Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats

Polyphenols from tea and other beverages such as red wine have been regarded with interest as possible chemopreventive agents against cancer. Here we report that red wine polyphenols (50 mg/kg) administered with the diet to F344 rats for 16 weeks inhibited colon carcinogenesis induced by azoxymethane (AOM, 7.4 mg/kg, total dose 74 mg/kg) or dimethylhydrazine (DMH, 30 mg/kg, total dose, 300 mg/kg). Polyphenol-treated animals had a consistently lower tumour yield compared to controls. In polyphenol-treated rats, the main bacterial strains in the faeces at sacrifice were Bacteroides, Lactobacillus and Bifidobacterium spp., whereas microorganisms predominantly identified in control-fed rats were Bacteroides, Clostridium and Propionibacterium spp. Wine polyphenols (57 mg/kg for 10 days, by gavage), administered to rats not treated with carcinogens, produced a significant decrease in the basal level of DNA oxidative damage of the colon mucosa as measured with the comet assay (average pyrimidine oxidation was reduced by 62% and purine oxidation by 57%, p<0.05). To further explore the molecular effects of wine polyphenols we used the microarray technology to study gene expression profiles: rats were treated with 50 mg/kg wine polyphenols for 14 days, mixed in the diet. Global expression analysis of 5707 genes revealed an extensive down-regulation of genes involved in a wide range of physiological functions, such as metabolism, transport, signal transduction and intercellular signalling. By analysing metabolic pathways with the GenMAPP software program we observed that two major regulatory pathways were down-regulated in the colon mucosa of polyphenols-treated rats: inflammatory response and steroid metabolism. We also found a down-regulation of many genes regulating cell surface antigens, metabolic enzymes and cellular response to oxidative stress. In conclusion, reduction of oxidative damage, modulation of colonic flora and variation in gene expression may all concur in the modulation of intestinal function and carcinogenesis by wine polyphenols.     

Focal adhesion molecules as potential target of lead toxicity in NRK-52E cell line

In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.     

Crystal structures of Escherichia coli ATP-dependent glucokinase and its complex with glucose

Intracellular glucose in Escherichia coli cells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E. coli O157:H7. The crystal structure of E. coli glucokinase has been determined to a 2.3-A resolution (apo form) and refined to final Rwork/Rfree factors of 0.200/0.271 and to 2.2-A resolution (glucose complex) with final Rwork/Rfree factors of 0.193/0.265. E. coli GlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small alpha/beta domain (residues 2 to 110 and 301 to 321) and a larger alpha+beta domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains. E. coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal Calpha shift of approximately 10 A. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the gamma-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.     

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15.     

Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.