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NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4 总被引:2,自引:0,他引:2
P M Shaw A Reiss M Adesnik D W Nebert J Schembri A K Jaiswal 《European journal of biochemistry》1991,195(1):171-176
NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate. 相似文献
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Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R-/- mice by 40-50%. On the other hand, renal COX-1 levels were similar in B2R-/- and +/+ mice. Renal renin protein was 61% lower in B2R-/- compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R-/- mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R-/- mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261+/-16 and 266+/-15fmol/g in B2R+/+ and B2R-/- mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R-/- mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression. 相似文献
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We cloned and sequenced the cDNA of a potent tumor transforming gene (TUTR1) from human testis and determined its primary structure. The TUTR1 cDNA is composed of 656 nucleotides and encodes a novel protein of 202 amino acids. The predicted TUTR1 protein is extremely hydrophilic and contains two proline-rich motifs at its C-terminus. Northern blot analysis of the mRNA from various human tissues and tumors revealed that TUTR1 mRNA is highly expressed in tumors of the pituitary gland, adrenal gland, ovary, endometrium, liver, uterus, and kidney as well as in cell lines derived from tumors of the pituitary, breast, endometrium, and ovary. With the exception of the testis, the levels of TUTR1 mRNA were either very low or undetectable in normal human tissues. Overexpression of TUTR1 in mouse fibroblasts (NIH 3T3) cells resulted in an increase in cell proliferation, induced cellular transformation in vitro, and promoted tumor formation in nude mice. These results suggest that TUTR1 is a novel and potent transforming gene, which may be involved in tumorigenesis in numerous different human tumors. 相似文献
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mRNA and protein levels of the stimulatory guanine nucleotide regulatory protein Gs are lower in the testis of obese (ob/ob) mice 总被引:1,自引:0,他引:1
Probing of total testis RNA with a cDNA corresponding to the alpha s subunit of the guanine nucleotide regulatory protein (G-protein) showed that levels of mRNA were markedly reduced in the ob/ob mouse compared to its +/+ control. The lowered level of mRNA resulted in lowered protein synthesis as shown by a marked decrease in both the 48,000 and 42,000 Mr peptides detected by (1) cholera toxin ribosylation, (2) immunodetection with an antibody specific for alpha s. This was not the result of overall lowered protein synthesis since the levels of alpha i2 and the beta subunits were unchanged. In the kidney there was no change in the levels of the alpha s subunit in obese membrane preparations. Lowered levels of alpha s were not correlated with changes in adenylyl cyclase activity. This suggests that in these membranes the G-protein subunit(s) are present in excess compared to the catalytic unit of adenylyl cyclase and that in the testis the G-proteins may be of more importance in other signalling pathways necessary for normal gonadal development and fertility. 相似文献
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Gaikwad A Long DJ Stringer JL Jaiswal AK 《The Journal of biological chemistry》2001,276(25):22559-22564
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that utilizes NAD(P)H as an electron donor, catalyzing the two-electron reduction and detoxification of quinones and their derivatives. NQO1-/- mice deficient in NQO1 activity and protein were generated in our laboratory (Rajendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J. P., Gonzalez, F. J., and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389). Mice lacking a functional NQO1 gene (NQO1-/-) were born normal and reproduced adeptly as the wild-type NQO1+/+ mice. In the present report, we show that NQO1-/- mice exhibit significantly lower levels of abdominal adipose tissue as compared with the wild-type mice. The NQO1-/- mice showed lower blood levels of glucose, no change in insulin, and higher levels of triglycerides, beta-hydroxy butyrate, pyruvate, lactate, and glucagon as compared with wild-type mice. Insulin tolerance test demonstrated that the NQO1-/- mice are insulin resistant. The NQO1-/- mice livers also showed significantly higher levels of triglycerides, lactate, pyruvate, and glucose. The liver glycogen reserve was found decreased in NQO1-/- mice as compared with wild-type mice. The livers and kidneys from NQO1-/- mice also showed significantly lower levels of pyridine nucleotides but an increase in the reduced/oxidized NAD(P)H:NAD(P) ratio. These results suggested that loss of NQO1 activity alters the intracellular redox status by increasing the concentration of NAD(P)H. This leads to a reduction in pyridine nucleotide synthesis and reduced glucose and fatty acid metabolism. The alterations in metabolism due to redox changes result in a significant reduction in the amount of abdominal adipose tissue. 相似文献
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Matsui H Takano N Takahashi T 《The international journal of biochemistry & cell biology》2005,37(11):2333-2343
Mouse kallikrein 24 is thought to encode a functional serine protease belonging to the mouse glandular kallikrein gene family. Preliminary results suggest that this kallikrein may play a role in testis function in adult mice. In order to obtain insights into its physiological functions, we undertook molecular and biochemical analyses of this enzyme. We cloned a cDNA for kallikrein 24 from the adult mouse testis cDNA library. Kallikrein 24 was expressed in the kidney, submandibular glands, ovary, epididymis, and testis of the mouse. In the testis, kallikrein 24 mRNA was detectable at 4 weeks of postnatal development, and became more prominent thereafter. The kallikrein 24 gene was expressed exclusively in the Leydig cells of adult mice. When Leydig cells isolated from a 2-week-old mouse testis were cultured in the presence of testosterone, kallikrein 24 expression was induced. Active recombinant enzyme showed trypsin-like specificity, favorably cleaving Arg-X bonds of synthetic peptide substrates. The enzymatic activity was strongly inhibited by typical serine protease inhibitors. Mouse kallikrein 24 degraded casein, gelatin, fibronectin and laminin. These results suggest that the enzyme may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis. The present findings should contribute to future physiological studies of this mouse testis protease. 相似文献
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M Kono M Sekine K Nakamura Y Hashimoto Y Seyama T Yamakawa A Suzuki 《Journal of biochemistry》1991,109(1):132-136
We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene 总被引:3,自引:0,他引:3
Tohru Kitada Shuichi Asakawa Shinsei Minoshima Yoshikuni Mizuno Nobuyoshi Shimizu 《Mammalian genome》2000,11(6):417-421
We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for
the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading
frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin
protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity =
89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading
frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing
variant by 3′-RACE method.
Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that
mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse
parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals.
Received: 5 May 1999 / Accepted: 11 February 2000 相似文献
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Hideo Satsu Emi Chidachi Yuto Hiura Haru Ogiwara Yusuke Gondo Makoto Shimizu 《Amino acids》2012,43(4):1547-1555
In this study, we examined the effects of 20 amino acids on the expression level of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human intestinal LS180 cells. Five amino acids were associated with significant increases in NQO1 mRNA expression; the most substantial increase was induced by cysteine, which markedly increased the NQO1 mRNA level in a time- and dose-dependent manner. Cysteine also increased the protein level of NQO1 and its enzymatic activity in LS180 cells. Furthermore, cysteine significantly up-regulated NQO1 promoter activity, and this induction was completely abolished by mutation of the antioxidant response element, a binding site of the nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown experiment using siRNA against Nrf2 showed the involvement of Nrf2 on cysteine-induced increase in NQO1 mRNA expression. Further, cysteine treatment increased the amount of Nrf2 protein in the nucleus and decreased the amount of Kelch-like ECH-associated protein 1 (a suppressor protein of Nrf2) in the cytosol, suggesting that Nrf2 was activated by cysteine. Oral administration of cysteine to mice significantly increased NQO1 mRNA levels in the mouse intestinal mucosa. These findings show that cysteine induces NQO1 expression in both in vitro and in vivo systems and also suggest that Nrf2 activation is involved in this induction. 相似文献
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Yamanaka M Koga M Tanaka H Nakamura Y Ohta H Yomogida K Tsuchida J Iguchi N Nojima H Nozaki M Matsumiya K Okuyama A Toshimori K Nishimune Y 《Biology of reproduction》2000,62(6):1694-1701
We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining. 相似文献
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We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice. 相似文献