Search PubMed⌕ Search

Biomedical subjects

H Ohkawa

Publications and source records attributed to H Ohkawa.

At least 109 records · Page 6Linked to original sources
PubMed1991

Rotation and interactions of genetically expressed cytochrome P-450IA1 and NADPH-cytochrome P-450 reductase in yeast microsomes.

Rat liver cytochrome P-450IA1 and/or yeast NADPH-cytochrome P-450 reductase was expressed genetically in yeast microsomes. The ratio of P-450IA1 to the reductase was about 17:1 and 1:2 without and with coexpression of the reductase, respectively. Rotational diffusion of P-450IA1 was examined by observing the flash-induced absorption anisotropy, r(t), of the heme.CO complex. In only P-450IA1-expressed microsomes, 28% of P-450IA1 was rotating with a rotational relaxation time (phi) of about 1200 microseconds. The mobile population was increased to 43% by the presence of the coexpressed reductase, while phi was not changed significantly. Increased concentration of KCl from 0 to 1000 mM caused considerable mobilization of P-450IA1. The results demonstrate a proper incorporation of P-450IA1 molecules into yeast microsomal membranes. The significant mobilization of P-450IA1 by the presence of reductase suggests a possible transient association of P-450IA1 with the reductase.

Animals↗
PubMed1991

Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.

Alcohol Dehydrogenase↗
PubMed1991

Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae.

Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.

Adrenodoxin↗
PubMed1991

Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase.

Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha). On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21. The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively. When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase. In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17. 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate. The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected. Androstenedione was further converted into testosterone by an unknown pathway present in S. cerevisiae cells.

17-alpha-Hydroxyprogesterone↗
PubMed1991

[In vivo assessment on the therapeutic effects of etoposide, vincristine and mitomycin C against human neuroblastoma].

Anti-tumor effects of etoposide (VP-16), vincristine and mitomycin C were evaluated with four human neuroblastoma xenograft, according to Battelle Columbus Laboratories protocol. Etoposide is one of the agents which has been reported to be effective against advanced neuroblastoma clinically, if combined with other agents. While vincristine was effective against 1 out 4 neuroblastoma xenografts, TS-N-2, with 58.1% maximum inhibition rate, etoposide was assessed ineffective as a single agent in all of the 3 xenografts used. Since etoposide had no effect on the weight gain in nude mice in this xenograft experiment, the dose of etoposide was increased two-fold against 2 xenografts, but found ineffective also in the increased dose. Mitomycin C, which has not been used in childhood malignant tumors, was effective against 2 out of 4 xenografts, TNB-9 and SK-N-AS, with 72.0% and 78.4% maximum inhibition rates, respectively.

Animals↗
PubMed1990

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria.

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

Amino Acid Sequence↗
PubMed1990

A new technique for the excision of the first branchial cleft fistula--turn-inside-out method of the fistula.

Conventional surgery of the first branchial cleft fistula has been highly complicated. Especially the management of the facial nerve and the complete removal of the fistula in the ear canal side were very difficult. According to the anatomical and embryological studies, we designed a new operative technique. We applied this method successfully in two cases, and we consider it to be very safe, easy and effective.

Branchioma↗
PubMed1990

Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae.

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.

17-alpha-Hydroxyprogesterone↗
PubMed1990

Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae.

The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae [Yabusaki et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.

Alcohol Dehydrogenase↗
PubMed1990

Construction of chimeric insecticidal proteins between the 130-kDa and 135-kDa proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship.

Eight chimeric insecticidal protein (IP) genes were constructed between the 130-kDa and 135-kDa IP genes of Bacillus thuringiensis subsp. aizawai, and expressed in Escherichia coli JM103 cells. The characterization of the produced chimeric IPs indicated that the variable region (VR1) in the amino-terminal half of the IPs is responsible for the insecticidal activity against larvae of Spodoptera litura and Plutella xylostella. The carboxy-terminal half of VR1 was important for the formation of the 60-kDa active fragment in the gut juice of S. litura larvae. Also, combination of the other two variable regions (VR2 and VR3), which were in the central and carboxy-terminal portions of the IPs, appeared to be related to the solubility of the IPs in the gut juice.

Animals↗
PubMed1990

[Present status of neuroblastoma mass screening in Japan. Neuroblastoma-Committee of the Japanese Childhood Cancer Society].

In 1973, mass screening program for 6-month old infants for early detection of neuroblastoma using a VMA spot test of a urine sample was initiated in Kyoto. In 1985, nation wide mass screening was initiated throughout the entire country and the Government has given the financial support to each district. In 1988, the Government recommended the institution of mass screening by quantitative measurements of VMA, HVA and creatinine using HPLC (high performance liquid chromatography), instead of the qualitative test of VMA alone. From 1974, at the time of initiation of mass screening for neuroblastoma to the end of October, 1989, 383 cases with this tumor have been discovered throughout the screening program. Three hundreds eighty three cases (88%) of them had been registered to the Neuroblastoma Committee of the Japanese Society of Pediatric Oncology. In this paper, the mass screening program was introduced and the 337 cases with this tumor detected by 6-month old screening were analyzed their clinical symptoms, findings, urinary VMA and HVA levels, primary sites, weights of primary tumor, histology, stages at diagnosis, metastatic sites, and the results of the treatment. Three hundreds twenty eight cases (97%) of them are expected to be cured. And we discussed clinical problems related to mass screening program for neuroblastoma, such as an increase of the incidence of infantile neuroblastomas detected by this program and the spontaneous regression.

Adolescent↗
PubMed1989

Computerized analysis of anorectal manometry.

A computer system was designed and applied to anorectal manometry that allows pressure data to be analysed in various ways. Two kinds of applications were presented. Image analysis enabled anorectal movements to be visualized as a three-dimensional image. This technique was shown to be useful in studying the pathophysiology of chronic constipation in children. Radial variation analysis demonstrated that there was no significant radial difference in the anal-canal pressure.

Anal Canal↗
PubMed1989

Clinical experience with a sucking sump catheter in the treatment of esophageal atresia.

This is an introduction to the sucking sump catheter as a method of continuous salivary suction from the oral cavity for the treatment of esophageal atresia and other diseases. Upper pouch suction on many occasions is not easy. The double lumen catheter was devised by Replogle to achieve better suction efficiency, but this is not always satisfactory. Therefore, we devised an oral suction catheter that is a sucking sump catheter. A no. 8 French Argyle Salem sump catheter was transformed; we added five to six holes, and the tip was bent into a double or triple circle. This is sucked on, as a nipple would be, by the infant. From 6 years of experience, we have determined that this works very well for continuous salivary aspiration from the oral cavity, that it causes no complications; this device can also contribute to early oral feeding after esophageal anastomosis.

Catheterization↗
PubMed1989

Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli.

Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.

Bacillus thuringiensis↗
PubMed1989

Spread of the single spike activity in the myenteric plexus of the guinea-pig duodenum.

The spontaneous single spikes in the myenteric plexus of the guinea-pig duodenum were recorded extracellularly. The myenteric plexus neurones in the cardiac region of the stomach also exhibited the spontaneous single spike activity. The frequency of these spikes was at low level. By the treatment with a high Mg and Ca-free Krebs solution, the single spikes were abolished. The generation of the single spikes was also inhibited by d-tubocurarine. These results suggest the presence of nicotinic neurotransmission in the myenteric plexus. The field stimulation could not evoke the single spike activity. The single spike activities of the myenteric plexus neurones were recorded by two electrodes situated in a ganglion or at two different ganglia. The simultaneous recordings by two different spots showed that there were corresponded and non-corresponded spikes. The former may be due to the conduction of excitation. The values of the correspondence ratio were calculated and the values ranged from 39.1% to 69.6% in a ganglion and ranged from 55.2% to 94.2% in different ganglia. The single spikes may spread over 10 mm in a longitudinal direction. These results suggest that the intra- and interganglionic interactions appear within the myenteric plexus and ganglia are interconnected into a functional integrative circuitry.

Action Potentials↗