Regulation of CREB-mediated gene expression by salt inducible kinase

Salt inducible kinase (SIK) was identified as a molecule induced in the adrenal glands of rats fed with a high-salt diet. A major downstream of SIK is regulation of camp-responsive element (CRE)-dependent gene expression. SIK represses the activity of CRE-binding protein (CREB) by phosphorylating a CREB-specific co-activator transducer of regulated CREB activity (TORC). When TORC is dephosphorylated it activates CREB in a CREB-phosphorylation independent manner. The importance of the dephosphorylation of TORC has been suggested by the fact that a kinase inhibitor staurosporine induces dephosphorylation of TORC and upregulates the gene expression of CYP11A, CYP11B1, CYP11B2 and StAR in adrenocortical cells. The identification of SIK caused a stir in the field of CREB studies and led to disclosure of cascades hidden behind the classical mechanism for CREB activity.     

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.     

磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

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。     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4–3A7 and 3A7–3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3′ end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.     

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.     

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.     

GST, CYP and PON1 polymorphisms in farmers attributing ill health to organophosphate-containing sheep dip

Previously we reported that in sheep dippers exposed to organophosphates the frequency of paraoxonase (PON1) polymorphisms differed between those with or without self-reported ill health. We have now examined whether polymorphisms in other genes involved in xenobiotic metabolism alter disease risk in this population. There were elevated but non-significant risks associated with the CYP2D6 WT genotype (odds ratio (OR) 1.47, 95% CI 0.83-2.60), or a GSTP1*B or *C allele (OR 1.37, 95% CI 0.88-2.01) or being GSTM1*2/GSTT1*2 homozygous (OR 1.61, 95% CI 0.74-3.48). Similar results were generally obtained after the exclusion of subjects to obtain a more homogenous case-referent population: for double null GSTM1 and GSTT1 homozygotes the OR was 2.06 (95% CI 0.85-2.04). In those also likely to have been exposed to diazinon, risks associated with a GSTP1*B or *C allele (OR 1.82, 95% CI 0.92-3.63) or a GSTM1*2/GSTT1*2 homozygous (OR 2.60, 95% CI 0.72-10.42) were elevated but not to a significant extent. Risk associated with PON1 genotype and phenotype varied with CYP2D6 and GSTP1 genotype but not consistently with a priori hypotheses. Further work is necessary to delineate more clearly pathways of organophosphate activation and non-PON1 pathways of detoxification and to confirm whether CYP and GST polymorphisms alter disease risk in populations exposed to organophosphates.     

Binding of Thermobifida fusca CDCel5A, CDCel6B and CDCel48A to easily hydrolysable and recalcitrant cellulose fractions on BMCC

The binding of the catalytic domain of Thermobifida fusca endoglucanase (EC 3.2.1.4) CD_Cel5A and exoglucanases (EC 3.2.1.91) CD_Cel6B and CD_Cel48A to BMCC was studied. At 5 °C, the binding of these CDs to BMCC is rapid with maximum binding occurring just after CD loading. An observed desorption of the bound CDs with time was attributed to the loss of binding sites due to hydrolysis of the easily hydrolysable BMCC. This conclusion was supported by prehydrolysis experiments where the easily hydrolysable BMCC fraction was removed, and binding to the recalcitrant fraction was observed. More of the CDs were bound to the recalcitrant fraction. This was especially true for the exoglucanases, CD_Cel6B and CD_Cel48A, where 81–76% of the total CD binding was to the recalcitrant BMCC fraction. The binding to the easily hydrolysable fraction for the endo CD_Cel5A was 1.5–4 times higher than that of the exo CD_Cel6B and CD_Cel48A at the same enzyme concentration. Although CD_Cel5A saturated the easily hydrolysable fraction of BMCC first, the ratio of the bound to the recalcitrant substrate was constant for CD_Cel6B and CD_Cel48A as 81 and 76%, respectively. As the reaction temperature was increased to 50 °C, CD_Cel5A and CD_Cel48A exhibited a rapid increase in the rate and extent of binding, while CD_Cel6B exhibited a decrease in the extent of binding.     

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).     

Phycomyces blakesleeanus car B mutants: Their use in assays of phytoene desaturase

Strains of car B (phytoene-accumulating) mutants of Phycomyces blakesleeanus have been characterized with respect to their carotene contents, in vitro formation of isoprenoids from [2-¹⁴C] mevalonic acid and their ability to produce [¹⁴C]phytoene in situ for use in coupled assays of phytoene desaturase activity. All strains produced predominantly (15-Z)-phytoene both in vivo and in vitro. Other isoprenoids were produced by cell extracts including squalene, sterols, prenyl diphosphates and prenyl alcohols. The addition of 1% Tween 60 to crude cell extracts of the mutants partially restored wild type carotenogenic activity and also altered the proportions of other isoprenoids formed. However, in a cytosolic fraction of the car B mutant, the addition of 1% Tween 60 did not result in the production of any carotenoid from phytoene. This fraction was the most effective source of [¹⁴C] phytoene for use in coupled assays of phytoene desaturase activity.     

The role of estrogen in the initiation of breast cancer

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17β-estradiol (E₂) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E₂, 4-OH-E₂, and 16--OH E₂), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E₂ induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER- and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E₂ induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E₂, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

Analysis of the SAG5 locus reveals a distinct genomic organisation in virulent and avirulent strains of Toxoplasma gondii

We have recently characterised, in the virulent strain RH of Toxoplasma gondii, three glycosylphosphatidylinositol-anchored surface antigens related to SAG1 (p30) and encoded by highly homologous, tandemly arrayed genes named SAG5A, SAG5B and SAG5C. In the present study, we compared the genomic organisation of the SAG5 locus in strains belonging to the three major genotypes of T. gondii. Southern blot analysis using a SAG5-specific probe produced two related but distinct hybridisation patterns, one exclusive of genotype I virulent strains, the other shared by avirulent strains of either genotype II or genotype III. To understand the molecular bases of this intergenotypic heterogeneity, we cloned and sequenced the SAG5 locus in the genotype II strain Me49. We found that in this isolate the SAG5B gene is missing, with SAG5A and SAG5C laying contiguously. This genomic arrangement explains the hybridisation profiles observed for all the avirulent strains examined and indicates that the presence of SAG5B is a distinctive trait of genotype I. Furthermore, we identified two novel SAG1-related genes, SAG5D and SAG5E, mapping respectively 1.8 and 4.0 kb upstream of SAG5A. SAG5D is transcribed in tachyzoites and encodes a polypeptide of 362 amino acids sharing 50% identity with SAG5A-C, whereas SAG5E is a transcribed pseudogene. We also evaluated polymorphisms at the SAG5 locus by comparing the coding regions of SAG5A-E from strains representative of the three archetypal genotypes. In agreement with the strict allelic dimorphism of T. gondii, we identified two alleles for SAG5D, whereas SAG5A, SAG5C and SAG5E were found to be three distinct nucleotide variants. The higher intergenotypic polymorphism of SAG5A, SAG5C and SAG5E suggests that these genes underwent a more rapid genetic drift than the other members of the SAG1 family. Finally, we developed a new PCR–restriction fragment length polymorphism method based on the SAG5C gene that is able to discriminate between strains of genotype I, II and III by a single endonuclease digestion.     

Synthesis and SAR of highly potent dual 5-HT1A and 5-HT1B antagonists as potential antidepressant drugs

Novel 5-HT₁ autoreceptor ligands based on the N-4-aryl-piperazinyl-N′-ethyl-5,6,7,8-tetrahydropyrido[4′, 3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one core are described. Aiming at antidepressants with a novel mode of action our objective was to identify potent antagonists showing balanced affinities and high selectivity for the 5-HT_1A and 5-HT_1B receptors. Strategies for the development of dual 5-HT_1A and 5-HT_1B antagonists based on 1 and 2 as leads and the corresponding results are discussed. Isoquinoline analogue 33 displayed high affinity and an antagonistic mode of action for the 5-HT_1A and the 5-HT_1B receptors and was characterized further with respect to selectivity, electrically stimulated [³H]5-HT release and in vivo efficacy.