细胞色素P4501B1基因多态性与乳腺癌易感性研究进展

细胞色素P450（cytochrome,CYP）1B1是P450超基因家族酶系的一个重要成员,广泛分布于肝外组织,其代谢受到外源性致癌物、雌激素等多种因素的调控。该基因存在遗传多态性,艮前已对CYP1B1基因多态性与乳腺癌易感性进行了多项研究。本文就CYP1B1基因的多态性、调控机制及其与乳腺癌的关系进行了综述。     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Sunflower cDNA Encoding New Cytochrome P450

The primary structure of the cDNA clone SF28 was determined in sunflower (Helianthus annuusL.) flowers. The clone comprises a 874-bp insert corresponding to 227 amino acid residues of the C-terminal part of the cytochrome P450 gene. The sunflower cytochrome P450 was considerably different from the already known plant and animal cytochromes P450.     

Natural allelic variations of xenobiotic-metabolizing enzymes affect sexual dimorphism in Oryzias latipes

Sexual dimorphisms, which are phenotypic differences between males and females, are driven by sexual selection. Interestingly, sexually selected traits show geographical variations within species despite strong directional selective pressures. This paradox has eluded many evolutionary biologists for some time, and several models have been proposed (e.g. ‘indicator model’ and ‘trade-off model’). However, disentangling which of these theories explains empirical patterns remains difficult, because genetic polymorphisms that cause variation in sexual differences are still unknown. In this study, we show that polymorphisms in cytochrome P450 (CYP) 1B1, which encodes a xenobiotic-metabolizing enzyme, are associated with geographical differences in sexual dimorphism in the anal fin morphology of medaka fish (Oryzias latipes). Biochemical assays and genetic cross experiments show that high- and low-activity CYP1B1 alleles enhanced and declined sex differences in anal fin shapes, respectively. Behavioural and phylogenetic analyses suggest maintenance of the high-activity allele by sexual selection, whereas the low-activity allele possibly has experienced positive selection due to by-product effects of CYP1B1 in inferred ancestral populations. The present data can elucidate evolutionary mechanisms behind genetic variations in sexual dimorphism and indicate trade-off interactions between two distinct mechanisms acting on the two alleles with pleiotropic effects of xenobiotic-metabolizing enzymes.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and -naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied. (Mol Cell Biochem120: 171–179, 1993)Thesis student of the Graduate School of Biomedical Sciences, the University of Texas Health Science Center at Houston     

Preparation of Cell Wall Fractions by Various Ways and Activity of Bound Saccharase in Sugar Beet Seedlings

Washed cells of facultative methylotrophs which have the serine pathway showed high activities for l-methionine formation from dl-homocysteine, in the presence of methanol as methyl donor. Strain FM 518, isolated from soil and identified as a bacterium belonging to the genus Pseudomonas, showed the highest activity for l-methionine formation and was used as the parental strain for breeding the l-methionine-producing mutants. An ethionine-resistant mutant, FE 244, derived from strain FM 518, accumulated 0.8 mg/ml l-methionine in a methanol-medium under optimum conditions.     

大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.     

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH⁺/CYP2E1⁺) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.     

Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: Cytochrome P450eryF

Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.