9,10-环甲基十七烷酸诱导灵芝三萜酸合成的信号转导和基因表达分析

为探究9,10-环甲基十七烷酸（9,10-CMA）诱导灵芝三萜酸合成的相关信号机制,分析了NO信号的介导作用。结果表明,在NO信号分子介导下,9,10-CMA可有效地刺激灵芝菌丝体中三萜合成关键酶细胞色素CYP450和苯丙氨酸解氨酶（PAL）的活化以及三萜酸的合成。NO可作为灵芝菌丝体中CYP450产生和PAL活化的上游分子发挥作用。在9,10-CMA诱导下,通过荧光定量PCR分析了6个关键酶基因在灵芝三萜酸合成过程的动态表达。结果表明,在9,10-CMA诱导下与灵芝三萜酸合成相关的角鲨烯合成酶基因sqs、细胞色素P450单加氧酶CYP5150L8基因和3‐羟基‐3‐甲基戊二酰辅酶A还原酶基因hmgr的上调最显著,提示这3个酶在三萜诱导过程中具有重要作用。     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。     

CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene-gene interactions

Gene-environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene-gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene-gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene-gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9-8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5-15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.     

Changes in gene expression during senescence of adrenocortical cells in culture

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B > CYP21 > CYP17 > CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at −2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.     

Molecular genetic studies on the biosynthesis of aldosterone in humans

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.     

Polymorphism of metabolic genes and susceptibility to occupational chronic manganism

In this study we investigated genetic polymorphisms of five metabolizing genes and their association with occupational chronic manganism. We recruited 49 patients with chronic manganism and 50 unrelated healthy control subjects who were welders and ferromanganese smelters and occupationally exposed to manganese dust and fume in the same workshops from three metallurgical industries. The controls were matched to the cases by sex, age, cigarette and alcohol intake, as well as the manganese exposure duration. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the cytochrome P450 2D6L gene (CYP2D6L) and the NAD(P)H:quinone oxidoreductase gene (NQO1). Allele-specific PCR was used to detect the cytochrome P450 1A1 gene (CYP1A1), and the glutathione-S-transferase mu and theta genes (GSTM and GSTT). The frequency of polymorphic alleles, a mutation of CYP2D6L, was significantly lower in patients with chronic manganism (16.3%) than in controls (29.0%). Individuals with the homozygote polymorphism (L/L) of CYP2D6 had a 90% decreased risk of chronic manganism compared with the wild-type (Wt/Wt) (odds ratio =0.10, 95% confidence interval = 0.01-0.82). A significant association between the CYP2D6 genotype subgroup and the latency of chronic manganese poisoning was also found. Patients who had homozygous (L/L) or heterozygous (Wt/L) mutant alleles developed manganism an average of 10 years later than those who were homozygous wildtype (Wt/Wt). However, the allele and genotype frequencies of CYP1A1 and NQO1 genes were distributed similarly in cases and controls. In addition, no difference in the frequencies of GSTM1 and GSTT1 null genotypes were observed between cases and controls. The results suggest that CYP2D6L gene polymorphism might influence susceptibility to manganese-induced neurotoxicity. However, because of limited sample size, our results should be validated in large-scale studies.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

Genetic analysis of mortality in murine angiostrongyliasis costaricensis using SMXA recombinant inbred mouse strains

The SMXA recombinant inbred mouse strain set was produced by systematic inbreeding from the F2 generation of a cross between two progenitor inbred strains, A/J and SM/J, which differed markedly with respect to the patterns of infection with Angiostrongylus costaricensis. We have applied this set to genetic analysis of mouse susceptibility to this nematode infection. The mortality was variable among substrains of the SMXA RI strains, indicating the involvement of multiple genes. Linkage analysis showed several chromosomal regions closely linked to mortality; chromosome 6 (D6Rik86, 87; P0.001), 10 (D10Rik66–D10Mit12; P=0.0058), 13 (D13Rik79, 80; P=0.0096) and 17 (D17Mit28–D17Rik76; P=0.0088).     

Effects of anastrozole on the intratumoral gene expression in locally advanced breast cancer

Intratumoral levels of E₁ (oestrone), E₁S (oestrone sulphate) and E₂ (oestradiol) are significantly reduced by treatment with the aromatase inhibitor anastrozole regardless of treatment response. The purpose of the present pilot study was to look for additional markers of biochemical response to aromatase inhibitors on mRNA expression level. Whole genome expression was studied using microarray analysis of breast cancer tissue from 12 patients with locally advanced tumors, both before and following 15 weeks of treatment with the aromatase inhibitor anastrozole (Arimidex^®). Intratumoral mRNA levels for a subset of genes coding for steroid metabolizing enzymes, hormone receptors and some growth mediators involved in cell cycle control were analysed by quantitative RT-PCR. There was a correlation between the two methods for some but not all genes. The mRNA expression levels of the different genes were correlated to each other and to the intratumoral levels of E₁, E₂ and E₁S, before and after the treatment. Notably, a correlation of the E₁/E₂ metabolic ratio to the mRNA levels of CYP19A1 was observed before treatment (r = 0.745, p < 0.005). Whole genome expression analysis of these 12 breast cancer patients revealed similar tumor classification to previously published larger studies. Tumors with no or low expression of ESR1 (oestrogen receptor) clustered together and were characterized by a strong basal-like signature highly expressing keratins 5/17, cadherin 3, frizzled and apolipoprotein D, among others. The luminal epithelial tumor cluster, on the other hand, highly expressed ESR1, GATA binding protein 3 and N-acetyl transferase. An evident ERBB2 cluster was observed due to the marked over-expression of the ERBB2 gene and GRB7 and PPARBP in this patient material). Using significance analysis of microarrays (SAM), we identified 298 genes significantly differently expressed between the partial response and progressive disease groups.     

Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution

The fugu (pufferfish) genome has been sequenced, and a second genome assembly was released 17 May 2002. Exhaustive searches were made to identify all P450 genes and pseudogenes from the earlier release of 26 October 2001. P450 genes assembled as completely as possible from these data were used to do additional searches of the newer assembly and all P450 genes and pseudogenes in the available fugu sequence data have been identified, compared to human P450s, and assigned names. There are 54 P450 genes in fugu and 1 nearly intact pseudogene (CYP3A50P). CYP1A is missing much of its N-terminal half; however, 45 P450 genes are completely assembled. Eight others are lacking only one or two exons or less. CYP2X4 is known only from an EST. This may be a 55th P450 gene if it represents an accurate sequence. In addition to 2X4, there are 16 other pseudogene fragments or small pieces of P450 genes. At the P450 family level, 17 of 18 mammalian families are found in fugu. CYP39 is the only CYP family missing and it is not seen in any other fish sequence data either. The CYP2 family shows the largest degree of divergence. In the CYP2 family, only CYP2R1 and CYP2U1 are conserved as recognizable subfamilies across species. Intron-exon boundaries are largely preserved across 420 million years of evolution.     

张掖湿地芨芨草叶大小和叶脉密度的权衡关系

叶大小-叶脉密度的权衡关系是植物叶经济谱理论的基础, 对理解资源竞争条件下植物叶片的物理构建与生理代谢的关系具有重要的意义。该文采用标准化主轴估计(standardized major axis estimation, SMA)的方法, 按芨芨草(Achnatherum splendens)株丛密度设置I (>12丛·m^-2)、II (8-12丛·m^-2)、III (4-8丛·m^-2)和IV (<4丛·m^-2) 4个密度梯度, 以叶面积和叶干质量分别表示叶大小, 对张掖洪泛平原湿地不同密度条件下芨芨草种群的叶大小和叶脉密度的关系进行研究。结果表明: 随着芨芨草株丛密度的降低, 湿地群落的土壤含水量逐渐减小、土壤电导率逐渐增加, 芨芨草的净光合速率(P_n)、蒸腾速率(T_r)和分枝数呈先增大后减小的趋势, 叶面积、叶干质量、比叶面积和株高呈逐渐减小趋势、光合有效辐射(PAR)和叶脉密度呈逐渐增加趋势; 芨芨草叶大小和叶脉密度在高密度(I)和低密度(IV)样地均呈极显著负相关关系(p < 0.01), 中密度(II、III)样地二者呈显著负相关关系(p < 0.05); 叶大小和叶脉密度回归方程的SMA斜率在不同密度样地均显著小于-1 (p < 0.05), 即芨芨草叶大小和叶脉密度呈“此消彼长”的权衡关系。在高密度湿地群落芨芨草倾向于大叶片低叶脉密度的叶片构建模式, 在低密度湿地群落选择小叶片高叶脉密度的异速生长模式, 体现了密度制约下湿地植物的生物量分配格局和资源利用对策。     

Trade-off between leaf size and vein density of Achnatherum splendens in Zhangye wetland

Aims Trade-offs between leaf size and vein density are the basis of the theory of leaf economics spectrum, and are to understand the relationship between the physical build and physiological metabolism of plant leaves under different degrees of competition for resources. Our objective was to study the changes in the relationship between leaf size and vein density (leaf dry biomass and leaf area) in Achnatherum splendens populations with four plant bundle densities located in the flood plain wetland of Zhangye. Methods The study site was located at floodplain wetlands of Zhangye, Gansu Province, China. Survey and sampling were carried out in the communities that A. splendens dominated. According to the plant bundle density, the A. splendens communities were divided into four density gradients with “bundle” for the sampling units, high density (I, > 12 bundle·m^-2), medium density (II, 8-12 bundle·m^-2), medium density (III, 4-8 bundle·m^-2) and Low density (IV, <4 bundle·m^-2). According to the density of each combination, we chose seven (5 m × 5 m) A. splendens samples, resulting in a total of 28 samples (4 × 7). The soil physical and chemical properties of four density gradients were investigated and six samples of A. splendens were used to measure the leaf area, leaf dry biomass and vein density in laboratory, and biomass of different organs was measured after being dried at 85 °C in an oven. 28 plots were categorized into three groups: high, medium and low density, and the standardized major axis (SMA) estimation method was used to examine the allometric relationships between leaf area, leaf dry biomass and vein density. Important findings The results showed that with the population density changed from high, medium, to low, the soil moisture decreased, and soil electric conductivityincreased. The leaf area, leaf biomass and height of A. splendens decreased, and the vein density, specific leaf area and photosynthetically active radiation (PAR) increased gradually. In addition, leaf net photosynthetic rate (P_n), transpiration rate (T_r) and twig number firstly increased then decreased. There was a highly significantly negative correlation (p < 0.01) between the leaf size and vein density on the high- and low-level densities (I, IV), whereas less significant (p < 0.05) on the level of medium density (II, III). The SMA slope of regression equation in the scaling relationships between leaf size and vein density was significantly smaller than -1 (p < 0.05).