Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid

A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.     

Integration of cell attachment,cytoskeletal localization,and signaling by integrin-linked kinase (ILK), CH-ILKBP,and the tumor suppressor PTEN

Cell attachment and the assembly of cytoskeletal and signaling complexes downstream of integrins are intimately linked and coordinated. Although many intracellular proteins have been implicated in these processes, a new paradigm is emerging from biochemical and genetic studies that implicates integrin-linked kinase (ILK) and its interacting proteins, such as CH-ILKBP (alpha-parvin), paxillin, and PINCH in coupling integrins to the actin cytoskeleton and signaling complexes. Genetic studies in Drosophila, Caenorhabditis elegans, and mice point to an essential role of ILK as an adaptor protein in mediating integrin-dependent cell attachment and cytoskeletal organization. Here we demonstrate, using several different approaches, that inhibiting ILK kinase activity, or expression, results in the inhibition of cell attachment, cell migration, F-actin organization, and the specific cytoskeletal localization of CH-ILKBP and paxillin in human cells. We also demonstrate that the kinase activity of ILK is elevated in the cytoskeletal fraction and that the interaction of CH-ILKBP with ILK within the cytoskeleton stimulates ILK activity and downstream signaling to PKB/Akt and GSK-3. Interestingly, the interaction of CH-ILKBP with ILK is regulated by the Pi3 kinase pathway, because inhibition of Pi3 kinase activity by pharmacological inhibitors, or by the tumor suppressor PTEN, inhibits this interaction as well as cell attachment and signaling. These data demonstrate that the kinase and adaptor properties of ILK function together, in a Pi3 kinase-dependent manner, to regulate integrin-mediated cell attachment and signal transduction.     

Helicobacter pylori and extragastric diseases -- other helicobacters

The role of Helicobacter pylori infection is explored in more and more extragastric diseases without definite proof in most of the studies, except possibly some hematologic diseases. In cardiovascular diseases, including stroke, the presence of CagA positive strains may be involved. The possible role of helicobacters in hepatobiliary diseases goes beyond that of H. pylori to involve enterohepatic helicobacters. New Helicobacter species are regularly described and molecular methods are developed to improve their detection. Helicobacter felis remains the major species to be used in animal models of Helicobacter infection.     

Linkage of one gene for familial glucocorticoid deficiency type 2 (FGD2) to chromosome 8q and further evidence of heterogeneity

In several cases of familial glucocorticoid deficiency (FGD), referred to as FGD type 1, mutations have been described in the coding exon of the adrenocorticotropin receptor (melanonocortin receptor type 2, MC2R) gene. However, for the majority of cases (FGD type 2), no mutations were found in this gene. In the more informative families, the involvement of the MC2R locus could be excluded by linkage or sequencing analysis and, as there was no obvious candidate gene, a genome linkage scan was performed. Fourteen families were studied in this report. Evidence of linkage was found with markers on chromosome 8q in three out of the 14 families (maximum heterogeneity LOD score of 2.81 at D8S1763). These three families were consanguineous and the gene could be located by homozygosity mapping between markers D8S285 and D8S1718 in a 8.8-cM region. No potential candidate genes were apparent in the region. Linkage to this region could be excluded in some families from our sample giving highly negative LOD scores with the markers of the region. This result suggests that at least one other gene, located on a different region, must be responsible for FGD in these families and provides new evidence of genetic heterogeneity of this disorder.     

Sequencing and expression of the CD3 gamma/delta mRNA in Pleurodeles waltl (urodele amphibian)

The CD3 complex is an essential component of the T-cell receptor (TCR) implicated in T-cell maturation and activation. This TCR has been identified in both cartilaginous and bony vertebrates. In different studies where the CD3 chains were cloned and sequenced, it appeared that the CD3 complex is composed of several chains, all susceptible to phosphorylation and able to transduce signals. Here, by an approach combining degenerative oligonucleotide primers and RACE-PCR, we report the cloning and sequencing of a CD3 cDNA from the salamander Pleurodeles waltl, highly homologous to the Xenopus and chicken CD3 gamma/delta cDNAs. Using semi-quantitative PCR and Northern blot analysis, we found the highest CD3 gamma/delta mRNA expression in the thymus; weaker expression was observed in the spleen and blood, followed by the intestine, therefore confirming the tissue and lymphoid specificities of this mRNA. The signals in the spleen, blood and intestine represented 55%, 33% and 16%, respectively, of the signal detected in the thymus. During the embryonic and larval stages of Pleurodeles waltl development, CD3 gamma/delta mRNA expression begins early at the neurula stage (stage 15, 69 h after laying), increases up to stage 33 (9 days after laying) and afterwards remains stable, at least until the larval stage 42 (28 days after laying). As the thymus primordium appears much later, the question of the formation and maturation of the first T-cell precursors outside this organ is posed.     

Elemane-Type Sesquiterpene,Acetonide Derived Polyacetylene and Other Constituents from the Whole Plant of Gymnanthemum theophrastifolium (Schweinf. ex Oliv. & Hiern) H.Rob. and Their Chemophenetic Significance

The chemical investigation of the methanol extract of the whole plant of Gymnanthemum theophrastifolium (Schweinf. ex Oliv. & Hiern) H.Rob. (Asteraceae) led to the isolation of a new elemane-type sesquiterpene ( 1 ), a new acetonide derived polyacetylene ( 2 ) and a naturally occurring compound ( 3 ) from the plant kingdom along with sixteen known compounds ( 4 – 19 ). Their structures were elucidated by extensive NMR and MS analysis. This is the first report on the chemical constituents of G. theophrastifolium. Furthermore, compounds 12 , 13 , and 14 are reported for the first time from the family Asteraceae, while compound 9 is reported for the first time from the genus Gymnanthemum. Thus, the present results provide valuable insights to the chemophenetic knowledge of G. theophrastifolium, which is also discussed in this work.     

Evidence that stimulation of glucose metabolism by insulin is not altered in isolated soleus muscle of pregnant rats

Various concentrations of insulin stimulated to the same extent glucose transport and metabolism in incubated soleus muscle of virgin and 19-day-pregnant rats. This suggests that the resistance to insulin that occurs during pregnancy in vivo does not result from an intrinsic defect in skeletal muscles.     

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.     

The choice of controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by ³²P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.