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Biomedical subjects

Y Mizukami

Publications and source records attributed to Y Mizukami.

At least 253 records · Page 14Linked to original sources
PubMed1983

Primary structure of human pepsinogen gene.

A recombinant clone, which covers the pepsinogen gene in a single insert, has been isolated by screening a library of human genomic DNA, using a swine pepsinogen cDNA as a probe. Sequence analysis of coding DNA segments of the clone revealed that the pepsinogen gene occupies approximately 9.4-kilobase pairs of the genomic DNA and is separated into nine exons by eight introns of various lengths. The predicted amino acid sequence of human pepsinogen consists of 373 residues and is 82% homologous with that of swine pepsinogen. In addition, the predicted sequence contained a single sequence of 15 amino acid residues at the NH2 terminus, showing that the protein is synthesized as prepepsinogen. The structure of the gene, in which two homologous sequences including the two active site aspartyl residues of pepsin are present in different coding segments, is in support of the view that the pepsinogen gene evolved by duplication of a shorter ancestral gene.

Amino Acid Sequence↗
PubMed1983

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450.

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.

Animals↗
PubMed1983

Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes.

NAD(P)H-oxidase activity was demonstrated in glutaraldehyde-fixed rat hepatocytes by a cerium technique. The activity was observed exclusively on the bile-canalicular plasma membrane of hepatocyte. No reaction product was formed in the absence of NAD(P)H as the substrate. The reaction was inhibited by pCMB (surface sulfhydryl group specific reagent), by heating, by anaerobic incubation and by catalase (H2O2 scavenger), but it was not inhibited by KCN or NaN3. The present results show that bile-canalicular plasma membrane produces H2O2 and the cerium technique for demonstration of H2O2 is therefore an useful method for the subcellular localization of NAD(P)H-oxidase activity in the glutaraldehyde-fixed hepatocyte.

Anaerobiosis↗
PubMed1983

Gene structure of a phenobarbital-inducible cytochrome P-450 in rat liver.

The gene structure of a phenobarbital-inducible rat liver cytochrome P-50 was elucidated by sequence analysis of two isolated genomic clones. The total length of the gene was approximately 14 kilobases and was separated into nine exons by eight intervening sequences. Nucleotide sequences of all exon/intron boundaries follow the G-T/A-G rule. A putative transcription initiation site was assigned to an A, 30 base pairs upstream from the initiation codon by S1 nuclease protection mapping. A possible "TATA" equivalent sequence, C-A-T-A-A-A, was found 27 base pairs further upstream from this initiation site. A poly(A) attachment site was determined to be 386 or 387 base pairs downstream from the termination codon by comparison with the cDNA sequence. Detailed comparison with two cDNA sequences determined previously showed the coding nucleotide sequence of the genomic clones to concur with that of the pcP-450pb2 cDNA clone coding for cytochrome P-450e except for three neutral base substitutions. Therefore, we conclude that the gene sequence determined here is for the cytochrome P-450e gene or a similar gene. On the other hand, 40 base substitutions were found in about 1,900 base pairs compared between the sequences of the genomic clone and the other cDNA clones (pcP-450pbl and 4) coding for cytochrome P-450b, and 15 of them result in 14 amino acid replacements in the total 491 amino acid residues. These base substitutions occur in relatively limited regions of the sequences. Most of them are found in exons 6, 7, 8, and 9; most frequently in exon 7.

Amino Acid Sequence↗
PubMed1983

Histochemical and biochemical study on adenylate cyclase and 5'-nucleotidase activity in thyroid glands with normal and various thyroid diseases.

The adenylate cyclase and 5'-nucleotidase activity was measured biochemically in the thyroid glands from patients with various thyroid diseases in comparison with normal thyroid. The basal adenylate cyclase activity in normal thyroid was 159.3 p-moles cAMP/min./g tissue. The activity was elevated to 230% of basal with 20 mM NaF and 190% of basal with 100 mU/ml TSH. These values in chronic thyroiditis and Graves' disease were not significantly different from the values of normal thyroid. In adenomatous goiter, adenoma and carcinoma, the basal adenylate cyclase activity was significantly higher than that of normal thyroid. Parallel to the biochemical determination of both enzyme activities, the distribution of histochemically demonstrable adenylate cyclase and 5'-nucleotidase activity was described in the follicular cells with normal and various thyroid diseases. The reaction product of adenylate cyclase and 5'-nucleotidase activity was restricted to the plasma membrane of the follicular cells. However, the distribution and intensity of the adenylate cyclase reaction varied in each thyroid disease, except for the absence of reaction product in the basal plasma membrane. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that the basal plasma membrane may not play an important role of TSH-reception.

5'-Nucleotidase↗
PubMed1983

A case report of abetalipoproteinemia (Bassen-Kornzweig syndrome)--the first case in Japan.

The clinical and pathophysiological features of a case of abetalipoproteinemia in a 34-year-old patient are described. This patient is the first case reported in Japan. The patient was diagnosed as abetalipoproteinemia by confirming the Apo-B deficiency in the patient's serum and the slightly high cholesterol level in his mother's and borderline normal level in his father's. The patient had remarkably low lipid levels, acanthocytosis, and lipid malabsorption. An unusual feature of this case was that the patient had no neuromuscular or ocular manifestations. This was possibly related to his normal plasma vitamin A and E levels.

Abetalipoproteinemia↗
PubMed1983

Changes in localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in human thyroid carcinoma cells.

K+-dependent, ouabain-sensitive nitrophenyl phosphatase (K+-NPPase) activity, which reflects the terminal dephosphorylation step of (Na+ + K+)-ATPase action, was studied histochemically in human thyroid normal follicular cells and in human thyroid carcinoma cells, using a newly developed one-step lead citrate method. In normal thyroid follicular cells, reaction product for K+-NPPase activity was found on the lateral plasma membrane and not on either the apical or basal plasma membrane. In thyroid carcinoma cells, a large amount of reaction product was observed on the lateral plasma membrane and also on the apical and basal plasma membrane. Appropriate control experiments indicated that the deposition of reaction product was K+ dependent and ouabain sensitive. Although there was some overlap in the distribution of reaction products for K+-NPPase and Mg2+-ATPase, significant differences were consistently observed. The biochemical findings indicated that the K+-NPPase activity per milligram of DNA in thyroid carcinoma cells was approximately 10 times higher than that in normal thyroid cells, and that a significant positive correlation exists between K+-NPPase and (Na+ + K+)-ATPase activity. The physiologic and pathologic implications of this localization for tracing the route of active Na+ transport, which might participate in the transport of iodide ion in both human thyroid normal follicular cells and human thyroid carcinoma cells, are discussed.

4-Nitrophenylphosphatase↗
PubMed1982

Localization of adenylate cyclase and 5'-nucleotidase activities in human thyroid follicular cells.

The localization of adenylate cyclase and 5'-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultaneous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the otherhand, the histochemical localization of 5'-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5'-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5'-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5'-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.

5'-Nucleotidase↗
PubMed1982

Primary structure of a cytochrome P-450: coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 cDNA from rat liver.

We determined the coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 mRNA of the rat by analysis of three cloned cDNAs and by primer extension methods. The deduced amino acid sequence of the cytochrome is composed of 491 amino acids, and its predicted molecular weight and amino acid composition concur with those determined with the purified protein. The sequence of one of the three cloned cDNAs is not identical with that of the other two. In their 922 overlapping nucleotides, 14 nucleotide substitutions occur, and 7 of them result in 6 amino acid changes, therefore indicating the presence of at least two similar but distinct mRNAs for phenobarbital-inducible cytochrome P-450. These substitutions occur in a limited portion of the sequence, apparently forming some sort of a "variable region."

Amino Acid Sequence↗
PubMed1982

Molecular cloning and coding nucleotide sequence of complementary DNA of cytochrome P-450 involved in metabolic activation of carcinogenic substances.

Complementary (c)DNA clones of phenobarbital-inducible cytochrome P-450 which has a strong activity toward metabolic activation of aflatoxin B1 were identified by a hybridization-arrested translation assay and a positive hybridization-translation assay. The coding nucleotide sequence of the cytochrome P-450 mRNA was determined by analysis of three cloned cDNAs and by primer extension method using a 5' terminal region fragment (62 bp) of a clone (pcP-450 pb-2) as a primer. The deduced amino acid sequence of the cytochrome is composed of 491 amino acid and its predicted molecular weight and amino acid composition concur with those determined with the purified protein. The sequence of one of the three cloned cDNAs is not completely the same as that of the other two. Fourteen nucleotide substitutions occur in their 922 overlapping nucleotides and 7 of them result in 6 amino acid replacements, therefore indicating the presence of at least two similar but distinct mRNAs for phenobarbital-inducible cytochrome P-450. These substitutions occur in a limited portion of the sequence, apparently forming some sort of a "variable regions."

Aflatoxin B1↗
PubMed1982

Black thyroid. Morphological, biochemical and geriatric studies on the brown granules in the thyroid follicular cells.

An autopsy case of a 73-year-old man with "black thyroid" was reported. Investigations by light and electron microscopy and histochemical study of the black thyroid material disclosed the massive deposition brown granules in the follicular cells, to be residual bodies containing lipids. Lipids in these granules were mainly composed of phospholipid by biochemical analysis. Concerning the thyroidal function of this case the value of T4-I in the serum was within normal range (4.8-7.5 micrograms/dl) and peroxidase, NADPH-cytochrome C reductase and acid phosphatase activity in the black thyroid material revealed no significant increase or decrease, compared with other 12 autopsied cases. These granules resembled so-called lipofuscin granules, and the frequency and grade of the deposition in autopsied cases increase with aging.

Acid Phosphatase↗
PubMed1981

Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver.

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.

Animals↗
PubMed1981

Correlation between thyroid peroxidase activity and histopathological and ultrastructural changes in various thyroid diseases.

A morphological and biochemical study was performed on thyroid tissue with various thyroid diseases. The thyroid peroxidase (TPO) activity of normal thyroid tissues ranged from 2.6 to 7.0 mGU/mg DNA. The activity was low in adenomas and extremely low in carcinomas, and there was no significant relationship between the histological subclassification of follicular adenomas (simple, colloid, oxyphil) and TPO activity. The activity was various in the cases of chronic thyroiditis, ranging from non-detectable to 9.8 mGU/mg DNA, and the TPO activity showed a close correlation with the degree of lymphoid cell infiltration of the diseases. In the seven cases of Graves' disease, the values were high, though the elevation was not so remarkable in three cases which had already been euthyroid or slightly hypothyroid after long-term treatment. By means of subcellular fractionation, more than 50% of peroxidase activity was shown to be localized in the microsomal pellets, and this result well coincided with the electron microscopic findings of prominent development of rER.

Adenoma↗