Journal Article Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase Get access Yumi Kanegae, Kanegae 1Laboaory of Molecular Genetics University Tokyo4-6-1 Shirokanedi, Minato-ku, Tokyo 108, Japan Search for other works this author on: Oxford Academic PubMed Google Scholar Gwang Lee, Lee Sato, Sato Mieko Tanaka, Tanaka Michio Nakal, Nakal Japan3Discovery Research Laboratories III, Sumitomo Pharmaceuticals Center3-1-98...

Intestinal cancer is one of the most common human cancers. Aberrant activation canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation β-catenin, resulting in initiation intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles regulating intracellular protein levels both as a ligand-activated transcription factor ligand-dependent E3 ubiquitin ligase. Here, we show that AhR...

UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation of a variety xenobiotics and endobiotics. UGTs are divided into two families, UGT1 UGT2. The purpose this study was to estimate the absolute expression levels each UGT isoform in human liver evaluate interindividual variability. Real-time reverse transcriptase-polymerase chain reaction analysis performed determine copy numbers nine functional UGT1A isoforms seven UGT2B isoforms. We noticed that not only primers but also templates...

Rat liver cytochrome P-450MC cDNA was inserted between the ADH1 promoter and terminator regions of yeast expression vector pAAH5. On introduction resulting recombinant plasmid pAMC1, Saccharomyces cerevisiae cells synthesized up to 8 × 105 molecules per cell protein, most which localized in microsomes. Approximately half contained heme enzyme molecule. These formed a functional electron-transport chain microsomes exhibited aryl hydrocarbon hydroxylase activity toward benzo[a]pyrene.

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. (designated CYP719) belonged to a novel family. Further, heterologous expression yeast confirmed it had same activity as methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes conversion (S)-tetrahydrocolumbamine ((S)-THC) (S)-tetrahydroberberine...

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D₃ metabolism. In this study, we show the inducible enzyme CYP3A4 a major source of oxidative metabolism 1α,25-dihydroxyvitamin [1,25(OH)₂D₃] human liver and small intestine could contribute adverse effect. Heterologously-expressed catalyzed 23- 24-hydroxylation 1,25(OH)₂D₃. No microsomal...

Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) development those disorders two individuals.Two patients elevated levels or nephrolithiasis were investigated at National Institutes Health (NIH) Clinical Center NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, metabolites, performing CYP24A1 mutation analysis.Both exhibited hypercalciuria,...

Streptomyces griseolus CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that wild type variants metabolize 12 types nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited high against many NSAIDs. We successfully crystallized complexes wild-type (WT) diclofenac (DIF) or flufenamic acid (FLF). WT, carboxyl group...

Recently, we demonstrated that the alopecia observed in vitamin D receptor gene-deficient (Vdr-KO) rats is not seen with a mutant VDR(R270L/H301Q), which lacks ligand-binding ability, suggesting ligand-independent action of VDR plays crucial role maintaining hair cycle. Since Vdr-KO also showed abnormalities skin, relationship between and skin was examined. To clarify mechanism actions protein composition, gene expression patterns were compared among Vdr-KO, Vdr-R270L/H301Q, wild-type (WT)...

Human 25‐hydroxyvitamin D 3 (25(OH)D ) 24‐hydroxylase (CYP24) cDNA was expressed in Escherichia coli , and its enzymatic spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. cells, adrenodoxin reductase examined for metabolism of 25(OH)D 1α,25(OH) 2 their related compounds. CYP24 demonstrated a remarkable consisting both C‐23 C‐24 hydroxylation pathways towards whereas rat showed almost no pathway [Sakaki, T. Sawada, N....

Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in liver. <i>WND</i> (<i>ATP7B</i>) gene, which encodes a putative transporting P-type ATPase, defective patients. To investigate <i>in vivo</i> function WND protein as well its intracellular localization, <i>WND</i>cDNA was introduced to Long-Evans Cinnamon rat, known rodent model for recombinant adenovirus-mediated gene delivery. An immunofluorescent study and subcellular...

The coding region of the cDNA for rat kidney calcidiol 24‐hydroxylase ( P 450cc24), which is involved in calcium homeostasis animals, was inserted into an expression vector pKK223‐3. recombinant plasmid formed a specific manner without deletion or substitution any parts cDNA. When resulting introduced Escherichia coli JM109, cells produced protein immunoreactive to antibody against 450cc24. cell‐free extract transformed incubated with together bovine adrenodoxin and NADPH‐adrenodoxin...

Recently, it was revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and 24R,25-dihydroxyvitamin (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 A-ring. We now report isolation structural assignment 3-epi-25-hydroxyvitamin (3-epi-25(OH)D3 as a major metabolite 25-hydroxyvitamin (25(OH)D3) further metabolism vitamin metabolites. When 25(OH)D3 incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, hepatoblastoma cell...

A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and NADPH-cytochrome P450 reductase was constructed by combining the with fragment protease-solubilized moiety reductase. The inserted between yeast alcohol dehydrogenase I promoter terminator expression vector pAAH5 to yield plasmid pAMP19. Saccharomyces cerevisiae AH22 cells transformed pAMP19 produced 130-kD protein reactive both anti-cytochrome Ig antireductase Ig. containing exhibited about four times higher...

Ethanol extracts from 15 kinds of marine algae collected the coast Noto Peninsula in Japan were examined for their inhibitory effects on human salivary α-amylase. Four significantly suppressed enzyme activity. An inhibitor was purified extract Sargassum patens. The compound a new phloroglucinol derivative, 2-(4-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy) benzene-1,3,5-triol (DDBT), which strongly hydrolysis amylopectin by and pancreatic α-amylases. 50% activity (IC50) α-amylase inhibition...

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the was constructed by insertion of corresponding cDNA into vector pNG01 under control cauliflower mosaic virus 35S promoter nopaline synthase gene terminator. integrated genomes Agrobacterium infection techniques. In transgenic plants, protein localized...

Recombinant plasmids for expression of bovine cytochrome P450c21 (pAγ2), both and yeast NADPH-cytochrome P450 reductase (pARγ1), P450c21/yeast fused enzymes (pAFγR1, pAFγR2, pAFγR20), reductase/P450c21 (pAFRγ1 pAFRγ2) were constructed by using vector pAAH5. The each introduced into the Saccharomyces cerevisiae AH22 cells. recombinant strains AH22/pAγ2 (Y21) AH22/pARγ1 (Y21R) produced 2–3 × 103 molecules per cell. cultures converted progesterone 17α-hydroxyprogesterone 11-deoxycorticosterone...

Vitamin D3 (VD3), a prohormone in mammals, plays crucial role the maintenance of calcium and phosphorus concentrations serum. Activation VD3 requires 25-hydroxylation liver 1α-hydroxylation kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts into 1α,25-dihydroxyvitamin (1α,25(OH)2D3) two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined crystal structures highly active mutant (R84A) from Streptomyces...

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon produced by cigarette combustion, is implicated as causative agent in smoking-related cancer and atherosclerosis. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], potent ligand for the nuclear receptor vitamin D (VDR), has been shown to decrease risk of osteoporosis, some types cardiovascular disease, suggesting an opposing effect smoking. In this study, we investigated effects BaP on signaling pathway. effectively enhanced 1,25(OH)2D3-dependent...