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Biomedical subjects

O Gotoh

Publications and source records attributed to O Gotoh.

At least 91 records · Page 5Linked to original sources
PubMed1985

Possible steroid binding site common to adrenal cytochrome P-450scc and prostatic steroid binding protein.

By searching the entire PIR-protein-sequence data base, we have found that a dodecapeptide sequence in bovine adrenal cytochrome P-450scc is closely related to that in rat prostatic steroid binding protein. The two proteins belong to unrelated protein families, but both have steroids as substrates or ligands. Thus, the dodecapeptides may be important for substrate/ligand recognition in the individual proteins.

Adrenal Glands↗
PubMed1985

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat.

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of a functional cDNA for human thymidylate synthase.

We have determined the nucleotide sequence of a cDNA clone, pcHTS-1, encoding human thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) which was previously isolated from a human fibroblast expressible cDNA library and functional in mouse cells. The 1.6 kilobase cDNA insert of pcHTS-1 encodes a subunit protein of 313 amino acid (Mr = 35,706) and its predicted amino acid sequence is highly conserved in many regions including folylpolyglutamate and 5-fluoro-2'-deoxyuridylate binding sites, when compared with those of Lactobacillus casei, Escherichia coli, and bacteriophage T4. The cDNA contains in its 5'-untranslated region a triple tandemly repeated sequence consisting of 90 nucleotides, which starts immediately upstream of the ATG initiator codon, is very high in G+C content (80%), and can form three possible interconvertible stem-loop structures.

Amino Acid Sequence↗
PubMed1985

Effect of pretreatment with the calcium antagonist nimodipine on local cerebral blood flow and histopathology after middle cerebral artery occlusion.

We used the [14C]iodoantipyrine autoradiography technique to study the effect of pretreatment with the calcium antagonist nimodipine on local cerebral blood flow (lCBF) in rats that underwent middle cerebral artery (MCA) occlusion. In untreated control animals there were profound localized reductions in 1CBF 30 minutes after MCA occlusion. These were most pronounced in neocortical areas and in the caudate nucleus ipsilateral to the MCA occlusion. In animals pretreated with nimodipine (1 microgram X kg-1 X min-1 for 30 minutes before and 30 minutes after MCA occlusion), the ipsilateral decrease in 1CBF in cortical regions was significantly less than that in control animals. The drug did not appear to alter 1CBF in the ipsilateral caudate nucleus. Neuropathological quantification of the ischemic damage present 3 hours after occlusion showed that nimodipine pretreatment reduced the volume and extent of cellular damage in the periphery but not in the core of the lesion.

Acid-Base Equilibrium↗
PubMed1985

Alterations of the eicosanoid synthetic capacity of rat brain microvessels following ischemia: relevance to ischemic brain edema.

To know the mechanism underlying ischemic brain edema, a time-course analysis of the eicosanoid synthetic capacity of brain microvessels was carried out using unilateral, middle cerebral artery (MCA)-occluded rats. Concomitant with the development of brain edema the synthetic capacity of all products, including cyclooxygenase and lipoxygenase products, increased significantly. Next the effects of 15-hydroperoxyarachidonic acid (15-HPAA) on the synthetic capacity of microvessels were examined. The drug caused a generalized increase of each product, the profile of which was similar to that obtained with ischemic hemispheres, although the ratios of each product differed somewhat among them. The enhanced synthesis of eicosanoids by 15-HPAA was markedly suppressed by radical scavengers such as alpha-tocopherol, hydroquinone, and 1,2-bis(nicotineamide)-propane. Furthermore, the evolution of brain edema was virtually suppressed by the systemic administration of 1,2-bis(nicotineamide)-propane. The above result suggests that the enzyme activity of the arachidonic acid (AA) cascade of microvessels is stimulated by its own products. Such a mechanism will form a vicious cycle that accelerates the accumulation of free radicals within microvessels and thus may play a role in the progressing disruption of the blood-brain barrier (BBB) following ischemia.

Animals↗
PubMed1985

[The pathomechanism underlying ischemic brain edema: the role of Na+, K+-ATPase of the brain microvessels].

In the present study, the anti-edema effect of AVS [1,2-bis (nicotineamide)-propane] was evaluated using the cat MCA occlusion model with or without recirculation. In the prolonged ischemia (PI) group, cortical edema as assessed by the changes in specific gravity, developed in those cortical areas where the mean 1-CBF was less than 25-30 ml/100 g/min during MCA occlusion (4 hours). In the recirculation group (2 hours' ischemia followed by 2 hours' recirculation: RC group), the ischemic threshold for edema development was almost the same as in the PI group. In both groups, the drop in cortical specific gravity was significantly suppressed by AVS. Regarding the time-course of 1-CBF, there was no difference between the PI-AVS-treated and PI-saline-treated groups. In the RC group, however, the postischemic hypoperfusion was significantly ameliorated by AVS. Based on the present and previous data showing the antiedema effect of AVS, the mechanism of action of AVS was discussed in relation to the pathomechanism underlying ischemic brain edema. Our new concept of ischemic brain edema is briefly stated below. Related in vitro studies have shown the followings: (i) the influx of sodium not of proteins is the principal cause of ischemic brain edema: (ii) the eicosanoid synthetic capacity of the brain microvessel (MV) is increased simultaneous to edema development (iii) an elevation in the level of hydroperoxides enhances the activities of Na+, K+-ATPase as well as the arachidonate cascade of MV. These data suggest that free fatty acids and free radicals liberated following cerebral ischemia stimulate the activity of the MV-Na+, K+-ATPase, which results in increased sodium influx across the BBB. AVS was shown to scavenge hydroxyl radicals and to inhibit the stimulatory effects of a lipid hydroperoxide (15-HPAA) on the activities of Na+, K+-ATPase and the arachidonate cascade of the MV. These actions of AVS may be linked to its antiedema effect.

Animals↗
PubMed1984

Titration of mRNAs for cytochrome P-450c and P-450d under drug-inductive conditions in rat livers by their specific probes of cloned DNAs.

By sequence analysis of the cloned cDNA or genomic DNA, we have recently deduced the complete primary structures of two forms of 3-methylcholanthrene-inducible cytochromes P-450 (P-450c and P-450d). Comparing these sequences, we identified two highly conserved regions, amino acid numbers from 35 to 200 and from 340 to 470. The nucleotide sequences corresponding to these homologous regions are also well conserved, whereas other regions have undergone considerable sequence divergence. In RNA blot analysis with unfractionated mRNA isolated from 3-methylcholanthrene-treated rat livers, Probe A (specific to P-450c sequence) hybridized with mRNA around 23 S, while Probe B (specific to P-450d sequence) hybridized with mRNA around 18 S. When common sequence between P-450c and P-450d was used as the probes (Probe C or D), two bands were clearly observed around 23 and 18 S mRNAs. With the common DNA sequence between P-450c and P-450d as a probe (93.7% homology), we studied the induction of specific mRNA for P-450c and P-450d by a single dose of several chemical compounds to rats. 3-Methylcholanthrene increased both P-450c and P-450d mRNA levels by 50 and 10 times above the control at 17 h after the administration, respectively. Despite the lower induction rates, the P-450d mRNA level was constantly higher than or at least similar to that of P-450c mRNA. beta-Naphthoflavone and Kaneclor KC 500 showed similar induction ability to 3-methylcholanthrene. On the other hand, isosafrole induced P-450d mRNA to a much greater extent than P-450c mRNA.

Animals↗
PubMed1984

Distinct organization of methylcholanthrene- and phenobarbital-inducible cytochrome P-450 genes in the rat.

The complete nucleotide sequence of the methylcholanthrene-inducible cytochrome P-450c gene was determined by sequence analysis of cloned genomic DNA and the sequence, consisting of 524 amino acids, of the protein was deduced therefrom. The gene for the cytochrome was approximately 6.0 kilobases long and was split into seven exons. Comparison of the gene with that of the phenobarbital-inducible cytochrome P-450e showed that the gene structures for the two types of cytochrome P-450 differ greatly; the location, number, and size of intervening sequences are very dissimilar. However, the sequence homology between the two types of cytochrome suggests that the two genes have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1984

Coding nucleotide sequence of 3-methylcholanthrene-inducible cytochrome P-450d cDNA from rat liver.

We determined the coding nucleotide sequence of the mRNA for a 3-methylcholanthrene-inducible cytochrome P-450, P-450d, of rat liver by sequence analysis of cloned cDNAs. The predicted amino acid sequence of the cytochrome is composed of 513 amino acids, and its NH2-terminal sequence of 30 amino acids completely coincides with that reported from analysis of the purified cytochrome P-450d. The amino acid composition of the deduced sequence also agrees well with that determined from the purified protein. Computer-aided analysis was carried out to compare the complete primary structures of five species of cytochrome P-450, two molecular species of phenobarbital-inducible rat liver cytochrome P-450 (P-450b and P-450e), phenobarbital-inducible rabbit liver cytochrome P-450LM2, 3-methylcholanthrene-inducible rat liver cytochrome P-450d, and camphor-hydroxylating P-450 of Pseudomonas putida (P-450CAM). It is concluded therefrom that the time of divergence between cytochrome P-450b (P-450e) and P-450d is much earlier than that of branching between phenobarbital-inducible cytochromes P-450 of rat and rabbit. One highly conserved cysteine-containing region that is close to the COOH terminus is found in all of these cytochrome P-450 sequences, indicating that the heme-binding site is the cysteine residue in this region.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of Dictyostelium small nuclear RNA Dd8 not homologous to any other sequenced small nuclear RNA.

The cellular slime mold Dictyostelium discoideum, a lower eukaryote, was shown to contain several species of small nuclear RNA (Takeishi, K., and Kaneda, S. (1981) J. Biochem. (Tokyo) 90, 299-308; Wise, J. A., and Weiner, A. M. (1981) J. Biol. Chem. 256, 956-963). One of these RNAs, Dd9 or D2, was sequenced and found to be homologous to mammalian nucleolar U3 RNA. In the present study, the nucleotide sequence of another Dictyostelium small nuclear RNA Dd8 was determined by direct analysis. The sequence is: (formula; see text) Dd8 RNA contains high proportions of A (34%) and U (30%). No modified nucleotide could be detected in the internal region. Computer-assisted analysis of sequence homology indicated that Dd8 RNA is not homologous to any other small nuclear RNA species sequenced so far.

Base Sequence↗
PubMed1983

Prediction of melting profiles and local helix stability for sequenced DNA.

The utility and limits of the current theory of helix-coil transition of DNA were intensively studied. Comparison of calculated and observed melting profiles affords solid criteria of a theoretical model and parameter values. Our model differs from others in that heterogeneity in stacking interactions is taken into consideration (Section II). The parameter values were estimated by fitting calculated to observed profiles for several known-sequenced DNA fragments (Section III). Calculated profiles agree quite well with observed, not only for DNAs used for the fitting but also for those not used. Such good agreement was never obtained with traditional two-term (A X T and G X C) stability assignment. It was shown that the all-or-none approximation for local melting transition (Azbel's model) provides informative but somewhat erroneous estimates of local stability. The stabilities of 10 kinds of stacking doublets estimated by the fitting accord well with known properties of DNA double helix (Subsection III-4); three-dimensional structure, calculated stacking energies, and binding specificity of some intercalative drugs are all consistent with the estimated order of stacking stabilities. Relationship between triplet stability and genetic code was discussed. The mechanisms of ionic-strength dependence of melting profiles were discussed in Section IV. Four different mechanisms were proposed and their relative contributions in various salt ranges were considered. It was suggested that at low ionic strengths melting processes deviate significantly from equilibrium, which fixes a limit to the applicability of the present theoretical mode. Locally unstable regions and frequently opening regions were located on several natural DNAs by calculating probabilities of individual base-pair doublets being in an unstacked state. With few exceptions, origins of DNA replication on various DNAs fall on frequently opening regions, indicating their functional importance. Positive correlations were also obtained between frequently opening regions and transcriptional promoters, terminators and gene boundaries (Section V).

Base Composition↗
PubMed1983

Structural characteristics of cytochrome P-450. Possible location of the heme-binding cysteine in determined amino-acid sequences.

Computer-aided analyses were made of the complete amino-acid sequences of two P-450 species, the phenobarbital-inducible major P-450 of rat liver microsomes (P-450PB) and camphor-hydroxylating P-450 of Pseudomonas putida (P-450cam). Statistically significant homology was recognized between the two P-450 sequences, but these sequences were not related to those of other groups of hemoproteins, such as hemoglobins, peroxidases, and cytochrome c's and b's. Two highly homologous regions, HR1 and HR2, and two other weakly homologous regions were found on optimally matched alignment of the P-450 sequences. The secondary structures of the two P-450's predicted by current prediction methods bear strong resemblance at these homologous regions. Both HR1 and HR2 contain a cysteine residue near the center of the homologous regions, and they are the only regions that show significant homology among all 48 combinations of local sequences around the cysteine residues (six on P-450PB and eight on P-450cam). HR1 is located in the N-proximal half of the molecule, is rich in hydrophilic residues, and is predicted to be helical. On the other hand, HR2 is close to the C-terminus, has intermediate hydrophobicity, and may take a complex secondary structure of a turn-sheet-helix. The amino-acid sequences around the HR1 and HR2 regions are also well conserved in another P-450 species, rabbit P-450LM2.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver.

Poly(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli X1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Mitochondrial DNA analysis of mouse-rat hybrid cells: effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs.

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAPr) and CAP-sensitive (CAPs) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAPr mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAPr parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAPr parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1-4 times that of rat mtDNAs inthe mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.

Animals↗
PubMed1982

Salt-concentration dependence of thermal denaturation of restriction fragment DNAs from phi X174.

High-resolution differential melting curves of phi X174 Y1 and Y2 restriction fragment DNAs, for which the base sequences were known, were measured at various sodium ion concentrations ranging from 195 to 2.3 mM. The curves were resolved into component peaks, and the change in the melting temperature, the change in the area, and the change in the breadth of each peak with change in salt concentration were examined. The locations of the melting regions corresponding to the peaks in the melting curves were assigned based on theoretical calculations of melting curves and stability maps. It was found that as the salt concentration was decreased from the high to the intermediate range, the breadths of the peaks on the low-temperature side decreased whereas those on the high-temperature side remained almost constant, and also the separation between the peaks along the temperature axis increased. Changes in the positions of peaks relative to one another were interpreted in terms of the difference in the free energy increase between a loop state and an end-coil state as the salt concentration decreased.

Bacteriophage phi X 174↗