = 1.99 A(3)Da(-1)). The crystals are stable to X-radiation and scatter to at least 2.8 A resolution.

Journal Article↗
PubMed1993

Heat shock promoter of thermophilic chaperonin operon.

The translation of a heat shock protein (HSP), TGroEL, of thermophilic bacterium PS3 increased within 10 minutes when the culture temperature was raised from 60 degrees C to 70 degrees C. In contrast to hyperthermophilic HSPs such as Pyrodictium ATPase, TGroEL is homologous to GroEL of E. coli (Tamada et al. (1991) Biochem. Biophys. Res. Commun. 197, 565-571). The promoter of the thermophilic heat shock gene (tgrE) was sequenced to analyze the heat shock response. The sequences of the promoter were CCTACTTGCA (-35 region) and GTTCATTAATA (-10 region), which are more heat labile than those of the sigma 32 recognition sites of GroEL. The expression of this homologous tgrE in E. coli may be lethal to the cells. The ATPase alpha subunit of this thermophile is also homologous to TGroEL.

Amino Acid Sequence↗
PubMed1993

Factors for transient entrainment of ventricular tachycardias by rapid atrial pacing.

Thirteen patients with sustained ventricular tachycardia (VT) were studied to elucidate predisposing factors for the development of constant and progressive fusion by rapid atrial pacing. All patients demonstrated transient entrainment by rapid ventricular pacing during VT. Constant and progressive fusion were observed in 7 patients (positive group) during rapid atrial pacing, but not in 6 (negative group). In the positive group, VT was induced by atrial pacing in 2 patients. The demonstration of constant and progressive fusion by atrial pacing was not dependent on QRS morphology or ventriculoatrial conduction during VT. VT cycle length in the positive group (363 +/- 59 ms) was longer than in the negative group (297 +/- 31 ms; p = 0.033). The maximal atrial pacing rate producing 1:1 atrioventricular (AV) conduction in the positive group was 171 +/- 18 beats/min compared with 125 +/- 22 beats/min in the negative group (p = 0.002). There were distinct differences between the positive and negative groups in the ratio of VT cycle length to minimal atrial cycle length causing 1:1 AV conduction (1.02 +/- 0.12 vs 0.61 +/- 0.12; p = 0.0001). It is concluded that AV conduction, VT cycle length and especially their ratio are important factors for the development of transient entrainment by rapid atrial pacing during VT. Therefore, atrial pacing can be used as an easy and useful method to examine transient entrainment during VT.

Adult↗
PubMed1993

Molecular basis of allosteric activation of bacterial L-lactate dehydrogenase.

The three-dimensional structure of allosteric L-lactate dehydrogenase from Bifidobacterium longum, the first example of a T-state structure of L-lactate dehydrogenase, has been determined to 2.0 A. A comparative study of this structure with the previously reported R-state structure from Bacillus stearothermophilus has revealed the allosteric activation mechanism of the bacterial L-lactate dehydrogenase. The fructose 1,6-bisphosphate-induced conformational change at the effector site and the substrate affinity change at the activity site are clearly shown at a molecular level. Coupling of these changes can be simply explained by a set of concerted rotations between subunits in the tetramer of the enzyme. This T to R transition is the first example for a tetrameric allosteric protein where the rotations occur around each of three axes of symmetry.

Allosteric Regulation↗
PubMed1993

Hyperphosphorylation of cytokeratins by okadaic acid class tumor promoters in primary human keratinocytes.

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are potent tumor promoters on mouse skin (H. Fujiki, M. Suganuma, S. Nishiwaki, S. Yoshizawa, J. Yatsunami, R. Matsushima, H. Furuya, S. Okabe, S. Matsunaga, and T. Sugimura. In: R. D'Amato, T. J. Slaga, W. Farland, and C. Henry (eds.), Relevance of Animal Studies to the Evaluation of Human Cancer Risk, pp. 337-350. New York: John Wiley and Sons, Inc., 1992). These tumor promoters, which are also inhibitors of protein phosphatases 1 and 2A, induced hyperphosphorylation of M(r) 60,000, M(r) 58,000, M(r) 56,000, M(r) 52,000, M(r) 42,000, and M(r) 27,000 proteins in PHK 16-I cells, human keratinocytes immortalized by human papillomavirus type 16. Except for the M(r) 27,000 protein, these hyperphosphorylated proteins were identified to be cytokeratin peptides (CK) CK 5, CK 6, CK 7, CK 16, and CK 19, by anti-cytokeratin antibodies. CK 5 and CK 6 were more strongly phosphorylated than CK 16 and CK 19. The in vitro hyperphosphorylation of these cytokeratins was also found by incubation with an enzyme fraction containing a mixture of protein phosphatase 2A and protein kinases isolated from mouse brain and various concentrations of dinophysistoxin-1. Indirect immunofluorescence microscopy with anti-cytokeratin antibodies revealed that the hyperphosphorylated cytokeratins had retracted to the perinuclear area. The hyperphosphorylated M(r) 27,000 protein was identified as a heat shock protein, HSP27. Hyperphosphorylation of HSP27 and intermediate filaments, such as cytokeratins, is one of the early biochemical changes, or pleiotropic effects, in cells induced by the okadaic acid class of tumor promoters.

Blotting, Western↗
PubMed1993

Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli. Evidence that the genes for phosphofructokinase and pyruvate kinase constitute an operon.

Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate. The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined. The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da. The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin. Similarly to the E. coli enzymes, the enzyme does not contain an N-terminal domain, in contrast to the eukaryotic pyruvate kinases. However, the Bacillus stearothermophilus enzyme had an extra C-terminal sequence consisting of about 110 amino acid residues. A phosphoenolpyruvate-binding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C-terminal sequence. There was an open reading frame upstream of the pyruvate kinase gene. The homology of the sequence showed that the gene encodes phosphofructokinase. Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes constitute an operon is presented.

Amino Acid Sequence↗
PubMed1993

Hyperphosphorylation of retinoblastoma protein and p53 by okadaic acid, a tumor promoter.

A potent tumor promoter, okadaic acid, induced hyperphosphorylation of tumor suppressor proteins, retinoblastoma protein and p53, by in vitro incubation with nuclei isolated from rat regenerating liver as well as by incubation with primary human fibroblasts. Most of the retinoblastoma protein migrated to a hyperphosphorylated position in electrophoresis. The phosphorylation of p53 was increased at a rate 8 times that in non-treated primary human fibroblasts. Hyperphosphorylation of tumor suppressor proteins, mediated through inhibition of protein phosphatases 1 and 2A, is involved in tumor promotion by okadaic acid. The significance of hyperphosphorylation of the retinoblastoma protein and p53 is discussed in relation to the regulation of the cell cycle.

Animals↗
PubMed1993

Perineural invasion of carcinoma of the pancreas and biliary tract.

Carcinoma of the pancreatobiliary system often produces perineural invasion extending to extrabiliary and extrapancreatic sites. A surgical technique has been developed to manage this invasion and has been used since 1974. Serial sections were prepared from 90 resected specimens and examined for perineural involvement of intramural and extramural biliary plexuses as well as the pancreatic nerve plexus. Perineural invasion was seen in 34 of 40 patients with carcinoma of the common bile duct, in ten of 14 with cancer of the gallbladder and in four of 15 with carcinoma of the papilla of Vater. Invasion extended to the extramural biliary or pancreatic nerve plexuses in 24 of 40 patients with carcinoma of the bile duct. Involvement of the intrapancreatic nerves was seen in all 21 patients with carcinoma of the pancreatic head and that of the pancreatic nerve plexus in 17 of 21. Perineural invasion was often found at the most progressive margin of the tumour. The survival rate of patients with perineural invasion was low compared with that of those without such invasion, although survival of patients with perineural invasion tended to be longer after extensive resection. The perineural space should be regarded as an important route for the spread of pancreatic and biliary carcinoma.

Biliary Tract↗
PubMed1993

Histological evaluation of the intrahepatic biliary tree in intrahepatic cholesterol stones, including immunohistochemical staining against apolipoprotein A-1.

Apolipoprotein A-1 is known to be one of inhibiting factors of cholesterol nucleation in bile, and decreased activity of apolipoprotein A-1 is considered to predispose cholesterol-supersaturated bile to formation of cholesterol crystals. To study the pathogenesis of the intrahepatic formation of cholesterol stones, we examined surgically resected liver specimens from six patients with intrahepatic cholesterol stones and compared the characteristic histopathological features with those of intrahepatic calcium bilirubinate stones, using morphological examination and immunohistochemical staining against apolipoprotein A-1. Morphologically, in all six patients with cholesterol stones the severity of chronic proliferative cholangitis with proliferation of the mucus-producing glandular elements in the walls of the large bile duct or periductal tissues was less extensive than that seen with calcium bilirubinate stones, and cholesterol crystals had formed in the septal and interlobular bile ducts. Immunohistochemically, unlike the normal liver and calcium bilirubinate stone-containing lobes, the hepatocytes and the epithelial lining of the bile ducts and peribiliary glands of the cholesterol stone-containing lobes did not react completely (some of the epithelial cells reacted only faintly) with apolipoprotein A-1 antibody. These findings suggest that an abundance of mucous substance and bacterial infection of the biliary tree may not be necessary for the formation of cholesterol stones, compared with findings in cases of calcium bilirubinate stones. We suggest that cholesterol crystals may be produced in the septal and interlobular bile ducts in the microenvironment of cholesterol-supersaturated bile and decreased activity of apolipoprotein A-1.

Adult↗
PubMed1993

Microdissection and microcloning of genomic DNA markers from human chromosomal region 11q23.

A human genomic DNA library was constructed by using a microdissection-microcloning procedure with polymerase chain reaction (PCR) techniques on DNA from the chromosome 11q23 region. A total of 450 recombinant pUC clones were isolated from the library. Their insert sizes ranged from 150 to 850 bp with a mean of 320 bp. Fifty pUC clones were randomly selected and analyzed in detail. Southern blot analyses showed that 21 (42%) clones were unique DNA sequences, 20 (40%) clones were repetitive sequences, and 9 (18%) clones had no detectable hybridization. The unique sequences were used further in a secondary screening of a partially digested human genomic DNA library constructed in phage vector, and 4 clones were isolated. The chromosomal locations of these phage clones were confirmed to be in the q23 region of chromosome 11 by fluorescence in situ suppression hybridization. These pUC microclones isolated from the chromosomal region-specific genomic DNA library will be useful in the construction of physical contig maps with yeast artificial chromosome and/or cosmid clones and in the positional cloning of disease-associated genes localized to the q23 region of chromosome 11.

Chromosome Mapping↗
PubMed1993

Isolation of region-specific cosmids by hybridization with microdissection clones from human chromosome 10q11.1-q21.1.

A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.

Base Sequence↗
PubMed1993

Voltage-dependent inhibition of the sodium pump by external sodium: species differences and possible role of the N-terminus of the alpha-subunit.

Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had alpha-subunits truncated before position Lys28 (alpha delta K28) or Thr29 (alpha delta T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent KI values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na(+)-K+ competition for external binding to the E2P form, apparent KI values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the KI value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysine-rich cluster of the alpha-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the KI value. For alpha delta K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If Lys28 is included in the truncation (alpha delta T29), the effective charge increases to 1.5 of an elementary charge and the apparent KI value is reduced to 107 mM. The KI values for pump inhibition by external Na+, calculated by taking into account Na(+)-K+ competition, are smaller than the K1/2 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an access channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.

Animals↗
PubMed1993

The origin of cytologically unidentifiable chromosome abnormalities: six cases ascertained by targeted chromosome-band painting.

De novo chromosome structural abnormalities cannot always be diagnosed by the use of standard cytogenetic techniques. We applied a previously developed chromosome-band-specific painting method to the diagnosis of such rearrangements. The diagnostic procedures consisted of microdissection of an aberrant chromosomal region of a given patient, polymerase chain reaction (PCR) amplification of the dissected chromosomal DNA, and subsequent competitive fluorescence in situ hybridization (FISH) using the PCR products as a probe pool on metaphase chromosomes from the patient and/or a karyotypically normal person. With this strategy, we studied 6 de novo rearrangements (6p+, 6q+, 9p+, 17p+, +mar, and +mar) in 6 patients. These rearrangements had been seen by conventional banding but their origin could not be identified. In all 6 patients, we successfully ascertained the origin. Using an aberrant region-specific probe pool, FISH signals appeared on both the aberrant region and a region of another specific chromosome pair. A reverse probe pool that was generated through the microdissection of normal chromosomes at a candidate region for the origin of the aberration hybridized with both the aberrant and the candidate regions. We thus diagnosed one patient with 17p+ as having trisomy for 14q32-qter, one with 9p+ as having trisomy for 12pter-p12, one with 6q+ as having a tandem duplication (trisomy) of a 6q23-q25 segment, one with 6p+ as having a tandem duplication (trisomy) of a 6p23-q21.3 segment, one with a supernumerary metacentric marker chromosome as having tetrasomy for 18pter-cen, and the last with an additional small marker chromosome as having trisomy for 18p11.1 (or p11.2)-q11.2. The present targeted chromosome-band-painting method provides the simple and rapid preparation of a probe pool for region-specific FISH, and is useful for the diagnosis of chromosome abnormalities of unknown origin.

Base Sequence↗
PubMed1993

The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

Adenosine Triphosphatases↗