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P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.  相似文献   

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Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.  相似文献   

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Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.  相似文献   

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Expression and function of the UM4D4 antigen in human thymus   总被引:3,自引:0,他引:3  
UM4D4 is a newly identified T cell surface molecule, distinct from the Ag receptor and CD2, which is expressed on 25% of peripheral blood T cells, resting or activated. Monoclonal anti-UM4D4 is mitogenic for T cells and T cell clones. Since alternative activation pathways independent of Ag/MHC recognition may be important in thymic differentiation, the expression and function of UM4D4 was examined in human thymus. UM4D4 was found on the surface of 6% of thymocytes. All thymocyte subsets contained UM4D4+ cells but expression was greatest on thymocytes that were CD1- (12%), CD3+ (11%) and especially CD4-CD8- (18%). CD3+CD4- CD8- cells, most of which bear the gamma delta-receptor, were greater than or equal to 50% + for UM4D4. Moreover, anti-UM4D4 was comitogenic for thymocytes together with PMA or IL-2. Anti-UM4D4 also reacted strongly with a subset of thymic epithelial cells in both cortex and medulla. Dual color fluorescence microscopy, with anti-UM4D4 and antibodies to other thymic epithelial Ag, showed UM4D4 expression on neuroendocrine thymic epithelium but not on thymic fibrous stroma. Thus, UM4D4 is expressed on, and represents an activation pathway for, a subset of thymic T cells. In addition, this determinant, initially identified as a novel T cell activating molecule, is broadly expressed by neuroendocrine thymic epithelium. Although the function of UM4D4 on the thymic epithelial cells is not yet clear, it is possible that UM4D4 represents a pathway for the functional activation of a subset of the thymic epithelium as well as a subset of thymocytes, thus playing a dual role in T cell differentiation.  相似文献   

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Role of IgE receptors in effector function of human eosinophils   总被引:24,自引:0,他引:24  
After analysis of the technical parameters of the rosette assay with human IgE-coated erythrocytes, Fc epsilon receptors for IgE (Fc epsilon R) on human peripheral blood eosinophils were compared to Fc epsilon R on lymphocytes and monocytes. Antibodies directed against Fc epsilon R on lymphocytes and monocytes inhibited the IgE rosettes formed by eosinophils from hypereosinophilic patients, which suggests that Fc epsilon R on eosinophils were antigenically related to Fc epsilon R on lymphocytes and monocytes. Fc epsilon R on human eosinophils were shown to participate in the killing effect of Schistosoma mansoni schistosomula in vitro in the presence of purified eosinophils from highly hypereosinophilic patients (blood counts greater than 3000/mm3) and anti-schistosomula IgE antibodies present in S. mansoni-infected patient sera. Similar levels of inhibition of cytotoxicity were obtained after preincubation of eosinophils with aggregated human IgE or with anti-Fc epsilon R antibodies, whereas preincubation with aggregated IgG or with anti-C3b receptor antibodies did not decrease the killing effect for schistosomula targets. This IgE-dependent cytotoxic capacity seemed restricted to eosinophils with an abnormally low density ("hypodense" cells) present only in highly hypereosinophilic patients. These observations might be related to nonparasitic situations in which increased levels of IgE and tissue or blood eosinophils are observed.  相似文献   

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Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.  相似文献   

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目的:研究CYP4F3基因单核苷酸多态性(SNP)在中国汉族人群中的分布,为进一步研究该基因群体遗传学特征及与疾病易感性的相关性提供更为详实的数据。方法:对CYP4F3基因进行重测序,构建连锁不平衡模式,选择标签SNP在192例北京和424例广州汉族个体中进行基因分型。结果:CYP4F3基因重测序共检出30个SNP,连锁不平衡分析显示广州和北京地区人群的连锁不平衡模式不同,但选择的8个标签SNP的等位基因和基因型频率分布在2个人群中的差异无统计学意义。结论:中国北京地区汉族与广州地区汉族人群CYP4F3基因多态性无显著差异,但不同种族间存在差异。  相似文献   

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In order to obtain cDNA clones coding for CYP4 proteins in frog Xenopus laevis, degenerate primers were designed utilizing the conserved sequences of known CYP4s and were used to amplify partial cDNA fragments from liver mRNA. Five new CYP genes were identified. Three of these genes, XL-1, -2 and -3, were assigned to the CYP4T subfamily found previously in fish and amphibians. The other two genes, XL-4 and XL-5, were quite similar to CYP4F and CYP4V subfamilies, respectively. Subsequently, two full-length cDNA clones corresponding to XL-4 and XL-5 were isolated and characterized. The resultant cDNAs, designated as CYP4F42 and CYP4V4, had open reading frames encoding proteins of 528 and 520 residues, respectively. RT-PCR analysis indicated that the expression of CYP4F42 was limited to the liver, kidney, intestine and brain. In contrast, CYP4V4 mRNA was expressed ubiquitously.  相似文献   

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Synthesis and release of leukotriene C4 by human eosinophils   总被引:13,自引:0,他引:13  
When human peripheral blood eosinophils isolated to 92.5% +/- 6.9 purity were stimulated with either the calcium ionophore A23187 or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), immunoreactive leukotriene C4 (LTC4) was initially localized intracellularly and was subsequently released to the external medium in kinetically distinguishable steps. Eosinophils were stimulated with 2.5 microM A23187 in the presence of 20 mM L-serine, a hypochlorous acid scavenger that prevents the oxidative metabolism of sulfidopeptide leukotrienes. Total production of immunoreactive LTC4, the sum of intra- and extracellular LTC4, was complete within 5 to 10 min. At 5, 10, and 30 min, 65.9% +/- 15.2, 42.3% +/- 24.3, and 5.5% +/- 3.9, respectively, of the total amount of LTC4 measured remained intracellular as detected after the media and cells were separated and the latter was extracted with methanol. The time course for the intracellular synthesis and extracellular release of immunoreactive LTC4 from eosinophils pretreated with 5 micrograms/ml cytochalasin B and stimulated with 0.5 microM FMLP was like that obtained with ionophore, although the total LTC4 production was only approximately 10%. The identity of the intracellular LTC4 was confirmed by elution with reverse-phase high pressure liquid chromatography followed by scanning UV spectroscopy, radioimmunoassay, and bioassay. Eosinophils that were stimulated with A23187 in the absence of L-serine metabolized newly synthesized LTC4 to 6-trans-LTB4 diastereoisomers and subclass-specific diastereoisomeric sulfoxides that were identified only in the extracellular medium. Thus the response of purified eosinophils to two different stimuli demonstrates a transient intracellular accumulation of biologically active LTC4, the distinct extracellular release, and the apparent limitation of oxidative metabolism to the extracellular location.  相似文献   

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We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.  相似文献   

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On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.  相似文献   

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Eosinophils are seen together with neutrophils at sites of inflammation. However, their roles are not clear. In addition, eosinophils infiltrate tumor tissue in some neoplastic diseases. In this study, we show that large amounts of the neutrophil-activating CXC chemokine growth-related oncogene (GRO)-alpha can be produced by human eosinophils. Eosinophils showed presence of preformed GRO-alpha in the crystalloid-containing specific granules (190 pg/2 x 10(6) cells). During incubation, a strong increase in GRO-alpha gene expression was seen. At a low cell density, addition of TNF-alpha or IL-1 beta increased the production of GRO-alpha in eosinophils, which was not the case at a higher cell density. Eosinophils can produce TNF-alpha themselves, and neutralizing Abs against TNF-alpha significantly inhibited GRO-alpha production. This suggests that autocrine and paracrine effects from TNF-alpha can be important when up-regulating GRO-alpha gene expression. In contrast, IFN-gamma, a prototypic Th1-cytokine, down-regulated expression of GRO-alpha. This may be important during resolution of inflammation but also suggests different roles for eosinophils depending on the inflammatory context. Tumor-infiltrating eosinophils in Hodgkin's disease of the nodular sclerosing type are associated with a poor prognosis. Eosinophils from such tumor tissue showed an abundant expression of GRO-alpha. The GRO-alpha receptor CXCR2 was also detected in tumor tissue, proposing interactions between eosinophils and the tumor. Our findings suggest that eosinophils can promote inflammation through recruitment of CXCR2-bearing cells. In addition, this feature of the eosinophils indicates a role for these cells in the biology of certain tumors.  相似文献   

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