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Biomedical subjects

H Takeya

Publications and source records attributed to H Takeya.

67 records · Page 4Linked to original sources
PubMed1989

Purification of a DNA replication terminus (ter) site-binding protein in Escherichia coli and identification of the structural gene.

In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.

Amino Acid Sequence↗
PubMed1989

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis.

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.

Amino Acid Sequence↗
PubMed1989

Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis.

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.

Amino Acid Sequence↗
PubMed1988

Bovine factor VII. Its purification and complete amino acid sequence.

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).

Amino Acid Sequence↗
PubMed1988

A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX.

A new trisaccharide sugar chain was identified in bovine blood coagulation factors VII and IX. A pentapeptide isolated from factor VII contained Ser-52, which could not be identified with a gas-phase sequencer, suggesting an unknown substituent on the serine residue (Takeya, H. et al. (1988) J. Biol. Chem., in press). The same results were obtained for a pentapeptide containing Ser-53 of factor IX. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was coupled with 2-aminopyridine. The fluorescent pyridylamino (PA-) derivative of the trisaccharide was purified by gel-filtration and reversed-phase HPLC. The sugar composition of the PA-trisaccharide was found to be 2 mol of xylose and 1 mol of PA-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII and IX.

Amino Acids↗
PubMed1985

[Experimental study on analysis of the cardiovascular function during carbon monoxide poisoning and resuscitation under hyperbaric oxygenation].

Experimental dogs were intoxicated with 0.5% carbon monoxide until the carboxyhemoglobin level reached 73--81%. Then they were resuscitated with 100% oxygen at normal atmospheric pressure (1ATA), or oxygen at three atmospheric pressure (3ATA). First, the time necessary for half clearance of blood carboxyhemoglobin was measured and compared between above two groups. In the group treated with oxygen at 3ATA (OHP), the time necessary for half clearance was 12 minutes. It was found that when oxygen at 3ATA was administered, carbon monoxide was eliminated 1.4 times faster than group which received oxygen at 1ATA. Secondary, in the present work, we have applied an analytical method for the cardiovascular function by thermodilution method using Swan-Ganz catheter proven under 1ATA to that under hyperbaric conditions. Summarizing the experimental results, it may be said that in CO poisoned dogs even the time when the CO-Hb level exceed 77% (mean-value), generally a slight inhibition of the circulatory system was seen. These changes showed an immediate return to control levels with the initiation of compression of oxygen inhalation. However, following the course for 45 minutes thereafter while almost no change was seen in the aortic pressure and heart rate, both the cardiac index and left ventricular work index showed a decreasing tendency while a prominent rise was seen in the systemic vascular resistance index. Generally in normal dogs under high pressure oxygen, as pointed out by my team, such tendencies as described above are seen. However, in CO poisoned dogs like wise, when the CO-Hb level returns to 10%, the facts that an inhibiting tendency is seen in the cardiovascular system seems to be a point of interest. I am of the opinion that this fact in actual clinical work should be kept in mind when therapy is required in CO poisoning in human subjects. Generally speaking it may be said that when hyperbaric oxygen therapy is indicated, there still remains various problems concerning optimum compression values, the duration of compression and in the compression-decompression pattern for the individual.

Animals↗
PubMed1981

Fast oscillatory EEG activity induced by analgesic concentrations of nitrous oxide in man.

EEGs during inhalation of analgesic concentrations of 30%, 50%, and 70% nitrous oxide in oxygen, in the absence of other medication were studied in man. Although nitrous oxide has been considered to produce no significant change in EEG activity, it was found to produce characteristic fast oscillatory activity (FOA) in a predictable fashion. FOA was associated with unconsciousness or unresponsiveness to stimuli and appeared predominantly in the frontal areas. Power spectrum analyses showed that the frequency of FOA was remarkably constant across subjects and across trials with a peak frequency of 34 Hz. Amplitude and quantity of FOA increased as the concentration of nitrous oxide increased approaching 100 microV and more than 90%, respectively, under 70% nitrous oxide. It took approximately 1 hour after cessation of nitrous oxide before EEGs had completely returned control pattern with little FOA in spite of expected rapid decrease of alveolar nitrous oxide concentration. FOA can serve as a clinical monitor of the effectiveness of nitrous oxide analgesia.

Adult↗