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.     

Gr-1+ myeloid cells lacking T cell protein tyrosine phosphatase inhibit lymphocyte proliferation by an IFN-gamma- and nitric oxide-dependent mechanism

The T cell protein tyrosine phosphatase is involved in the immune system regulation, as evidenced by defective function and development of several hemopoietic cell populations in T cell protein tyrosine phosphatase (TC-PTP)-deficient mice. In particular, B and T cell proliferation is greatly inhibited when total splenocytes are stimulated by LPS or anti-CD3 mAb. To define the functional defect of TC-PTP(-/-) lymphocytes, we isolated T and B cells from the spleen of TC-PTP(-/-) mice. We show that the proliferative response of lymphocytes was greatly increased when cultured as a purified population, indicating that an inhibitory population is present in TC-PTP(-/-) spleen. However, TC-PTP(-/-) lymphocytes have a 2- to 3-fold lower proliferation rate compared with TC-PTP(+/+) lymphocytes, suggesting that, as shown previously in embryonic fibroblasts, TC-PTP is involved in the control of cell cycle in lymphocytes. We have characterized phenotypically and functionally the inhibitory population present in the spleen of TC-PTP(-/-) mice. We show that a Gr-1(+)-enriched cell population isolated from TC-PTP(-/-) mice suppresses the CD3-induced proliferation of T cells in coculture in vitro. The specific inhibition of NO synthesis with N(G)-monomethyl-L-arginine.monoacetate restored splenocyte responses, and there is a strict correlation between NO levels and the degree of suppression. Neutralization of IFN-gamma with specific mAb almost completely abolished the inhibitory activity of Gr-1(+) cells and concomitantly high levels of NO secretion. Moreover, inhibition of lymphocyte proliferative responses required cell-cell contact to achieve sufficient levels of NO. These findings demonstrate an important function of TC-PTP in the induction of the NO pathway that mediates inhibition of T cell proliferation.     

Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.     

Connecting cell adhesion to the actin polymerization machinery: vinculin as the missing link?

Cell migration is essential to many physiological and pathological processes such as embryogenesis, wound healing or metastasis. This complex process involves a tight coordination between three essential steps - protrusion, adhesion and retraction. Although historically protrusion and adhesion have been linked through structural protein-protein interactions, a direct functional link between the two has long eluded biologists. Recent work from the Burridge laboratory now suggests that vinculin, a cytoskeletal protein involved in the building of the adhesion scaffold, could be the missing link that connects early adhesion sites to the actin-driven protrusive machinery.     

Refolding from denatured inclusion bodies,purification to homogeneity and simplified assay of MGDG synthases from land plants

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.     

Translational regulation of prostaglandin endoperoxide H synthase-1 mRNA in megakaryocytic MEG-01 cells. Specific protein binding to a conserved 20-nucleotide CIS element in the 3'-untranslated region

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [(35)S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3'-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level.     

Internalization and transcytosis of pancreatic enzymes by the intestinal mucosa.

As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.     

The ultrastructure of micropropagated and greenhouse rose plant stomata

Stomata of leaves from in vitro grown rose plantlets remain opened in the dark. The ultrastructure of their guard cells was studied after a 7 h light and a 7 h dark period, and compared to that of functional stomata from plants which have been acclimatized to greenhouse conditions. Qualitative and quantitative observations concerning the shape of the guard cells, mitochondria, plastids and starch grains, demonstrated the similarity in guard cell ultrastructure. The peculiarity of guard cell ultrastructure of in vitro cultured plants was the inability to close in the dark; vacuolar area was 40% of the whole guard cell area during both light and dark period whereas, in guard cells from greenhouse plants, the vacuolar area was 40% of the whole guard cell area during the light and only 25% during the dark period. These results indicate that stomata from in vitro plants are duly developed and possess an ultrastructure suitable for a typical functioning. The inability to close in the dark results from atypical water relation.