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Biomedical subjects

K Sogawa

Publications and source records attributed to K Sogawa.

At least 91 records · Page 5Linked to original sources
PubMed1992

Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

Animals↗
PubMed1992

Elevation of the level of lipid peroxidation associated with hepatic injury in LEC mutant rat.

Long-Evans Cinnamon (LEC) mutant rat, which spontaneously develops a chronically necrotizing hepatic injury at 4-5 months of age, exhibits an excess hepatic copper accumulation. The hepatic injury was completely correlated to the excess hepatic copper accumulation in backcross progenies, supporting the previous hypothesis that the copper cytotoxicity causes the hepatic injury in LEC rat liver. The levels of the lipid peroxidation in symptomatic LEC rats at 4 months of age were significantly higher than those of age-matched asymptomatic LEC and normal rats. These results suggest that excessively accumulated copper provokes hepatic injury through initiating the lipid peroxidation.

Animals↗
PubMed1991

A novel species of cytochrome P-450 (P-450ib) specific for the small intestine of rabbits. cDNA cloning and its expression in COS cells.

We have isolated a cDNA clone for a P-450, designated P-450ib (Ichihara, K., Kusunose, E., Kaku, M., Yamamoto, S., and Kusunose, M. (1985) Biochim. Biophys. Acta 831, 99-105), from a cDNA library of rabbit small intestine mucosa by using synthetic DNA fragment by the polymerase chain reaction, as a hybridization probe. The cDNA with a 1,829-base pair insert encodes a polypeptide of 501 amino acids. The deduced amino acid sequence contains all of the sequences of the NH2-terminal and 14 tryptic fragments from purified P-450ib. As the NH2-terminal methionine was not found in the sequence from the purified protein, the apoprotein of P-450ib is composed of 500 amino acids with a molecular weight of 57,193. P-450ib shows 35-41% sequence similarity with several members of 8 subfamilies in the P-450 II family, whereas it has a less than 30% sequence similarity with other P-450 families, suggesting that this P-450 is the first member of a novel subfamily within the P-450 II family. RNA blot analysis shows that mRNA hybridized to the cDNA is expressed in the small intestine, but not significantly in other tissues including liver, colon, kidney, lung, spleen, brain, stomach, and cecum, indicating that P-450ib is a P-450 specific to the small intestine. The protein expressed in COS-7 cells using the cDNA in an expression vector, pKCRH2, shows benzphetamine N-demethylase activity and gives a band identical with that of P-450ib in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1991

cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity.

We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.

Amino Acid Sequence↗
PubMed1991

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Somatostatin gene expression in the developing monkey frontal and cerebellar cortices.

Somatostatin (SRIF) mRNA was determined in the developing monkey frontal and cerebellar cortices by the dot blot and the northern blot analyses at embryonic day 120 (E120), embryonic day 140 (E140), newborn stage (Nb), postnatal day 60 (P60) and adult stage (Ad.) At E120, at which time the migration of the cortical neurons had already been completed, SRIF mRNA was detectable with 50% of the maximal value at E140 in the cerebral frontal cortex (von Bonin and Bailey's area FD). After E140, the level of mRNA gradually declined to the adult level by P60 with 25% of the maximal value. In the cerebellum, SRIF mRNA was highly expressed at E120. The level decreased to 18% of the maximum at E140. Between the newborn and adult stages, there existed no positive signal of the mRNA. In contrast, both fetal and adult liver tissues contained no amounts of SRIF mRNA. We discussed the physiological meanings of the enhanced SRIF gene expression in the developing monkey cerebral and cerebellar cortices.

Aging↗
PubMed1990

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression.

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.

Amino Acid Sequence↗
PubMed1990

A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

Animals↗
PubMed1990

Functional expression of microsomal and mitochondrial cytochrome P-450 (d and SCC) in COS-7 cells from cloned cDNA.

Using full length cDNA introduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7 cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. We have succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochrome P-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COS cells remain to be studied. These results show the applicability of the COS-7 expression system to investigations of the functions of members of the P-450 superfamily whose cDNA has been newly isolated.

Animals↗
PubMed1990

Transcriptional regulation of 3-methylcholanthrene-inducible P-450 gene responsible for metabolic activation of aromatic carcinogenes.

We have investigated the transcriptional regulation of 3-methylcholanthrene (3MC)-inducible P-450c gene which is involved in the metabolic activation of polycyclic aromatic carcinogens. Reverse genetic study using the fusion gene composed of the 5' upstream sequence of P-450c gene and the structure gene for bacterial chloramphenicol acetyltransferase (CAT) and a cultured cell line of Hepa-1 cells localized two kinds of cis-acting regulatory DNA elements. One is designated XRE or xenobiotic responsive element which is responsible for the inducibility of the gene and is distributed 5 times in the region from -3.0 to -0.5 kb. The other is BTE or basal transcription element whose deletion reduces a low level of the constitutive CAT expression to a background level, and which is localized immediately upstream of the TATA sequence. Both kinds of regulatory elements are necessary for a high level of inducible expression. Gel mobility shift assay strongly suggests that the binding protein to the XRE is an Ah receptor with a specific affinity for 3MC or 2,3,7,8,tetrachlorodibenzo-p-dioxin (TCDD). Without inducer treatment, cryptic form of the binding protein occurs only in the cytoplasm of the Hepa-1 cells. Upon treatment with the inducer, the cryptic form of the binding protein exhibits binding activity to XRE and, at the same time, translocates to the nuclei. The BTE-binding protein is an ubiquitous nuclear factor and its cDNA cloning reveals the DNA-binding feature with zinc finger motifs.

Animals↗
PubMed1989

Two forms of omega-hydroxylase toward prostaglandin A and laurate. cDNA cloning and their expression.

We have isolated cDNA clones for two forms of P-450s, P-450ka-1 and P-450ka-2, from a rabbit kidney cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase (Matsubara, S., Yamamoto, S., Sogawa, K., Yokotani, N., Fujii-Kuriyama, Y., Haniu, M., Shively, J.E., Gotoh, O., Kusunose, E., and Kusunose, M. (1987) J. Biol. Chem. 262, 13366-13371), as a hybridization probe. The cDNAs for P-450ka-1 and P-450ka-2 encode polypeptides of 510 and 511 amino acids, respectively, with sequence similarity of 85% and 87% to P-450p-2. The two deduced primary structures have 87% identity. RNA blot analysis demonstrated that the mRNAs for P-450ka-1 and P-450ka-2 formed single bands at approximately 3.0- and 2.6-kilobase positions, respectively. The mRNA for P-450ka-1 was expressed only in the liver and kidney and was increased remarkably in these tissues by the administration of clofibrate. In contrast, the mRNA for P-450ka-2 was expressed constitutively in the liver, kidney, and small intestine, but its transcription was enhanced only in the liver by clofibrate treatment. Thus, in spite of their high sequence similarity, these P-450 species have different modes of regulatory expression. Comparison of the nucleotide sequences among P-450ka-1, P-450ka-2, and P-450p-2 shows about 90% overall sequence similarity in any pair of the three sequences. Nucleotide replacements are not evenly distributed, but are rather biased. There is a region of approximately 500 base pairs of exceptionally high homology among the three sequences. These results indicate that the gene conversion event occurred during the evolutionary process of these genes.

Amino Acid Sequence↗
PubMed1989

Primary structure and transcriptional regulation of rat pepsinogen C gene.

The entire rat pepsinogen C gene has been isolated from a rat genomic library, using the rat pepsinogen C cDNA as a probe. Southern blot analysis showed that there exists at least two rat pepsinogen C genes. The nucleotide sequences of the coding regions and the 5'- and 3'-flanking regions of one of the rat pepsinogen C genes have been determined. This gene is split into 9 exons interrupted by eight intervening sequences. The 5'-flanking region is similar to that of the human pepsinogen C gene, but only the former has the core sequence of the Sp1 binding site. The amount of transcripts of the rat pepsinogen C genes was found to increase during development, and a similar increase was shown to be induced by injection of hydrocortisone. As a candidate of a factor which regulates the transcription, we found a 25-kDa protein by Southwestern blotting. It binds to a specific site in the 5'-flanking region of the gene only in the presence of Mg2+ ion, and it is present in the nuclear fraction of the gastric mucosa but not of the liver.

Amino Acid Sequence↗
PubMed1989

Repression of cytochrome P-450c gene expression by cotransfection with adenovirus E1a DNA.

Gene expression of rat cytochrome P-450c (P-450c) depends upon inducible enhancers scattered in the 5'-upstream region of the gene. We show that expression of the P-450c gene is repressed by contransfection with adenovirus E1a DNA, regardless of the presence or absence of inducers, in a transient expression system of HeLa cells. Since cotransfection of either 13S or 12S E1a cDNA was effective in the repression, the region necessary for repression could be separated from that of transactivation of other adenovirus early genes. Moreover, we investigated the regions responsible for the inhibitory activity using in-frame deletion mutants lacking internal or external portions of the E1a proteins. The sequence responsible for the repression was located in the amino-terminal half of the E1a proteins. The inducible expression of the chimeric plasmid containing a 24-base-pair enhancer sequence of the P-450c gene placed in a heterologous promoter of SV40 was repressed by cotransfection with E1a DNA, suggesting strongly that the inhibitory effect of the E1a proteins upon P-450c gene expression was caused by blocking the enhancer activity.

Adenoviridae↗
PubMed1989

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions.

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.

Aging↗
PubMed1988

DNA methylation and expression of the rat pepsinogen gene in embryonic, adult, and neoplastic tissues.

The relationship between methylation and expression of rat pepsinogen 1 (Pg1) genes was investigated in various tissues. On Northern blotting with a Pg1 complementary DNA probe, Pg1 mRNA was detected only in the glandular stomach of normal rats. Methylation analysis with Msp1/HpaII and Hha1 revealed tissue specific methylation patterns of Pg1 genes with less methylated in the stomach than in other normal tissues not expressing the genes. During stomach development, there was a progressive increase in the Pg1 mRNA level that almost coincided with change in the mucosal pepsinogen level and progressive demethylation after the onset of transcription. Thus, there was an inverse correlation between methylation and expression of Pg1 genes, suggesting a role of DNA methylation in Pg1 gene regulation during normal differentiation, although not its primary role in gene activation. There was no detectable Pg1 mRNA in either primary or transplanted stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine. The methylation patterns of Pg1 genes were different from those of normal tissues that expressed the gene and of those that did not and no simple correlation was observed between methylation and expression of Pg1 genes. This result is consistent with a previous finding that DNA methylation is deranged in tumor cells.

Animals↗