In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.     

Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Cholesteryl ester flux from HDL to VLDL-1 is preferentially enhanced in type IIB hyperlipidemia in the postprandial state

Postprandial triglyceride-rich lipoproteins (TRL) exert proatherogenic effects at the arterial wall, including lipid deposition. Following consumption of a mixed meal (1200 kcal), plasma-mediated cellular free cholesterol (FC) efflux, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) activities were determined in subjects (n = 12) displaying type IIB hyperlipidemia and compared with those in a normolipidemic control group (n = 14). The relative capacity of plasma to induce FC efflux from Fu5AH cells via the SR-BI receptor was significantly increased 4 h postprandially (+23%; P < 0.005) in the type IIB group, whereas it remained unchanged for postprandial plasma from normolipidemic subjects. LCAT activity was significantly elevated 2 h postprandially in both the IIB and control groups, (+46% and +36%, respectively; P < 0.005 vs. respective baseline value). In type IIB subjects, total cholesteryl ester (CE) mass transfer from HDL to total TRL [chylomicrons (CMs) + VLDL-1 + VLDL-2 + IDL] increased progressively from 15 +/- 2 micro g CE/h/ml at baseline to 28 +/- 2 micro g CE transferred/h/ml (+87%; P = 0.0004) at 4 h postprandially. CE transfer to CMs and VLDL-1 was preferentially stimulated (2.6-fold and 2.3-fold respectively) at 4 h in IIB subjects and occurred concomitantly with elevation in mass and particle number of both CMs (2.3-fold) and VLDL-1 (1.3-fold). Furthermore, in type IIB subjects, CETP-mediated total CE flux over the 8 h postprandial period from HDL to potentially atherogenic TRL was significantly enhanced, and notably to VLDL-1 (32-fold elevation; P < 0.005), relative to control subjects. Such CE transfer flux was reflected in a significant postprandial increase in CE-TG ratio in both CMs and VLDL-1 in type IIB plasmas. In conclusion, HDL-CE is preferentially targeted to VLDL-1 via the action of CETP during alimentary lipemia, thereby favoring formation and accumulation of atherogenic CE-rich remnant particles.     

Transcytosis of iota-toxin across polarized CaCo-2 cells

Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.     

Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

Regulation of Protrusion Shape and Adhesion to the Substratum during Chemotactic Responses of Mammalian Carcinoma Cells

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J. E. Segallet al.,1996,Clin. Exp. Metastasis14, 61–72), we analyzed the relationship between adhesion and chemoattractant-stimulated protrusion. Exposure to EGF led to a rapid remodeling of the adhesive contacts on adherent cells, in synchrony with extension of a flat lamellipod over the substratum. EGF-stimulated lamellipods still extended in the presence of adhesion-blocking peptides or over nonadhesive surfaces. They were, however, slightly shorter and retracted rapidly under those conditions. The major protrusive structure observed on well-spread, adherent cells, after EGF stimulation was a flat broad lamellipod, whether or not in contact with the substratum, while cells in suspension showed transient protrusive activity over the entire cell surface. We conclude that the initial adhesive status of the cell conditions the shape of the outcoming protrusion. Altogether our results suggest that, although adhesive contacts are not necessary for lamellipod extension, they play a role in stabilizing the protrusion as well as in the control of its final shape and amplitude.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Profiling of sugar transporter genes in grapevine coping with water deficit

The profiling of grapevine (Vitis vinifera L.) genes under water deficit was specifically targeted to sugar transporters. Leaf water status was characterized by physiological parameters and soluble sugars content. The expression analysis provided evidence that VvHT1 hexose transporter gene was strongly down-regulated by the increased sugar content under mild water-deficit. The genes of monosaccharide transporter VvHT5, sucrose carrier VvSUC11, vacuolar invertase VvGIN2 and grape ASR (ABA, stress, ripening) were up-regulated under severe water stress. Their regulation in a drought-ABA signalling network and possible roles in complex interdependence between sugar subcellular partitioning and cell influx/efflux under Grapevine acclimation to dehydration are discussed.