75 results on '"Steroid Hydroxylases immunology"'
Search Results
2. Regulating Innate and Adaptive Immunity for Controlling SIV Infection by 25-Hydroxycholesterol.
- Author
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Wu T, Ma F, Ma X, Jia W, Pan E, Cheng G, Chen L, and Sun C
- Subjects
- Animals, CD4-Positive T-Lymphocytes pathology, CD4-Positive T-Lymphocytes virology, Cell Line, Female, Macaca, Macrophages pathology, Macrophages virology, Mice, Mice, Knockout, Simian Acquired Immunodeficiency Syndrome genetics, Simian Acquired Immunodeficiency Syndrome pathology, Steroid Hydroxylases genetics, Steroid Hydroxylases immunology, CD4-Positive T-Lymphocytes immunology, Hydroxycholesterols immunology, Immunity, Cellular, Immunity, Innate, Macrophages immunology, Simian Acquired Immunodeficiency Syndrome immunology, Simian Immunodeficiency Virus immunology
- Abstract
Persistent inflammation and extensive immune activation have been associated with HIV-1/SIV pathogenesis. Previously, we reported that cholesterol-25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25-HC) had a broad antiviral activity in inhibiting Zika, Ebola, and HIV-1 infection. However, the underlying immunological mechanism of CH25H and 25-HC in inhibiting viral infection remains poorly understood. We report here that 25-HC effectively regulates immune responses for controlling viral infection. CH25H expression was interferon-dependent and induced by SIV infection in monkey-derived macrophages and PBMC cells, and 25-HC inhibited SIV infection both in permissive cell lines and primary monkey lymphocytes. 25-HC also strongly inhibited bacterial lipopolysaccharide (LPS)-stimulated inflammation and restricted mitogen-stimulated proliferation in primary monkey lymphocytes. Strikingly, 25-HC promoted SIV-specific IFN-γ-producing cellular responses, but selectively suppressed proinflammatory CD4+ T lymphocytes secreting IL-2 and TNF-α cytokines in vaccinated mice. In addition, 25-HC had no significant immunosuppressive effects on cytotoxic CD8+ T lymphocytes or antibody-producing B lymphocytes. Collectively, 25-HC modulated both innate and adaptive immune responses toward inhibiting HIV/SIV infection. This study provides insights into improving vaccination and immunotherapy regimes against HIV-1 infection.
- Published
- 2018
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3. Interferons: Reprogramming the Metabolic Network against Viral Infection.
- Author
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Raniga K and Liang C
- Subjects
- Acetyltransferases immunology, Animals, Humans, Indoleamine-Pyrrole 2,3,-Dioxygenase immunology, Macaca mulatta, Mice, SAM Domain and HD Domain-Containing Protein 1 immunology, Steroid Hydroxylases immunology, Virus Replication, Immunity, Innate, Interferons immunology, Metabolic Networks and Pathways immunology, Signal Transduction immunology, Virus Diseases immunology
- Abstract
Viruses exploit the host and induce drastic metabolic changes to ensure an optimal environment for replication and the production of viral progenies. In response, the host has developed diverse countermeasures to sense and limit these alterations to combat viral infection. One such host mechanism is through interferon signaling. Interferons are cytokines that enhances the transcription of hundreds of interferon-stimulated genes (ISGs) whose products are key players in the innate immune response to viral infection. In addition to their direct targeting of viral components, interferons and ISGs exert profound effects on cellular metabolism. Recent studies have started to illuminate on the specific role of interferon in rewiring cellular metabolism to activate immune cells and limit viral infection. This review reflects on our current understanding of the complex networking that occurs between the virus and host at the interface of cellular metabolism, with a focus on the ISGs in particular, cholesterol-25-hydroxylase (CH25H), spermidine/spermine acetyltransferase 1 (SAT1), indoleamine-2,3-dioxygenase (IDO1) and sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1), which were recently discovered to modulate specific metabolic events and consequently deter viral infection., Competing Interests: The authors declare no conflict of interests.
- Published
- 2018
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4. Cholesterol metabolism and immunity.
- Author
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Simon A
- Subjects
- Animals, Hydroxycholesterols metabolism, Inflammation metabolism, Interferon Type I immunology, Mevalonate Kinase Deficiency immunology, Mevalonate Kinase Deficiency metabolism, Mice, Mice, Knockout, Phosphotransferases (Alcohol Group Acceptor) genetics, Steroid Hydroxylases genetics, Hydroxycholesterols immunology, Inflammation immunology, Interleukin-1beta antagonists & inhibitors, Steroid Hydroxylases immunology
- Abstract
A study in mice implicates a cholesterol derivative in damping down the inflammatory response mediated by interleukin-1β and explaining, at least in part, the immunosuppressive effect of type I interferon, which is used in the treatment of multiple sclerosis.
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- 2014
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5. Inflammation. 25-Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon.
- Author
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Reboldi A, Dang EV, McDonald JG, Liang G, Russell DW, and Cyster JG
- Subjects
- Animals, Encephalomyelitis, Autoimmune, Experimental genetics, Encephalomyelitis, Autoimmune, Experimental immunology, Feedback, Physiological, Inflammasomes genetics, Inflammasomes immunology, Inflammation immunology, Inflammation microbiology, Interleukin-1 immunology, Macrophage Activation, Macrophages immunology, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Response Elements genetics, Shock, Septic genetics, Shock, Septic immunology, Steroid Hydroxylases genetics, Hydroxycholesterols metabolism, Inflammation genetics, Interferon Type I immunology, Steroid Hydroxylases immunology
- Abstract
Type I interferon (IFN) protects against viruses, yet it also has a poorly understood suppressive influence on inflammation. Here, we report that activated mouse macrophages lacking the IFN-stimulated gene cholesterol 25-hydroxylase (Ch25h) and that are unable to produce the oxysterol 25-hydroxycholesterol (25-HC) overproduce inflammatory interleukin-1 (IL-1) family cytokines. 25-HC acts by antagonizing sterol response element-binding protein (SREBP) processing to reduce Il1b transcription and to broadly repress IL-1-activating inflammasomes. In accord with these dual actions of 25-HC, Ch25h-deficient mice exhibit increased sensitivity to septic shock, exacerbated experimental autoimmune encephalomyelitis, and a stronger ability to repress bacterial growth. These findings identify an oxysterol, 25-HC, as a critical mediator in the negative-feedback pathway of IFN signaling on IL-1 family cytokine production and inflammasome activity., (Copyright © 2014, American Association for the Advancement of Science.)
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- 2014
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6. Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell-autonomous activation of vitamin D in dendritic cells.
- Author
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Kundu R, Chain BM, Coussens AK, Khoo B, and Noursadeghi M
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- Dendritic Cells cytology, Female, Homeostasis physiology, Humans, Macrophages cytology, Macrophages immunology, Male, Monocytes cytology, Monocytes immunology, T-Lymphocytes cytology, Vitamin D3 24-Hydroxylase, 25-Hydroxyvitamin D3 1-alpha-Hydroxylase immunology, Calcitriol immunology, Dendritic Cells immunology, Lymphocyte Activation physiology, Steroid Hydroxylases immunology, T-Lymphocytes immunology
- Abstract
The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]₂ D) potently inhibits DC priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]₂ D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1, and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25-dihydroxyvitamin D to 1,25[OH]₂ D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]₂ D, upregulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]₂ D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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7. Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses.
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Yi T, Wang X, Kelly LM, An J, Xu Y, Sailer AW, Gustafsson JA, Russell DW, and Cyster JG
- Subjects
- 3-Hydroxysteroid Dehydrogenases genetics, 3-Hydroxysteroid Dehydrogenases immunology, 3-Hydroxysteroid Dehydrogenases metabolism, Animals, Bone Marrow Cells immunology, Bone Marrow Cells metabolism, Cells, Cultured, Cytochrome P450 Family 7, Female, Flow Cytometry, Gene Expression, HEK293 Cells, Humans, Hydroxycholesterols metabolism, Lymphocyte Activation immunology, Lymphoid Tissue immunology, Lymphoid Tissue metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled immunology, Receptors, G-Protein-Coupled metabolism, Reverse Transcriptase Polymerase Chain Reaction, Steroid Hydroxylases genetics, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Stromal Cells metabolism, B-Lymphocytes immunology, Cell Movement immunology, Hydroxycholesterols immunology, Immunity, Humoral immunology, Stromal Cells immunology
- Abstract
7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
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8. Steroid requirements and immune associations with vitamin D are stronger in children than adults with asthma.
- Author
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Goleva E, Searing DA, Jackson LP, Richers BN, and Leung DY
- Subjects
- Adolescent, Adult, Age Factors, Cells, Cultured, Child, Dexamethasone therapeutic use, Female, Humans, Immunoglobulin E blood, Interleukin-13 genetics, Interleukin-13 metabolism, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Male, Middle Aged, Prospective Studies, Steroid Hydroxylases genetics, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism, Vitamin D3 24-Hydroxylase, Young Adult, Adrenal Cortex Hormones administration & dosage, Asthma blood, Asthma drug therapy, Asthma immunology, Vitamin D blood
- Abstract
Background: The effects of serum vitamin D status on atopy, steroid requirement, and functional responsiveness to corticosteroids in children versus adults with asthma have not been studied systematically., Objective: We sought to explore the age-specific effects of vitamin D in asthmatic patients., Methods: Serum vitamin D levels were examined in a prospective study of adults and children (102 healthy control subjects and 103 asthmatic patients). PBMCs were cultured for 3 hours with or without 100 nmol/L dexamethasone, and the expression of corticosteroid-regulated genes was detected by using real-time PCR. Serum IgE levels were measured, and information about asthmatic patients' steroid requirements was collected., Results: Deficient serum vitamin D levels (<20 ng/mL) were found in 47.6% of asthmatic patients and 56.8% of healthy control subjects, with means ± SDs of 20.7 ± 9.8 and 19.2 ± 7.7 ng/mL, respectively. In multivariate regression models a significant positive correlation between serum vitamin D levels and the expression of vitamin D-regulated targets, cytochrome P450, family 24, subfamily a (cyp24a) expression by PBMCs (P = .0084, pediatric asthma group only) and serum LL-37 levels (P = .0006 in the pediatric group but P = .0067 in the adult asthma group), was found. An inverse association between vitamin D and serum IgE levels was observed in the pediatric (P = .006) asthma group. Serum vitamin D level (P = .05), as well as PBMC cyp24a expression (P = .0312), demonstrated a significant inverse relationship with daily inhaled corticosteroid dose in the pediatric asthma group only. Cyp24a expression in PBMCs correlated positively with in vitro suppression of TNF-α by dexamethasone (P = .05) and IL-13 (P = .0094) in PBMCs in the pediatric asthma group only., Conclusions: This study demonstrated significant associations between serum vitamin D status and steroid requirement and in vitro responsiveness to corticosteroids in the pediatric but not the adult asthma group. Vitamin D was also related to IgE levels in children but not in adults., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)
- Published
- 2012
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9. Immunochemical detection of cytochrome P450 enzymes in liver microsomes of 27 cynomolgus monkeys.
- Author
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Uehara S, Murayama N, Nakanishi Y, Zeldin DC, Yamazaki H, and Uno Y
- Subjects
- Animals, Anticoagulants metabolism, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Coumarins metabolism, Cytochrome P-450 CYP3A immunology, Cytochrome P-450 CYP3A metabolism, Cytochrome P-450 Enzyme System biosynthesis, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System immunology, Enzyme Assays, Female, Humans, Hydroxylation, Immunoblotting, Inactivation, Metabolic, Macaca fascicularis, Male, Microsomes, Liver metabolism, Oxazines metabolism, Sensitivity and Specificity, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver enzymology
- Abstract
The cynomolgus monkey is widely used as a primate model in preclinical studies because of its evolutionary closeness to humans. Despite their importance in drug metabolism, the content of each cytochrome P450 (P450) enzyme has not been systematically determined in cynomolgus monkey livers. In this study, liver microsomes of 27 cynomolgus monkeys were analyzed by immunoblotting using selective P450 antibodies. The specificity of each antibody was confirmed by analyzing the cross-reactivity against 19 CYP1-3 subfamily enzymes using recombinant proteins. CYP2A, CYP2B6, CYP2C9/19, CYP2C76, CYP2D, CYP2E, CYP3A4, and CYP3A5 were detected in all 27 animals. In contrast, CYP1A, CYP1D, and CYP2J were below detectable levels in all liver samples. The average content of each P450 showed that among the P450s analyzed CYP3A (3A4 and 3A5) was the most abundant (40% of total immunoquantified P450), followed by CYP2A (25%), CYP2C (14%), CYP2B6 (13%), CYP2E1 (11%), and CYP2D (3%). No apparent sex differences were found for any P450. Interanimal variations ranged from 2.6-fold (CYP3A) to 11-fold (CYP2C9/19), and most P450s (CYP2A, CYP2D, CYP2E, CYP3A4, and CYP3A5) varied 3- to 4-fold. To examine the correlations of P450 content with enzyme activities, metabolic assays were performed in 27 cynomolgus monkey livers using 7-ethoxyresorufin, coumarin, pentoxyresorufin, flurbiprofen, bufuralol, dextromethorphan, and midazolam. CYP2D and CYP3A4 contents were significantly correlated with typical reactions of human CYP2D (bufuralol 1'-hydroxylation and dextromethorphan O-deethylation) and CYP3A (midazolam 1'-hydroxylation and 4-hydroxylation). The results presented in this study provide useful information for drug metabolism studies using cynomolgus monkeys.
- Published
- 2011
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10. Calcitriol inhibits TNF-alpha-induced inflammatory cytokines in human trophoblasts.
- Author
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Díaz L, Noyola-Martínez N, Barrera D, Hernández G, Avila E, Halhali A, and Larrea F
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- 3-Hydroxysteroid Dehydrogenases antagonists & inhibitors, 3-Hydroxysteroid Dehydrogenases metabolism, Aromatase genetics, Aromatase immunology, Aromatase metabolism, Calcitriol analogs & derivatives, Calcitriol metabolism, Calcitriol pharmacology, Cells, Cultured, Chorionic Gonadotropin genetics, Chorionic Gonadotropin immunology, Chorionic Gonadotropin metabolism, Dose-Response Relationship, Immunologic, Female, Gene Expression Regulation, Developmental drug effects, Gene Expression Regulation, Developmental immunology, Humans, Immune Tolerance drug effects, Interferon-gamma genetics, Interferon-gamma metabolism, Interleukin-6 genetics, Interleukin-6 metabolism, Pregnancy, Progesterone Reductase genetics, Progesterone Reductase immunology, Progesterone Reductase metabolism, Receptors, Calcitriol antagonists & inhibitors, Steroid Hydroxylases genetics, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Trophoblasts drug effects, Trophoblasts metabolism, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha immunology, Vitamin D3 24-Hydroxylase, Calcitriol immunology, Placental Hormones immunology, Pregnancy Complications immunology, Trophoblasts immunology, Tumor Necrosis Factor-alpha metabolism
- Abstract
Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-alpha (TNF-alpha)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-alpha. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and TNF-alpha were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-alpha on CYP24A1, chorionic gonadotropin (hCG), 3beta-hydroxysteroid dehydrogenase (HSD3B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-alpha stimulated IL-6, IFN-gamma and its own expression more than 3-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-alpha also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-alpha induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-alpha inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.
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- 2009
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11. [Influence repeated stress on immune responciveness and monooxigenase activity of normotensive and hypertensive rats].
- Author
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Tseĭlikman VE, Kozochkin DA, Markel' AL, Tseĭlikman OB, Sibiriak SV, Sinitskiĭ AI, Sysakov DA, and Simbitrsev AS
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Cytochrome P-450 CYP1A1 immunology, Cytochrome P-450 CYP1A1 metabolism, Cytochrome P-450 CYP2B1 immunology, Cytochrome P-450 CYP2B1 metabolism, Hypersensitivity, Delayed enzymology, Hypertension enzymology, Mixed Function Oxygenases metabolism, Rats, Rats, Wistar, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Stress, Physiological enzymology, Hypersensitivity, Delayed immunology, Hypertension immunology, Mixed Function Oxygenases immunology, Stress, Physiological immunology
- Abstract
Repeated stress led to antipodal directions in immune system and cytochrome P450 activities of normotensive and hypertensive rats. Enhancement of the Reaction of Delayed Hypersensitivity, suppression of cytochrome P450-mediated monooxigenase activities were observed in Wistar rats. On the contrary, in the NISAG decrease of the Reaction Delayed Hypersensitivity, elevation of cytochrome P450-mediated monooxigenase activities were observed, as comparison with Wistar rats.
- Published
- 2008
12. Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain.
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Ramirez DM, Andersson S, and Russell DW
- Subjects
- Animals, Cells, Cultured, Cholesterol 24-Hydroxylase, Endoplasmic Reticulum metabolism, Enzyme-Linked Immunosorbent Assay, Female, Humans, Immunoblotting, Immunohistochemistry, Mice, Mice, Inbred BALB C, Mice, Knockout, Rats, Recombinant Proteins immunology, Steroid Hydroxylases immunology, Transfection, Antibodies, Monoclonal immunology, Antibody Specificity immunology, Brain metabolism, Neurons metabolism, Steroid Hydroxylases metabolism
- Abstract
Cholesterol 24-hydroxylase is a cytochrome P450 (CYP46A1) that is selectively expressed in the brain and is responsible for the majority of cholesterol turnover in the central nervous system. Mice deficient in 24-hydroxylase exhibit impaired learning and defective hippocampal long-term potentiation, suggesting that the metabolism of cholesterol by this enzyme is required for learning and memory formation. To determine where in the neuron cholesterol turnover was taking place, monoclonal antibodies directed against 24-hydroxylase were generated by immunization of mice with recombinant protein and used to detect the enzyme in brain homogenates, cultured neurons, and histological sections. 24-Hydroxylase was localized to the endoplasmic reticulum and was distributed throughout the cell bodies and dendrites of multiple types of neurons; the enzyme was not detected in axon terminals or in the cells of 24-hydroxylase knockout mice. 24-Hydroxylase was highly expressed in pyramidal neurons of the hippocampus and cortex, in Purkinje cells of the cerebellum, and in hippocampal and cerebellar interneurons. Within the retina, 24-hydroxylase was detected in ganglion cells and some but not all cells of the inner nuclear layer. These findings reveal the microsomal localization of 24-hydroxylase and provide subcellular insight into cholesterol turnover in the brain., ((c) 2008 Wiley-Liss, Inc.)
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- 2008
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13. The controversial role of vitamin D in the skin: immunosuppression vs. photoprotection.
- Author
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Kuritzky LA, Finlay-Jones JJ, and Hart PH
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- Animals, DNA radiation effects, DNA Damage immunology, Dose-Response Relationship, Immunologic, Humans, Immune Tolerance drug effects, Immune Tolerance physiology, Langerhans Cells drug effects, Langerhans Cells immunology, Langerhans Cells radiation effects, Mice, Skin immunology, Skin radiation effects, Steroid Hydroxylases adverse effects, Steroid Hydroxylases immunology, Vitamin D pharmacology, Vitamin D physiology, Vitamin D analogs & derivatives
- Abstract
Vitamin D is produced in the skin by ultraviolet (UV) B radiation (290-320 nm). The active metabolite 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is made systemically by hydroxylation of vitamin D in the liver and the kidney, but also locally in the epidermis, which suggests that 1,25(OH)(2)D(3) may have important functions in the skin. 1,25(OH)(2)D(3) has opposing effects: it can mimic immunosuppressive effects caused by UV irradiation in some models, or reverse UV-induced DNA damage and immunosuppression in other models. 1,25(OH)(2)D(3) exerts effects on Langerhans cells that are characteristic of those associated with UV radiation (UVR)-induced suppression of contact hypersensitivity, and topical application of the vitamin D analogue calcipotriene suppresses contact hypersensitivity in human subjects to a similar extent as UVR. However, 1,25(OH)(2)D(3) decreases DNA damage both in vitro when added to human skin cells in culture before and after UVR, and in vivo when applied to mouse skin after UVR. Furthermore, topical 1,25(OH)(2)D(3) applied to mouse skin after UVR reversed the immunosuppressive effect of UVR in a contact hypersensitivity model. This review will discuss the role of 1,25(OH)(2)D(3) as either a mediator of UVR-induced immune suppression or as a photoprotective molecule against UVR-induced DNA damage and immune suppression.
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- 2008
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14. CYP7B expression and activity in fibroblast-like synoviocytes from patients with rheumatoid arthritis: regulation by proinflammatory cytokines.
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Dulos J, van der Vleuten MA, Kavelaars A, Heijnen CJ, and Boots AM
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- Cell Line, Cytochrome P-450 Enzyme System immunology, Cytochrome P450 Family 7, Cytokines immunology, Cytokines physiology, Dehydroepiandrosterone metabolism, Down-Regulation, Humans, Interleukin-1 physiology, Interleukin-10 physiology, Steroid Hydroxylases immunology, Synovial Fluid metabolism, Synovial Membrane cytology, Transforming Growth Factor beta physiology, Tumor Necrosis Factor-alpha physiology, Up-Regulation, Arthritis, Rheumatoid immunology, Cytochrome P-450 Enzyme System biosynthesis, Dehydroepiandrosterone analogs & derivatives, Dehydroepiandrosterone immunology, Steroid Hydroxylases biosynthesis, Synovial Membrane immunology
- Abstract
Objective: The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen-induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7alpha-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA)., Methods: The presence of 7alpha-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7alpha-OH-DHEA with the use of high-performance liquid chromatography., Results: The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested., Conclusion: Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.
- Published
- 2005
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15. Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.
- Author
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Trap C, Nato F, Chalbot S, Kim SB, Lafaye P, and Morfin R
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- Amino Acid Sequence, Animals, Antibodies, Monoclonal biosynthesis, Antibodies, Monoclonal metabolism, Antibody Specificity, Binding Sites, Antibody, Catalysis, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Cytochrome P450 Family 7, DNA, Complementary immunology, Dehydroepiandrosterone antagonists & inhibitors, Dehydroepiandrosterone metabolism, Hippocampus enzymology, Hippocampus immunology, Humans, Immunoglobulin M metabolism, Immunohistochemistry, Injections, Intramuscular, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Steroid Hydroxylases genetics, Steroid Hydroxylases metabolism, Vaccines, DNA administration & dosage, Vaccines, DNA immunology, Antibodies, Monoclonal analysis, Cytochrome P-450 Enzyme System analysis, Cytochrome P-450 Enzyme System immunology, DNA, Complementary administration & dosage, Steroid Hydroxylases analysis, Steroid Hydroxylases immunology
- Abstract
The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues.
- Published
- 2005
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16. Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.
- Author
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Lytton SD, Berg U, Nemeth A, and Ingelman-Sundberg M
- Subjects
- Adolescent, Adult, Antibody Specificity, Azathioprine administration & dosage, Azathioprine therapeutic use, Blotting, Western, Chemical and Drug Induced Liver Injury etiology, Chemical and Drug Induced Liver Injury immunology, Child, Child, Preschool, Cyclosporine administration & dosage, Cyclosporine pharmacokinetics, Cyclosporine therapeutic use, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP2E1 immunology, Cytochrome P-450 CYP3A, Drug Therapy, Combination, Epitopes immunology, Female, Graft Rejection prevention & control, Humans, Immunosuppressive Agents administration & dosage, Immunosuppressive Agents pharmacokinetics, Immunosuppressive Agents therapeutic use, Infant, Kidney Diseases chemically induced, Kidney Diseases immunology, Kidney Transplantation immunology, Liver Cirrhosis, Biliary immunology, Liver Transplantation immunology, Male, Middle Aged, Mixed Function Oxygenases immunology, Postoperative Complications chemically induced, Prednisone administration & dosage, Prednisone therapeutic use, Recombinant Fusion Proteins immunology, Steroid Hydroxylases immunology, Tacrolimus administration & dosage, Tacrolimus pharmacokinetics, Tacrolimus therapeutic use, Aryl Hydrocarbon Hydroxylases, Autoantibodies immunology, Autoantigens immunology, Cyclosporine adverse effects, Cytochrome P-450 Enzyme System immunology, Graft Rejection immunology, Immunoglobulin G immunology, Immunosuppressive Agents adverse effects, Isoenzymes immunology, Steroid 16-alpha-Hydroxylase, Tacrolimus adverse effects
- Abstract
Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.
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- 2002
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17. Monoclonal antibodies specific and inhibitory to human cytochromes P450 2C8, 2C9, and 2C19.
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Krausz KW, Goldfarb I, Buters JT, Yang TJ, Gonzalez FJ, and Gelboin HV
- Subjects
- Animals, Antibodies, Monoclonal isolation & purification, Antibodies, Monoclonal metabolism, Binding Sites, Antibody, Cytochrome P-450 CYP2C19, Cytochrome P-450 Enzyme System metabolism, Enzyme Inhibitors metabolism, Humans, Hybridomas, Mice, Mice, Inbred BALB C, Microsomes, Liver enzymology, Microsomes, Liver immunology, Microsomes, Liver metabolism, Mixed Function Oxygenases metabolism, Steroid Hydroxylases metabolism, Antibodies, Monoclonal pharmacology, Antibody Specificity, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Enzyme Inhibitors pharmacology, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases immunology, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases antagonists & inhibitors, Steroid Hydroxylases immunology
- Abstract
Hybridomas were isolated that produce 13 monoclonal antibodies (mAbs) that are specific and highly inhibitory to members of the human P450 2C subfamily, 2C8, 2C9, 2C9*2, and 2C19. Many of the mAbs to P450 2C8, 2C9, and 2C19 are specific and exhibit potent inhibitory activity (85-95%). mAb 281-1-1 specifically binds, immunoblots, and strongly inhibits the activity of P450 2C8. mAb 763-15-5 specifically binds and strongly inhibits the activity of P450 2C9. mAb 1-7-4-8 specifically binds and strongly inhibits the activity of P450 2C19. The other mAbs bind and inhibit sets and subsets of the P450 2C family. The single and the combinatorial use of the mAbs can "reaction phenotype", i.e., determine the metabolic contribution and interindividual variation of a P450 isoform for the metabolism of a drug or nondrug xenobiotic in human liver microsomes. The utility of the mAb-based analytic system was examined with the model substrates Taxol (paclitaxel), diazepam, tolbutamide, diclofenac, mephenytoin, and imipramine. The mAb system can identify drugs metabolized by a common P450 or several P450s and polymorphic P450s. The mAb system identifies drugs or drug metabolic pathways that are catalyzed by a single P450 and thus may be used for in vivo phenotyping. The mAb system can identify whether a particular drug is metabolized by a single P450 that may exhibit polymorphic expression in humans. The mAb system offers large potential for studies of cytochrome P450 function useful in drug discovery and reduces the possibility of adverse drug reactions due to polymorphisms and drug interactions.
- Published
- 2001
18. Induction of intestinal cytochrome P450 (CYP3A) by rifampicin in beagle dogs.
- Author
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Kyokawa Y, Nishibe Y, Wakabayashi M, Harauchi T, Maruyama T, Baba T, and Ohno K
- Subjects
- Administration, Oral, Animals, Cytochrome P-450 Enzyme System immunology, Dogs, Enzyme Induction, Enzyme-Linked Immunosorbent Assay, Female, Immune Sera pharmacology, Immunoblotting, Male, Microsomes, Liver drug effects, Microsomes, Liver enzymology, Rifampin administration & dosage, Steroid Hydroxylases immunology, Cytochrome P-450 Enzyme System biosynthesis, Intestine, Small drug effects, Intestine, Small enzymology, Rifampin pharmacology, Steroid Hydroxylases biosynthesis
- Abstract
Both male and female beagle dogs (four dogs/sex) were orally treated with rifampicin (Rif) at the dose of 10 mg/kg/day for 7 days and an additional eight dogs (four dogs/sex) were used as a control. The inducible effect of Rif on intestinal cytochrome P450, especially CYP3A enzyme, was investigated by measuring microsomal testosterone 6beta-hydroxylation (6beta-OHT) activity, immunoblot and ELISA analysis. In male dogs, microsomal 6beta-OHT activity in the duodenum, upper, middle and lower part of the jejunum and the ileum of the control was 229, 204, 194, 129 and 57 pmol/min/mg protein, while the activity of the Rif-treated dogs significantly increased to 456, 486, 430, 192 and 138 pmol/min/mg protein, respectively. The activity of intestinal 6beta-OHT in the control and Rif-treated female dogs showed almost similar levels to those observed in the corresponding male dogs. The activity of intestinal 6beta-OHT in both control and Rif-treated dogs was specifically inhibited by anti-CYP3A12 antiserum. The apparent K(m) value for 6beta-OHT activity in all sections of the small intestine was comparable with that in the liver, and no significant changes were observed in between control and Rif-treated dogs. In both control and Rif-treated dogs, immunoblotting of intestinal microsomes with anti-CYP3A12 antiserum produced a band indistinguishable from that of purified CYP3A12 or of immunoreactive CYP3A12 in liver microsomes. A significant increase in intestinal CYP3A content by Rif treatment was quantitatively verified by the ELISA analysis and the magnitude of its increase correlated well with that of 6beta-OHT activity elevation. Furthermore, the results of immunohistochemistry using the anti-CYP3A12 antiserum indicated that CYP3A protein was specifically distributed in epithelial cells throughout the small intestine and appeared to be predominant at the apical side of villus cells. These results demonstrate that Rif induces not only hepatic CYP3A12 but also intestinal CYP3A in dogs.
- Published
- 2001
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19. Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies.
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Hamaoka N, Oda Y, Hase I, and Asada A
- Subjects
- Antibodies, Monoclonal immunology, Cytochrome P-450 CYP1A2, Cytochrome P-450 CYP2B6, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System metabolism, Enzyme Inhibitors pharmacology, Humans, Hydroxylation, Microsomes, Liver metabolism, Orphenadrine pharmacology, Oxidoreductases, N-Demethylating antagonists & inhibitors, Oxidoreductases, N-Demethylating immunology, Oxidoreductases, N-Demethylating metabolism, Steroid Hydroxylases antagonists & inhibitors, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Troleandomycin pharmacology, Anesthetics, Intravenous metabolism, Anti-Anxiety Agents metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System physiology, Midazolam metabolism, Oxidoreductases, N-Demethylating physiology, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases physiology
- Abstract
We determined the contribution of cytochrome P450 (CYP) isoforms to the metabolism of midazolam by kinetic analysis of human liver microsomes and CYP isoforms and by examining the effect of chemical inhibitors and monoclonal antibodies against CYP isoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam (1'-OH MDZ) by human liver microsomes with a Michaelis-Menten constant (Km) of 4.1 (1.0) (mean (SD)) micromol litre(-1) and a maximum rate of metabolism (Vmax) of 5.5 (1.1) nmol min(-1) mg protein(-1) (n = 6). Of the nine representative human liver CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylation activity, with Kms of 40.7, 1.7 and 3.0 micromol litre(-1), respectively, and Vmax values of 12.0, 3.3 and 13.2 nmol min(-1) nmol P450(-1), respectively (n = 4). Midazolam 1'-hydroxylation activity of human liver microsomes correlated significantly with testosterone 6beta-hydroxylation activity, a marker of CYP3A activity (r2 = 0.77, P = 0.0001), but not with S-mephenytoin N-demethylation activity, a marker of CYP2B6 activity (r2 < 0.01, P = 0.84) (n = 11). Troleandomycin and orphenadrine, chemical inhibitors of CYP isoforms, inhibited the formation of 1'-OH MDZ by human liver microsomes. Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ by 79%, whereas monoclonal antibody against CYP2B6 had no effect on midazolam 1'-hydroxylation by human liver microsomes (n = 5). These results indicate that only CYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolism of midazolam in vitro.
- Published
- 2001
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20. A potential role for sterol 27-hydroxylase in atherogenesis.
- Author
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Shanahan CM, Carpenter KL, and Cary NR
- Subjects
- Actins immunology, Actins metabolism, Adolescent, Adult, Aged, Antibodies analysis, Antigens, CD immunology, Antigens, Differentiation, Myelomonocytic immunology, Aorta enzymology, Aorta pathology, Arteriosclerosis pathology, Biomarkers, Cells, Cultured, Child, Child, Preschool, Cholestanetriol 26-Monooxygenase, Coronary Vessels enzymology, Coronary Vessels pathology, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System immunology, DNA Probes chemistry, DNA, Complementary analysis, Female, Gene Expression, Humans, Hydroxycholesterols metabolism, In Situ Hybridization, Macrophages enzymology, Macrophages immunology, Male, Middle Aged, Muscle, Smooth, Vascular enzymology, Muscle, Smooth, Vascular pathology, RNA, Messenger genetics, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Steroid Hydroxylases genetics, Steroid Hydroxylases immunology, Tunica Intima enzymology, Tunica Intima pathology, Arteriosclerosis enzymology, Cytochrome P-450 Enzyme System metabolism, Steroid Hydroxylases metabolism
- Abstract
Objective: 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression., Methods: Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells., Results: Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core., Conclusions: 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.
- Published
- 2001
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21. Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages.
- Author
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Monkawa T, Yoshida T, Hayashi M, and Saruta T
- Subjects
- Antibodies, Antineoplastic Agents pharmacology, Blotting, Northern, Blotting, Western, Calcitonin pharmacology, Calcitriol pharmacology, Calcium Channel Agonists pharmacology, Cell Differentiation physiology, Cell Line, Cholestanetriol 26-Monooxygenase, Cloning, Molecular, Cyclic AMP pharmacology, DNA, Complementary, Dactinomycin pharmacology, Gene Expression Regulation, Enzymologic drug effects, Humans, Interferon-gamma pharmacology, Macrophages cytology, Nucleic Acid Synthesis Inhibitors pharmacology, Parathyroid Hormone pharmacology, RNA, Messenger analysis, Ribonucleases, Steroid Hydroxylases analysis, Steroid Hydroxylases immunology, Gene Expression Regulation, Enzymologic physiology, Macrophages enzymology, Steroid Hydroxylases genetics
- Abstract
Background: The 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) is almost exclusively expressed in the kidney. However, 1alpha-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1alpha-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1alpha-hydroxylase at a molecular level., Methods: We used the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells by treatment with phorbol ester. The expression of 1alpha-hydroxylase in THP-1 cells was examined by Northern blotting and immunoblotting using an antibody raised against a synthetic peptide corresponding to the 14 C-terminal amino acids of 1alpha-hydroxylase. We investigated the regulation of 1alpha-hydroxylase mRNA expression by RNase protection assay., Results: Northern blot and immunoblot analyses confirmed the expression of 1alpha-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1alpha-hydroxylase mRNA in THP-1 cells (198 +/- 9%). 1,25-(OH)2D3, known as a suppressor of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-gamma (2000 IU/mL) increased the expression of 1alpha-hydroxylase mRNA in differentiated THP-1 cells (922 +/- 25%)., Conclusions: The present results suggest that 1alpha-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney. Interferon-gamma treatment increases macrophage 1alpha-hydroxylase levels via directly increasing gene expression of this enzyme.
- Published
- 2000
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22. An inhibitory monoclonal antibody to human cytochrome P450 that specifically binds and inhibits P4502C9II, an allelic variant of P4502C9 having a single amino acid change Arg144 Cys.
- Author
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Krausz KW, Goldfarb I, Yang TJ, Gonzalez FJ, and Gelboin HV
- Subjects
- Alleles, Arginine genetics, Arginine metabolism, Cysteine genetics, Cysteine metabolism, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Diclofenac metabolism, Enzyme-Linked Immunosorbent Assay, Genetic Variation genetics, Humans, Hybridomas, Isoenzymes antagonists & inhibitors, Isoenzymes genetics, Isoenzymes immunology, Isoenzymes metabolism, Phenanthrenes metabolism, Polymorphism, Genetic genetics, Polymorphism, Genetic immunology, Protein Binding genetics, Protein Binding immunology, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins immunology, Recombinant Proteins metabolism, Steroid Hydroxylases genetics, Steroid Hydroxylases metabolism, Tolbutamide metabolism, Amino Acid Substitution genetics, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Antibody Specificity, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases antagonists & inhibitors, Steroid Hydroxylases immunology
- Abstract
A monoclonal antibody (MAb 292-2-3) has been isolated that binds specifically to a single allele of three expressed human cytochrome P4502C9 alleles. The MAb binds to 2C9Cys144 (II), and does not bind to the wild-type 2C9Arg144 (I), or the third allele 2C9Ile-->Leu359 (III) and thus the MAb detects an allele with > 99% homology and differing from the wild-type 2C9Arg144 (I) by a single amino acid. The MAb 292-2-3 does not bind to the other 2C isoforms (2C8, 2C18, 2C19) or the other human cytochrome P450s, 1A1, 1A2, 2A6, 2B6, 2C8, 2D6, 2E1 or 3A4/5. MAb 292-2-3 inhibits the metabolism of tolbutamide, diclofenac and phenanthrene by the target 2C9Cys144 (II) allele by > 90% and does not inhibit the catalytic activity of the wild-type 2C9Arg144 (I), or 2C9Ile-->Leu359 (III) the other 2C isoforms 2C8, 2C18, 2C19, or the other non-2C human P450s listed above. The MAb 292-2-3 is thus a prototype of an ideal and extraordinarily specific reagent for the detection and measurement of the metabolic role of highly related isoforms and polymorphic alleles of human cytochrome P450s. MAbs of high specificity can also determine the amount of phenotypic expression of polymorphic alleles and their metabolic role in drug and non-drug xenobiotic metabolism in heterozygote individuals. The inhibitory MAb might also identify allele-specific substrates of polymorphic human cytochrome P450s.
- Published
- 2000
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23. CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes.
- Author
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Wester MR, Lasker JM, Johnson EF, and Raucy JL
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antibodies, Monoclonal isolation & purification, Antibodies, Monoclonal pharmacology, Cross Reactions, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C8, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System immunology, Diclofenac metabolism, Humans, Hydroxylation drug effects, Male, Mephenytoin metabolism, Microsomes, Liver drug effects, Mixed Function Oxygenases immunology, Rabbits, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver metabolism, Mixed Function Oxygenases metabolism, Steroid 16-alpha-Hydroxylase, Tolbutamide metabolism
- Abstract
Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.
- Published
- 2000
24. Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms.
- Author
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Perloff MD, von Moltke LL, Court MH, Kotegawa T, Shader RI, and Greenblatt DJ
- Subjects
- Animals, Antibodies pharmacology, Biotransformation, Blotting, Western, Chromatography, High Pressure Liquid, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Dose-Response Relationship, Drug, Humans, Immunochemistry, In Vitro Techniques, Inhibitory Concentration 50, Ketoconazole pharmacology, Mice, Mixed Function Oxygenases immunology, Protein Isoforms physiology, Species Specificity, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System physiology, Microsomes, Liver metabolism, Midazolam pharmacokinetics, Oxidoreductases, N-Demethylating physiology, Steroid 16-alpha-Hydroxylase, Triazolam pharmacokinetics
- Abstract
Midazolam (MDZ) and triazolam (TRZ) hydroxylation, reactions considered to be cytochrome P-4503A (CYP3A)-mediated in humans, were examined in mouse and human liver microsomes. In both species, alpha- and 4-hydroxy metabolites were the principal products. Western blotting with anti-CYP3A1 antibody detected a single band of immunoreactive protein in both human and mouse samples: 0.45 +/- 0. 12 and 2.02 +/- 0.24 pmol/mg protein (mean +/- S.E., n = 3), respectively. Ketoconazole potently inhibited MDZ and TRZ metabolite formation in human liver microsomes (IC(50) range, 0.038-0.049 microM). Ketoconazole also inhibited the formation of both TRZ metabolites and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) range, 0.0076-0.025 microM). However, ketoconazole (10 microM) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Anti-CYP3A1 antibodies produced concentration-dependent inhibition of MDZ and TRZ metabolite formation in human liver microsomes and of TRZ metabolite and 4-OH-MDZ formation in mouse liver microsomes to less than 20% of control values but reduced alpha-OH-MDZ formation to only 66% of control values in mouse liver microsomes. Anti-CYP2C11 antibodies inhibited alpha-OH-MDZ metabolite formation in a concentration-dependent manner to 58% of control values in mouse liver microsomes but did not inhibit 4-OH-MDZ formation. Thus, TRZ hydroxylation appears to be CYP3A specific in mice and humans. alpha-Hydroxylation of MDZ has a major CYP2C component in addition to CYP3A in mice, demonstrating that metabolic profiles of drugs in animals cannot be assumed to reflect human metabolic patterns, even with closely related substrates.
- Published
- 2000
25. Immunoreactivity to various human cytochrome P450 proteins of sera from patients with autoimmune hepatitis, chronic hepatitis B, and chronic hepatitis C.
- Author
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Miyakawa H, Kitazawa E, Kikuchi K, Fujikawa H, Kawaguchi N, Abe K, Matsushita M, Matsushima H, Igarashi T, Hankins RW, and Kako M
- Subjects
- Adult, Animals, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP2D6 genetics, Cytochrome P-450 CYP2E1 immunology, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System blood, Cytochrome P-450 Enzyme System genetics, Cytochromes, Electrophoresis, Polyacrylamide Gel, Female, Fluorescent Antibody Technique, Hepatitis B, Chronic blood, Hepatitis B, Chronic immunology, Hepatitis C, Chronic blood, Hepatitis C, Chronic immunology, Hepatitis, Autoimmune blood, Hepatitis, Autoimmune immunology, Humans, Male, Middle Aged, Mixed Function Oxygenases immunology, Rats, Sodium Dodecyl Sulfate, Steroid Hydroxylases immunology, Tumor Cells, Cultured, beta-Galactosidase immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 CYP2D6 immunology, Cytochrome P-450 Enzyme System immunology, Hepatitis B, Chronic enzymology, Hepatitis C, Chronic enzymology, Hepatitis, Autoimmune enzymology, Steroid 16-alpha-Hydroxylase
- Abstract
Numerous human Cytochrome P450 enzymes (CYPs) associated with 'phase I' drug metabolism have been identified. Among them, CYP2D6 is thought to be the major target autoantigen to anti-liver kidney microsome (LKM)-1 autoantibody, a characteristic feature of autoimmune hepatitis (AIH) type II. In this study, we were able to clone CYP2D6 cDNA from a human liver cDNA library and express the CYP2D6 recombinant protein, and also to prepare four other representative human CYP proteins (CYP1A2, 2C9, 2E1, and 3A4). These preparations were used to assay the immunoreactivity of patients with AIH type I (n=35) and type II (n=9). As comparison groups, sera from patients with chronic hepatitis B (n=15), chronic hepatitis C (n=55; 24 anti-LKM-1-positive, 31 anti-LKM-1-negative), and from normal controls (n=30) were included. The five CYP proteins did not react with sera from normal controls nor from patients with chronic hepatitis B. CYP2D6 reacted with sera from 100% (9/9) of AIH type II patients, 79% (19/24) of patients with anti-LKM-1-positive chronic hepatitis C, and 6.5% (2/31) of patients with anti-LKM-1-negative chronic hepatitis C. In contrast, CYP1A2 reacted with serum from one patient with AIH type I, CYP2E1 reacted with sera from two patients with AIH type I, one patient with anti-LKM-1-positive chronic hepatitis C, and two patients with anti-LKM-1-negative chronic hepatitis C, and CYP3A4 reacted with sera from one patient with AIH type II and one patient with anti-LKM-1-positive chronic hepatitis C. CYP2C9 did not react with any of the sera included in this study. From these results, it is suggested that CYPs other than CYP2D6 can function as immunotargets in certain disease conditions.
- Published
- 2000
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26. Evidence for involvement of polymorphic CYP2C19 and 2C9 in the N-demethylation of sertraline in human liver microsomes.
- Author
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Xu ZH, Wang W, Zhao XJ, Huang SL, Zhu B, He N, Shu Y, Liu ZQ, and Zhou HH
- Subjects
- Antibodies, Monoclonal immunology, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C8, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System metabolism, Enzyme Inhibitors pharmacology, Humans, Kinetics, Methylation, Mixed Function Oxygenases immunology, Mixed Function Oxygenases metabolism, Selective Serotonin Reuptake Inhibitors metabolism, Sertraline analogs & derivatives, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System genetics, Microsomes, Liver enzymology, Mixed Function Oxygenases genetics, Polymorphism, Genetic, Sertraline metabolism, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases genetics
- Abstract
Aims: The present study was designed to define the kinetic behaviour of sertraline N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P450 involved in this metabolic pathway., Methods: The kinetics of the formation of N-demethylsertraline were determined in human liver microsomes from six genotyped CYP2C19 extensive (EM) and three poor metabolisers (PM). Selective inhibitors of and specific monoclonal antibodies to various cytochrome P450 isoforms were also employed., Results: The kinetics of N-demethylsertraline formation in all EM liver microsomes were fitted by a two-enzyme Michaelis-Menten equation, whereas the kinetics in all PM liver microsomes were best described by a single-enzyme Michaelis-Menten equation similar to the low-affinity component found in EM microsomes. Mean apparent Km values for the high-and low-affinity components were 1.9 and 88 microm and V max values were 33 and 554 pmol min-1 mg-1 protein, respectively, in the EM liver microsomes. Omeprazole (a CYP2C19 substrate) at high concentrations and sulphaphenazole (a selective inhibitor of CYP2C9) substantially inhibited N-demethylsertraline formation. Of five monoclonal antibodies to various cytochrome P450 forms tested, only anti-CYP2C8/9/19 had any inhibitory effect on this reaction. The inhibition of sertraline N-demethylation by anti-CYP2C8/9/19 was greater in EM livers than in PM livers at both low and high substrate concentrations. However, anti-CYP2C8/9/19 did not abolish the formation of N-demethylsertraline in the microsomes from any of the livers., Conclusions: The polymorphic enzyme CYP2C19 catalyses the high-affinity N-demethylation of sertraline, while CYP2C9 is one of the low-affinity components of this metabolic pathway.
- Published
- 1999
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27. Role of CYP3A in bromperidol metabolism in rat in vitro and in vivo.
- Author
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Watanabe M, Tateishi T, Tanaka M, Kumai T, and Kobayashi S
- Subjects
- Animals, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System drug effects, Cytochrome P-450 Enzyme System immunology, Dexamethasone pharmacology, Enzyme Inhibitors pharmacology, Erythromycin pharmacology, Half-Life, Haloperidol metabolism, Haloperidol pharmacokinetics, Immune Sera pharmacology, Male, Microsomes, Liver drug effects, Oxidoreductases, N-Demethylating drug effects, Propionates metabolism, Quinine pharmacology, Rats, Rats, Sprague-Dawley, Steroid Hydroxylases antagonists & inhibitors, Steroid Hydroxylases immunology, Theophylline analogs & derivatives, Theophylline pharmacology, Troleandomycin pharmacology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Haloperidol analogs & derivatives, Microsomes, Liver metabolism, Oxidoreductases, N-Demethylating metabolism
- Abstract
1. The aim was to identify whether CYP3A metabolizes bromperidol (BP), an antipsychotic drug, to form 4-fluorobenzoyl-propionic acid (FBPA) in hepatic microsomes from 8-week-old male Sprague-Dawley rats and to investigate whether an inhibitor or an inducer of CYP3A affects BP pharmacokinetics in rat. 2. In an in vitro study, only troleandomycin showed marked inhibition of FBPA formation among several specific CYP isozyme inhibitors studied including troleandomycin, diethyldithiocarbamate, furafylline and quinine. Anti-rat CYP3A2 serum inhibited FBPA formation by 80%, whereas other anti-rat CYP sera (1A1, 1A2, 2B1, 2C11, 2E1) only slightly inhibited it. 3. In a pharmacokinetic study, BP half-life was prolonged to 137% of the average control value by 7-day treatment with erythromycin, a CYP3A inhibitor, and shortened to 58% of the control by 2-day treatment with dexamethasone, a CYP3A inducer. BP clearance was reduced to 68% of the control by erythromycin and was increased to 145% of control by dexamethasone. 4. These results suggested that BP biotransformation is catalysed mainly by CYP3A to form FBPA in rat and that the modification of this enzyme activity would affect the pharmacokinetics of BP.
- Published
- 1999
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28. Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19.
- Author
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Yasumori T, Chen LS, Li QH, Ueda M, Tsuzuki T, Goldstein JA, Kato R, and Yamazoe Y
- Subjects
- Antibodies immunology, Anticonvulsants metabolism, Cells, Cultured, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System immunology, Humans, Hydroxylation, Japan, Kinetics, Mixed Function Oxygenases immunology, Phenytoin analogs & derivatives, Saccharomyces cerevisiae, Stereoisomerism, Steroid Hydroxylases immunology, Substrate Specificity, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver metabolism, Mixed Function Oxygenases metabolism, Phenytoin metabolism, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases metabolism
- Abstract
Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4'- hydroxylation were approximately 3-fold higher than those of 3'-hydroxylation, and not significantly different in EM and PM. Formation of 4'-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4'-(S)-, 3'-(R)-, and 3'-(S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4'-(R)- and 4'-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4'-(S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4'-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4'-hydroxylation with a different chiral preference from CYP2C9.
- Published
- 1999
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29. Immunohistochemical study of cytochrome P450 2C and 3A in human non-neoplastic and neoplastic tissues.
- Author
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Yokose T, Doy M, Taniguchi T, Shimada T, Kakiki M, Horie T, Matsuzaki Y, and Mukai K
- Subjects
- Antibodies, Monoclonal analysis, Antigen-Antibody Reactions, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Frozen Sections, Humans, Immunohistochemistry, Liver Neoplasms enzymology, Liver Neoplasms pathology, Microsomes, Liver enzymology, Neoplasms pathology, Organ Specificity, Oxidoreductases, N-Demethylating immunology, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System analysis, Neoplasms enzymology, Oxidoreductases, N-Demethylating analysis, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases analysis
- Abstract
Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.
- Published
- 1999
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30. Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21.
- Author
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Nakamura A, Hirota T, Morino A, Imaoka S, Funae Y, Yamamoto Y, Tasaki T, Masuda M, Kazusaka A, and Fujita S
- Subjects
- Animals, Antibodies pharmacology, Calcium Channel Blockers chemistry, Cholinergic Antagonists chemistry, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System immunology, Cytochrome P450 Family 2, Enzyme Inhibitors pharmacology, Humans, Isoenzymes genetics, Isoenzymes metabolism, Liver enzymology, Lymphocytes metabolism, Male, Microsomes, Liver drug effects, Microsomes, Liver metabolism, Phenylacetates chemistry, Quinidine pharmacology, Quinine pharmacology, Rats, Rats, Sprague-Dawley, Recombinant Proteins genetics, Recombinant Proteins metabolism, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Troleandomycin pharmacology, Aryl Hydrocarbon Hydroxylases, Calcium Channel Blockers metabolism, Cholinergic Antagonists metabolism, Cyclohexanes metabolism, Cytochrome P-450 Enzyme System metabolism, Phenylacetates metabolism, Steroid 16-alpha-Hydroxylase
- Abstract
1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the N-deethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS-21, and the cyclohexane-4-cis- and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.
- Published
- 1999
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31. Effect of 3-methylcholanthrene administration on expression of cytochrome P-450 isoforms induced by phenobarbital in rat hepatocytes.
- Author
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Mino K, Watanabe J, and Kanamura S
- Subjects
- Animals, Blotting, Northern, Cytochrome P-450 CYP1A1 metabolism, Cytochrome P-450 CYP1A2 metabolism, Cytochrome P-450 CYP2B1 analysis, Cytochrome P-450 CYP2B1 immunology, Cytochrome P-450 Enzyme System analysis, Cytochrome P-450 Enzyme System immunology, Cytoplasm chemistry, Immunoenzyme Techniques, In Situ Hybridization, Liver drug effects, Male, Microsomes, Liver immunology, RNA, Messenger analysis, Rats, Rats, Sprague-Dawley, Steroid Hydroxylases analysis, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 CYP2B1 metabolism, Cytochrome P-450 Enzyme System metabolism, Liver metabolism, Methylcholanthrene pharmacology, Phenobarbital pharmacology, Steroid Hydroxylases metabolism
- Abstract
The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.
- Published
- 1998
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32. N-oxidation of irsogladine by the CYP2C subfamily in the rat, dog, monkey and man.
- Author
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Nakamura A, Hirota T, Morino A, Shimada T, and Uematsu T
- Subjects
- Animals, Anti-Ulcer Agents pharmacokinetics, Antibodies pharmacology, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System immunology, Cytochrome P450 Family 2, Dogs, Half-Life, Humans, Hydroxylation drug effects, Kinetics, Lymphocytes enzymology, Macaca mulatta, Male, Microsomes, Liver enzymology, NADPH-Ferrihemoprotein Reductase immunology, NADPH-Ferrihemoprotein Reductase metabolism, Oxidation-Reduction, Rats, Rats, Sprague-Dawley, Species Specificity, Steroid Hydroxylases immunology, Triazines pharmacokinetics, Anti-Ulcer Agents metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases metabolism, Triazines metabolism
- Abstract
1. The metabolism of irsogladine (ISG) was studied in hepatic microsomes from the rat, dog, monkey and man, and marked species differences were observed in N-oxidation of ISG. The rank order of the activity of the N-oxidation was shown to be man < monkey < dog < rat. 2. Anti-NADPH-P450 reductase antibody inhibited the formation of the N-oxidized metabolite of ISG (ISG-N-oxide) in hepatic microsomes from rats by 74%. Anti-CYP2C11 antibody also inhibited the formation of ISG-N-oxide in hepatic microsomes from rat by 73%, whereas anti-CYP2E1, 3A2 and 4A1 antibody did not inhibit N-oxidation. Thus, CYP2C11 in the rat is at least partially responsible for the N-oxidation of ISG in the rat. 3. Anti-CYP2C11 antibody also inhibited the formation of ISG-N-oxide in hepatic microsomes from the dog and monkey by 61 and 46% respectively. Therefore, a isoform(s) similar to CYP2C11 partially contributed to the N-oxidation of ISG in the dog and monkey. In contrast, human CYP2C9, a member of the human CYP2C subfamily, did not catalyse the N-oxidation of ISG. 4. These findings show that the marked species difference in the N-oxidation of ISG is caused by the difference in the catalytic properties of CYP2C among the species examined.
- Published
- 1997
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33. Comparison of CYP3A activities in a subclone of Caco-2 cells (TC7) and human intestine.
- Author
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Raeissi SD, Guo Z, Dobson GL, Artursson P, and Hidalgo IJ
- Subjects
- Antibodies immunology, Caco-2 Cells, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Enzyme Inhibitors pharmacology, Humans, Hydroxylation, Intestinal Mucosa ultrastructure, Jejunum enzymology, Jejunum ultrastructure, Ketoconazole pharmacology, Microsomes enzymology, Microsomes, Liver enzymology, Oxidation-Reduction, Oxidoreductases, N-Demethylating antagonists & inhibitors, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Terfenadine metabolism, Terfenadine pharmacokinetics, Troleandomycin pharmacology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Intestinal Mucosa enzymology, Oxidoreductases, N-Demethylating metabolism
- Abstract
Purpose: To compare the activity of the CYP3A enzyme expressed by TC7, a cell culture model of the intestinal epithelial cell, to the activity of human intestinal CYP3A4, using terfenadine as a substrate., Methods: The metabolism of terfenadine was investigated in intact cells and microsomal preparations from TC7, human intestine, and liver. The effect of two CYP3A inhibitors, ketoconazole and troleandomycin (TAO), on the metabolism of terfenadine was also examined., Results: Only hydroxy-terfenadine was detected in TC7 microsomal incubations. In contrast, azacyclonol and hydroxy-terfenadine were detected in human intestinal and hepatic microsomal incubations. The Km values for hydroxy-terfenadine formation in TC7 cells, intestine and liver microsomes were 1.91, 2.5, and 1.8, microM respectively. The corresponding Vmax values were 2.11, 61.0, and 370 pmol/min/mg protein. Km values for azacyclonol in intestinal and hepatic samples were 1.44 and 0.82 microM and the corresponding Vmax values were 14 and 60 pmol/min/mg protein. The formation of hydroxy-terfenadine was inhibited by ketoconazole and TAO in human intestine and TC7 cell microsomes. The Km and Vmax values for terfenadine metabolism in intact TC7 cells were similar to those from TC7 cell microsomes., Conclusions: Our results indicate that TC7 cells are a potentially useful alternative model for studies of CYP3A mediated drug metabolism. The CYP3A expressed by TC7 cells is not CYP3A4, but probably CYP3A5, making this cell line suitable for studies of colonic drug transport and metabolism.
- Published
- 1997
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34. Stereoselective N-demethylation of chlorpheniramine by rat-liver microsomes and the involvement of cytochrome P450 isozymes.
- Author
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Nomura A, Sakurai E, and Hikichi N
- Subjects
- Animals, Antibodies immunology, Cytochrome P-450 CYP1A1 immunology, Cytochrome P-450 CYP1A1 metabolism, Cytochrome P-450 CYP2B1 immunology, Cytochrome P-450 CYP2B1 metabolism, Cytochrome P-450 CYP2E1 immunology, Cytochrome P-450 CYP2E1 metabolism, Cytochrome P-450 Enzyme System immunology, Cytochrome P450 Family 2, In Vitro Techniques, Isoenzymes immunology, Male, Methylcholanthrene pharmacology, Microsomes, Liver drug effects, Phenobarbital pharmacology, Rats, Rats, Wistar, Stereoisomerism, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Aryl Hydrocarbon Hydroxylases, Chlorpheniramine pharmacokinetics, Cytochrome P-450 Enzyme System metabolism, Histamine H1 Antagonists pharmacokinetics, Isoenzymes metabolism, Microsomes, Liver enzymology, Steroid 16-alpha-Hydroxylase
- Abstract
Previous studies have suggested that degradation of the two stereoisomers of chlorpheniramine in the liver might be catalysed by different types of cytochrome P450. Stereoselective N-demethylation of chlorpheniramine and the involvement of cytochrome P450 (CYP) isozymes have, therefore, been investigated in the liver microsomes of eight-week-old male rats. Incubation of racemic chlorpheniramine with liver microsomes from the male rat resulted in the formation of both enantiomers of monodesmethylchlorpheniramine (DMChp). Further metabolism of DMChp to didesmethylchlorpheniramine (DDMChp) did not, however, occur. The S/R enantiomeric ratio for intrinsic clearance (Vmax/Km) was approximately 2.0, suggesting that the N-demethylation was stereoselective for S-(+)-chlorpheniramine. On the other hand, although the Vmax/Km value for the formation of S-(+)- and R-(-)-DMChp increased with phenobarbitone-inducible rat-liver microsomes, there was no difference between the rates of N-demethylation of the enantiomers. In contrast, 3-methylcholanthrene reduced the intrinsic clearance of S-(+)-chlorpheniramine by N-demethylation and increased its value for R-(-)-chlorpheniramine, showing no stereoselectivity for the N-demethylation of chlorpheniramine. The difference between the intrinsic clearance of the two enantiomers by N-demethylation was because of differences in affinity for the catalysing enzyme. This is indicative of stereoselective involvement of the main enzyme concerned in the N-demethylation of the enantiomers, considered to be CYP 2C11. Anti-CYP 2C11 also partially inhibited the N-demethylation of racemic chlorpheniramine in rat-liver microsomes exposed to phenobarbitone and 3-methylcholanthrene. That CYP 2B1 was involved in the N-demethylation of both enantiomers was also supported by results from an experiment using phenobarbitone-inducible rat-liver microsomes. CYP1A1 did not, however, catalyse the N-demethylation of either enantiomer. These results indicate that N-demethylation of the S-(+)-enantiomer of chlorpheniramine occurs preferentially in the microsomes, demonstrating the stereoselective contribution of CYP2C11. Immunoinhibition studies suggest, moreover, that the N-demethylation of both chlorpheniramine enantiomers is catalysed by CYP2B1, but not by CYP1A1.
- Published
- 1997
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35. Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.
- Author
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Almadhidi J, Moslemi S, Drosdowsky MA, and Séralini GE
- Subjects
- Androstenedione analogs & derivatives, Androstenedione pharmacology, Animals, Aromatase immunology, Aromatase Inhibitors, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Fadrozole pharmacology, Immune Sera pharmacology, Kinetics, Male, Miconazole pharmacology, Microsomes enzymology, Placenta enzymology, Steroid Hydroxylases antagonists & inhibitors, Steroid Hydroxylases immunology, Substrate Specificity, Testis enzymology, Testosterone metabolism, Aromatase metabolism, Cytochrome P-450 CYP1A1, Cytochrome P-450 Enzyme System metabolism, Estradiol metabolism, Horses metabolism, Steroid Hydroxylases metabolism
- Abstract
Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.
- Published
- 1996
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36. Tienilic acid-induced autoimmune hepatitis: anti-liver and-kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9.
- Author
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Lecoeur S, André C, and Beaune PH
- Subjects
- Animals, Cytochrome P-450 CYP2C9, Humans, Infant, Rabbits, Aryl Hydrocarbon Hydroxylases, Autoantibodies immunology, Autoimmune Diseases immunology, Chemical and Drug Induced Liver Injury immunology, Cytochrome P-450 Enzyme System immunology, Epitope Mapping, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases immunology, Ticrynafen toxicity
- Abstract
Tienilic acid-induced hepatitis is characterized by the presence of anti-liver and -kidney microsomal (anti-LKM2) autoantibodies in patient sera. Cytochrome P4502C9(CYP2C9), involved in the metabolism of tienilic acid, was shown to be a target for tienilic acid-reactive metabolites and for autoantibodies. To further investigate the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, the specificity of anti-LKM2 autoantibodies toward CYP2C9 was first determined, and the antigenic sites on CYP2C9 were localized. By constructing several deletion mutants derived from CYP2C9 cDNA and by probing the corresponding proteins with different anti-LKM2 sera, we defined three regions (amino acids 314-322, 345-356, and 439-455); they interacted to form a major conformational autoantibody binding site. This binding site was immunoreactive with 100% of sera and allowed removal of the entire reactivity of the sera tested by immunoblotting. Epitope mapping studies have been performed for CYP2D6, CYP17, CYP21A2, and, recently, CYP3A. Those data were compared with the results obtained in the current study with CYP2C9 in an attempt to elucidate one of the mechanisms by which CYP becomes immunogenic.
- Published
- 1996
37. A possible role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.
- Author
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Araya Z, Norlin M, and Postlind H
- Subjects
- 25-Hydroxyvitamin D3 1-alpha-Hydroxylase immunology, 25-Hydroxyvitamin D3 1-alpha-Hydroxylase isolation & purification, Animals, Antibodies, Monoclonal, Blotting, Western, Cholestanetriol 26-Monooxygenase, Chromatography, Ion Exchange, Cloning, Molecular, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System isolation & purification, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Humans, Kinetics, Molecular Weight, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Steroid Hydroxylases immunology, Steroid Hydroxylases isolation & purification, Swine, 25-Hydroxyvitamin D3 1-alpha-Hydroxylase metabolism, Cytochrome P-450 Enzyme System metabolism, Kidney Cortex enzymology, Mitochondria enzymology, Mitochondria, Liver enzymology, Steroid Hydroxylases metabolism
- Abstract
A mitochondrial cytochrome P450 fraction catalyzing 1 alpha- and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney. The ratio between the 1 alpha- and 27-hydroxylase activities was the same in all purification steps including a side fraction. Attempts to separate the 1 alpha- and 27-hydroxylase activities were unsuccessful. A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M(r) and immunoprecipitated both the 1 alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25-hydroxyvitamin D3 hydroxylases in kidney mitochondria. Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1 alpha-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 in kidney. In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.
- Published
- 1996
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38. Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes.
- Author
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Transon C, Lecoeur S, Leemann T, Beaune P, and Dayer P
- Subjects
- Adult, Age Factors, Anesthetics, Intravenous metabolism, Animals, Antitussive Agents metabolism, Cyclooxygenase Inhibitors metabolism, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP2D6 immunology, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Dextromethorphan metabolism, Diclofenac metabolism, Female, Humans, Hydroxylation, Isoenzymes immunology, Male, Methylation, Mice, Midazolam metabolism, Mixed Function Oxygenases immunology, Rabbits, Regression Analysis, Sex Factors, Steroid Hydroxylases immunology, White People, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 CYP2D6 metabolism, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, Microsomes, Liver enzymology, Mixed Function Oxygenases metabolism, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases metabolism
- Abstract
Objective: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies)., Methods: Diclofenac 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6., Results: Diclofenac 4'-hydroxylation exhibited Michaelis-Menten kinetics with kM = 3.4 mumol.l-1 and Vmax = 45 nmole.mg-1 P.h-1. Relative immunoreactivity of CYP2C9 was correlated with Vmax and CL(int). Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with kM = 4.4 mumol.l-1 and Vmax = 5.0 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP2D6 was correlated with Vmax and CL(int). Midazolam 1'-hydroxylation also exhibited Michaelis-Menten kinetics with kM = 3.3 mumol.l-1 and Vmax = 35 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP3A4 was correlated with Vmax and CL(int). Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes., Conclusion: The velocity of metabolite formation (Vmax) by the three major human drug metabolising P450 monoxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for Vmax than kM. Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers.
- Published
- 1996
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39. Antigenic mapping of bacterial and animal cytochromes P-450.
- Author
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Kolesanova EF, Kiselar JG, Jung C, Kozin SA, Hui Bon Hoa G, and Archakov AI
- Subjects
- Animals, Camphor 5-Monooxygenase chemistry, Chickens, Cytochrome P-450 CYP1A2 chemistry, Cytochrome P-450 Enzyme System chemistry, Mice, Pseudomonas putida chemistry, Rabbits, Steroid Hydroxylases chemistry, Aryl Hydrocarbon Hydroxylases, Camphor 5-Monooxygenase immunology, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 Enzyme System immunology, Epitope Mapping, Epitopes, B-Lymphocyte chemistry, Epitopes, B-Lymphocyte immunology, Pseudomonas putida enzymology, Steroid Hydroxylases immunology
- Abstract
A peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. The antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in the protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.
- Published
- 1996
- Full Text
- View/download PDF
40. Cytochrome P4502D and -2C enzymes catalyze the oxidative N-demethylation of the parkinsonism-inducing substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in rat liver microsomes.
- Author
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Narimatsu S, Tachibana M, Masubuchi Y, and Suzuki T
- Subjects
- Animals, Antibodies, Monoclonal pharmacology, Catalysis, Cytochrome P-450 Enzyme System immunology, Female, Inactivation, Metabolic, Male, Microsomes, Liver ultrastructure, Rats, Rats, Wistar, Sex Factors, Species Specificity, Steroid Hydroxylases immunology, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System pharmacology, Microsomes, Liver enzymology, Microsomes, Liver metabolism, Oxidoreductases, N-Demethylating metabolism, Parkinson Disease etiology, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases pharmacology
- Abstract
We have examined the oxidative N-demethylation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonism-inducing neurotoxin, in liver microsomes from adult Wistar and Dark Agouti (DA) rats. The oxidation of MPTP to 4-phenyl-1,2,3,6-tetrahydropyridine (PTP) in these preparations required NADPH as a cofactor and was significantly inhibited by SKF 525-A (2 mM). MPTP N-demethylation exhibited biphasic kinetics, consistent with two enzymes, a low Km system (Km1, 10.0 +/- 2.2 microM; Vmax1, 0.048 +/- 0.009 nmol/(min.mg of protein)) and a high Km system (Km2, 1180 +/- 91 microM; Vmax2, 4.80 +/- 0.75 nmol/(min.mg of protein)). We thus employed two substrate concentrations, 5 microM and 5 mM, for the low and high Km system, respectively, to assay enzyme activity in subsequent experiments. The oxidation activity was significantly decreased by pretreatment of rats with phenobarbital and beta-naphthoflavone. Furthermore, marked strain (Wistar > DA) and sex (male > female) differences were observed at low (5 microM) and high (5 mM) substrate concentrations, respectively. Reconstitution experiments with cytochrome P450BTL, which belongs to the P4502D subfamily, and P450m1 (P4502C11) demonstrated that MPTP N-demethylase occurs at concentrations of 5 microM and 5 mM. At 5 mM the male-specific P450m1 showed a remarkably high activity, over 400-fold that of P450BTL. Polyclonal antibodies against P450BTL and P450m1 effectively suppressed the activity at the low (5 microM) and the high (5 mM) substrate concentrations, respectively. These results suggest that, in the microsomal preparations used, MPTP N-demethylation is mainly mediated by P4502D enzyme(s) at lower substrate concentrations and by P4502C11 at higher substrate concentrations.
- Published
- 1996
- Full Text
- View/download PDF
41. Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extrahepatic tissues.
- Author
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Debri K, Boobis AR, Davies DS, and Edwards RJ
- Subjects
- Amino Acid Sequence, Animals, Antibodies immunology, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Enzyme-Linked Immunosorbent Assay, Female, Immunoblotting, Immunohistochemistry, Intestine, Small enzymology, Kidney enzymology, Lung enzymology, Male, Microsomes, Liver enzymology, Mixed Function Oxygenases immunology, Molecular Sequence Data, Pregnenolone Carbonitrile pharmacology, Rats, Rats, Wistar, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System biosynthesis, Liver enzymology, Mixed Function Oxygenases biosynthesis, Steroid Hydroxylases biosynthesis
- Abstract
Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.
- Published
- 1995
- Full Text
- View/download PDF
42. Production of a form-specific, inhibitory antibody against rat cytochrome P450 2B1 using a synthetic peptide antigen against a putative substrate binding site.
- Author
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Charnecki J, Putt D, Kim EY, and Kim H
- Subjects
- Amino Acid Sequence, Animals, Antibody Specificity, Antigens chemistry, Binding Sites, Blotting, Western, Cytochrome P-450 CYP2B1, Cytochrome P-450 Enzyme System immunology, Immunoglobulin G immunology, Male, Microsomes, Liver enzymology, Molecular Sequence Data, Oxidoreductases antagonists & inhibitors, Peptides chemistry, Protein Structure, Secondary, Rabbits, Rats, Rats, Sprague-Dawley, Steroid Hydroxylases immunology, Antibodies pharmacology, Antigens immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme Inhibitors, Peptides immunology, Steroid Hydroxylases antagonists & inhibitors
- Abstract
Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1. This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody. The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats. These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes.
- Published
- 1995
- Full Text
- View/download PDF
43. Dog liver microsomal P450 enzyme-mediated toluene biotransformation.
- Author
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Hanioka H, Hamamura M, Kakino K, Ogata H, Jinno H, Takahashi A, Nishimura T, and Ando M
- Subjects
- Animals, Antibodies pharmacology, Benzyl Alcohol, Benzyl Alcohols metabolism, Biotransformation, Cresols metabolism, Cytochrome P-450 Enzyme System immunology, Dogs, Female, Kinetics, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, Microsomes, Liver enzymology, Toluene pharmacokinetics
- Abstract
1. We studied toluene metabolism in dog liver microsomes and the major metabolite was benzyl alcohol with o- and p-cresol as minor metabolites. 2. The enzyme kinetics of toluene biotransformation were examined by means of Lineweaver-Burk analyses. The Michaelis-Menten values differed among the three pathways, the order being; Km, o-cresol > p-cresol > benzyl alcohol; Vmax, benzyl alcohol > o-cresol > p-cresol; and Cl(int), benzyl alcohol > p-cresol > o-cresol. 3. The formation of benzyl alcohol, o- and p-cresol from toluene was substantially inhibited by the P4502E inhibitors such as DDC (diethyldithiocarbamate) and 4-methylpyrazole in all pathways, with IC50's in the range of 0.02-0.59 mM. The P4502B inhibitors, metyrapone and secobarbital also inhibited benzyl alcohol and p-cresol formation, whereas o-cresol was not inhibited by these latter compounds. 4. Anti-rat P4502E1 antibodies inhibited benzyl alcohol, o- and p-cresol formation from 26 to 30% 0.2 ml serum/mg microsomal protein. Furthermore, anti-rat P4502B1/2 antibody inhibited benzyl alcohol and p-cresol formation (47 and 44% respectively), but not that of o-cresol. Anti-rat P4502C11/6 antibody also inhibited benzyl alcohol and p-cresol formation 31 and 24% respectively in a similar manner to that by the anti-rat P4502B1/2 antibody. 5. These results suggested that the P4502B, 2C and 2E isozymes in dog liver contribute to the formation of benzyl alcohol and p-cresol from toluene, and 2E isozyme preferentially contributes to the formation of o-cresol.
- Published
- 1995
- Full Text
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44. Induction of cytochrome P-4502B1-related mouse cytochrome P-450 and regulation of its expression by epidermal growth factor/transforming growth factor alpha in primary hepatocyte culture.
- Author
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Aubrecht J, Hirsch-Ernst KI, Becker-Rabbenstein V, Kahl GF, Taniguchi H, and Höhne MW
- Subjects
- Animals, Antibodies, Monoclonal, Cells, Cultured, Cytochrome P-450 CYP2B1, Cytochrome P-450 Enzyme System immunology, Enzyme Induction, Epidermal Growth Factor pharmacology, Gene Expression Regulation, Enzymologic drug effects, Male, Mice, Mice, Inbred C57BL, Oxidoreductases biosynthesis, Phenobarbital, RNA, Messenger biosynthesis, Rats, Rats, Wistar, Steroid Hydroxylases immunology, Time Factors, Transforming Growth Factor alpha pharmacology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System biosynthesis, Liver enzymology, Steroid Hydroxylases biosynthesis
- Abstract
Phenobarbital-dependent induction of mouse cytochrome P-450 (Cyp) orthologous to rat CYP2B1 and its modulation by hepatotrophic growth factors were examined in primary hepatocyte cultures. Compared to rat hepatocytes, induction in mouse hepatocytes was more rapid and effective. Ligands of the EGF receptor, epidermal growth factor, and transforming growth factor alpha inhibited induction on the basis of protein expression and CYP2B-associated 7-pentoxyresorufin-O-depentylase activity. Furthermore, EGF led to repression of accumulation of corresponding mRNA under phenobarbital, an effect not blocked by inhibition of protein synthesis under cycloheximide. Ligands of the EGF receptor may contribute towards the decrease in hepatic CYP expression observed during (pre)neoplastic development and regeneration.
- Published
- 1995
- Full Text
- View/download PDF
45. Purification and characterization of constituent testosterone 2 alpha-hydroxylase (cytochrome P450(2)alpha) from mouse liver.
- Author
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Sharma MC and Shapiro BH
- Subjects
- Amino Acid Sequence, Androgens metabolism, Androstenedione metabolism, Animals, Cross Reactions, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System isolation & purification, Cytochrome P450 Family 2, Female, Hydroxylation, Isoenzymes immunology, Isoenzymes isolation & purification, Mice, Molecular Sequence Data, Sequence Homology, Amino Acid, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases immunology, Steroid Hydroxylases isolation & purification, Substrate Specificity, Xenobiotics metabolism, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, Microsomes, Liver enzymology, Steroid Hydroxylases metabolism, Testosterone metabolism
- Abstract
Hepatic microsomal testosterone/androstenedione 2 alpha-hydroxylase (i.e., cytochrome P450(2)alpha) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2 alpha-hydroxylase activity. The catalytic activity of the purified cytochrome P450(2)alpha, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2 alpha-hydroxylation of testosterone and androstenedione; representing > 96% and > 92% of these respective metabolites. Polyclonal antibodies against cytochrome P450(2)alpha exhibited a dose-dependent and very selective inhibition of testosterone 2 alpha-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P450(2)alpha fraction was 12.06 nmol/mg protein. The specific testosterone 2 alpha-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P450(2)alpha was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P450(2)alpha is a member of the murine cytochrome P450 2d family.
- Published
- 1995
- Full Text
- View/download PDF
46. [Surface of cytochrome P-450 2B4: structure and function].
- Author
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Uvarov VIu, Kolesanova EF, Apletalina EV, Kozin SA, and Archakov AI
- Subjects
- Amino Acid Sequence, Epitopes, Humans, Molecular Sequence Data, Peptide Fragments, Surface Properties, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System physiology, Steroid Hydroxylases chemistry, Steroid Hydroxylases immunology, Steroid Hydroxylases physiology
- Abstract
The review is devoted to the identification and structure of one of the cytochromes P450s-cytochrome P-450 2B4 derived from the rabbit liver endoplasmic reticulum. A critical review is made of the data on this enzyme membrane topology, its active site's structure and localization of its membrane and water-exposed regions. The paper is based on the data available in the literature and the authors' own findings. Various experimental and calculating methods used to identify the topography of cytochrome P450 are covered in the paper.
- Published
- 1995
47. Cloning and sequencing of the major rainbow trout constitutive cytochrome P450 (CYP2K1): identification of a new cytochrome P450 gene subfamily and its expression in mature rainbow trout liver and trunk kidney.
- Author
-
Buhler DR, Yang YH, Dreher TW, Miranda CL, and Wang JL
- Subjects
- Aging, Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Cross Reactions, Cytochrome P-450 Enzyme System biosynthesis, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System classification, Cytochrome P-450 Enzyme System immunology, Cytochrome P450 Family 2, Female, Gene Expression Regulation, Male, Molecular Sequence Data, RNA, Messenger analysis, Restriction Mapping, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Sex Characteristics, Steroid Hydroxylases biosynthesis, Steroid Hydroxylases chemistry, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System genetics, Fish Proteins, Kidney enzymology, Liver enzymology, Oncorhynchus mykiss genetics, Steroid Hydroxylases genetics
- Abstract
We have cloned and sequenced a full-length cDNA encoding the major constitutive rainbow trout liver cytochrome P450 that we had earlier designated as cytochrome P450 LMC2 (Arch. Biochem. Biophys. 268, 227, 1989). The 1859-bp cDNA was isolated from a male rainbow trout liver cDNA library and contained an open reading frame encoding for a protein of 504 amino acids and having a calculated molecular weight of 56,795. From nucleotide and amino acid sequence comparisons, the trout P450 LMC2 has been assigned to a new cytochrome P450 gene subfamily designated P4502K1 or CYP2K1. Marked differences in the sex- and tissue-specific expression of this gene were found at both the transcriptional and translational level in sexually mature rainbow trout liver and trunk kidney. Transcriptional expression investigated by Northern analysis of total RNA using a 440-bp 3'-terminal cDNA probe (2K1,7c) showed sexual and organ differences. Mature male trunk kidney expressed 2K1,7c-hybridizable mRNA to a substantially greater extent than did female trunk kidney, with multiple mRNA bands appearing in approximately the 2.8- and 1.9-kb region. While the livers of some males displayed separate 2.8-kb hybridizable bands, such bands were generally undetectable in female liver samples. By contrast, 1.9-kb mRNA bands were found at generally similar concentrations in the livers of both sexes. Taking into consideration the individual biological variabilities, it was apparent that mature male trout expressed the 2.8-kb mRNA much more strongly in trunk kidney than in liver. Translational expression, analyzed by Western blotting of microsomes separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with rabbit polyclonal antibody and two different monoclonal antibodies prepared against P450 LMC2, demonstrated corresponding sex- and organ-related differences in P450 protein expression. Southern analysis of sexually mature male trout genomic DNA suggested that CYP2K1 is not a single copy gene, thus providing additional evidence for the complexity of the CYP2K1 system in rainbow trout.
- Published
- 1994
- Full Text
- View/download PDF
48. Membrane topology of liver microsomal cytochrome P450 2B4 determined via monoclonal antibodies directed to the halt-transfer signal.
- Author
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Black SD, Martin ST, and Smith CA
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal, Blotting, Western, Cytochrome P-450 Enzyme System immunology, Enzyme-Linked Immunosorbent Assay, Male, Mice, Molecular Sequence Data, Protein Sorting Signals chemical synthesis, Protein Sorting Signals immunology, Rabbits, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System chemistry, Intracellular Membranes enzymology, Microsomes, Liver enzymology, Protein Sorting Signals chemistry, Steroid Hydroxylases chemistry
- Abstract
The membrane topology of cytochrome P450 2B4 from the endoplasmic reticulum has been studied with highly-purified liver microsomes in a site-directed immunochemical approach. Microsomes were prepared from phenobarbital-induced rabbits, and the resulting microsomal fraction was washed 6 additional times with 0.1 M pyrophosphate buffer to effect removal of significant quantities of adventitiously-bound protein. Monoclonal antibodies were prepared against residues 18-29 of P450 2B4 (Leu18-Leu-Phe-Arg-Gly-His-Pro-Lys-Ala-His-Gly-Arg29), essentially corresponding to the halt-transfer signal. This region was chosen due to its mutually-exclusive location in the two alternative membrane topology models currently tenable [Black, S.D. (1992) FASEB J.6, 680-685]. Model "A" contains a single transmembrane anchor peptide with the amino terminus projecting into the lumen of the endoplasmic reticulum, while model "B" exhibits a hairpin loop of the first approximately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer; the halt-transfer signal peptide would be located at the cytosolic surface of the membrane in model "A" or as a loop on the lumenal side of the membrane in model "B". Nine antibodies, denoted as MmAbA, MmAbC, MmAbD, MmAbF, MmAbH, MmAbI, MmAbK, MmAbL, and MmAbP, were produced, and all were identified as IgM/kappa subtypes. Western blotting demonstrated that the antibodies could readily recognize P450 2B4 in microsomes. ELISA assays showed that all of the antibodies exhibited strong binding to intact microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
49. Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis.
- Author
-
Chen JH, Hara T, Fisher MJ, and Rees HH
- Subjects
- Adrenodoxin immunology, Adrenodoxin metabolism, Animals, Antibodies immunology, Blotting, Western, Cattle, Cholesterol Side-Chain Cleavage Enzyme immunology, Cholesterol Side-Chain Cleavage Enzyme metabolism, Cytochrome P-450 CYP11B2, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System metabolism, Fat Body enzymology, Ferredoxin-NADP Reductase immunology, Ferredoxin-NADP Reductase metabolism, Larva enzymology, Mitochondria enzymology, Moths embryology, Steroid 11-beta-Hydroxylase immunology, Steroid 11-beta-Hydroxylase metabolism, Steroid Hydroxylases drug effects, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Moths enzymology, Steroid Hydroxylases biosynthesis
- Abstract
The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.
- Published
- 1994
- Full Text
- View/download PDF
50. Epitope mapping of cytochrome P450 2B4 by peptide scanning.
- Author
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Kolesanova EF, Kozin SA, Lemeshko AO, and Archakov AI
- Subjects
- Amino Acid Sequence, Animals, Cats, Cytochrome P-450 Enzyme System immunology, Dogs, Enzyme-Linked Immunosorbent Assay, Epitopes analysis, Humans, Mice, Molecular Sequence Data, Peptide Fragments chemical synthesis, Peptide Mapping, Rabbits, Rats, Sequence Homology, Amino Acid, Steroid Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases, Cytochrome P-450 Enzyme System chemistry, Epitopes chemistry, Peptide Fragments chemistry, Steroid Hydroxylases chemistry
- Abstract
Overlapping hexapeptides covering the whole sequence of the cytochrome P450 2B4 have been synthesized on the solid supports and tested by ELISA using the polyclonal antiserum against cytochrome 2B4. 70 hexapeptide fragments have been found to interact specifically with the antiserum, i.e. to possess antigenic activity. The mapped linear epitopes occupy about 43% of the whole sequence of 2B4. They presumably form clusters in the regions of No. 60-150, 210-300, 390-430 and 465-486 amino acid residues. The use of cytochrome P450 DataBase has allowed to classify the revealed antigenic determinants into absolutely specific for 2B4, specific only for 2B4 and 2B5, characteristic for 2B subfamily and widely distributed in family 2.
- Published
- 1994
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