Maturation and release of interleukin-1beta by lipopolysaccharide-primed mouse Schwann cells require the stimulation of P2X7 receptors

The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1beta maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1beta but did not release detectable amounts of pro or mature IL-1beta. ATP on its own had no effect on the synthesis of pro-IL-1beta, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1beta by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1beta-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1beta in IL-1beta, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1beta. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1beta, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1beta release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1beta by immune-challenged Schwann cells.     

Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.     

Characterization of the immune response to Leishmania infantum recombinant antigens

Leishmaniases have a high prevalence in tropical countries. In order to improve existing diagnostic systems based on total Leishmania proteins, and to identify antigen candidates for vaccine development, an intensive search for the identification of antigens was performed using molecular biology techniques. In this study, the immune response to three L. infantum recombinant antigens was evaluated. Upon stimulation with KMP11, mononuclear cells from leishmaniasis patients produced high levels of IL-10, while a predominant IFN-gamma production could be observed in cultures stimulated with H2A and soluble Leishmania antigen. All the recombinant antigens induced very little IL-5. KMP11 decreased IFN-gamma production by 48% in cultures of peripheral blood mononuclear cells from cutaneous leishmaniasis patients who had been stimulated with soluble Leishmania antigen. Furthermore, antibodies to KMP11 were detected in the sera from all patients with visceral leishmaniasis and in the majority of the sera from patients with cutaneous leishmaniasis or individuals with asymptomatic L. chagasi infection. Thus, KMP11 is recognized by cells and sera of patients with different clinical forms of leishmaniasis, and KMP11, through IL-10 production, proved to be a potent antigen in modulating type 1 immune response.     

Fused RolA protein enhances beta-glucoronidase activity 50-fold: implication for RolA mechanism of action

We report that the plant oncoprotein RolA from Agrobacterium rhizogenes acts to stabilize beta-glucoronidase (Gus) when the two proteins are expressed as a fusion protein in transformed tobacco. The observed 50-fold increase of Gus activity was shown to be related to protein accumulation, with no significant changes in mRNA abundance, kinetic properties of the enzyme and thermostability. The entire RolA sequence is essential to achieve the full effect since both the N-terminal region, spanning a putative reverse signal-anchor and nuclear targeting sequences, or the contiguous C-terminal portion were shown to increase Gus activity only 10-fold. A possible interference of RolA in the protein degradation pathway regulated by auxin is discussed.     

Quinolizidine alkaloid profiles of two taxa of Teline maderensis

The alkaloid composition of the aerial parts of two taxa of Teline maderensis was studied by capillary GLC and GLC-MS. N-Methylcytisine was the major alkaloid found in both plants. Contents of cytisine and lupanine were higher in T. maderensis var. paivae while anagyrine content was more pronounced in T. maderensis var. maderensis. The alkaloids dehydrocytisine, N-acetylcytisine and epibaptifoline appeared only in T. maderensis var. maderensis and N-formylcytisine was identified as a minor constituent in T. maderensis var. paivae, and detected only in trace amounts in the other variety of the plant.     

Alkylaryl-amino derivatives of 3-hydroxy-4-pyridinones as aluminium chelating agents with potential clinical application

The neurotoxicity of aluminium is well established and so strategies for suitable aluminium chelating therapies, aimed at the treatment and/or amelioration of some neurological disorders, are of current interest. The present work describes a set of new bifunctional compounds containing a 3-hydroxy-4-pyridinone (3,4-HP) unit, as the aluminium chelating moiety, which is extra-functionalised with different alkyl-arylamine molecular segments, to account for the improvement on the biodistribution specificity of the chelating agents or the corresponding complexes. Besides the synthetic scheme, studies are performed to assess the properties of these compounds, namely in terms of lipophilicity, Al-chelating ability, speciation and in vivo 67Ga biodistribution. These studies show that the extrafunctional groups fortunately have small effects on the high Al chelating affinity of the 3,4-HP units, over a wide range of pH, but they lead to favourable changes on the lipo-hydrophilic balance of the ligands and on the complex speciation. Differences found in the biodistribution, namely the decrease of the blood-clearance rate and increase of the bone retention or the hepatobiliary excretion, seem to be mostly rationalized in terms of the increasing lipophilic character of the ligands.     

Evaluation of a microarray for genotyping polymorphisms related to xenobiotic metabolism and DNA repair

We present an oligonucleotide microarray ("MetaboChip") based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for cancer susceptibility and pharmacogenetics. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5' end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxynucleotide complementary to the template reveals the polymorphism. Ninety-three SNPs in 42 genes were selected among those resequenced in the context of the SNP500 project, using a set of 102 reference DNA samples from the Coriell Biorepository. Selected SNPs belong to the following genes: ADH1B, ALDH2, APEX, CDKN2A, COMT, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C19, CYP2C9, CYP2E1, CYP3A4, DRD2, DRD4, EPHX1, ERCC1, ERCC2, ERCC4, ERCC5, GRPR, GSTA4, GSTM3, GSTP1, GSTT2, LIG3, MDM2, MGMT, MPO, NAT1, NAT2, NQO1, OGG1, PCNA, POLB, SLC6A3, SOD2, TP53, XRCC1, XRCC2, XRCC3, and XRCC9. We assessed the performance of APEX by comparing the results obtained with MetaboChip against those reported by the SNP500. Among 88 SNPs that yielded signals, 6 showed less than 99% of concordance, whereas 82 performed accurately, showing that APEX is a reliable and sensitive genotyping method.     

Investigations on the reaction pattern of photosystem II in leaves from Arabidopsis thaliana wild type plants and mutants with genetically modified lipid content

The role of digalactosyldiacylglycerol (DGDG) for the functional competence of photosystem II (PS II) has been analyzed in leaves of Arabidopsis thaliana plants where the lipid composition was selectively modified by genetic mutations. Measurements with a newly developed laser flash fluorometer and data evaluation within the framework of an extended "3-quencher" model lead to the following results: (i) the normalized fluorescence transients F(t)/F(0) induced by an actinic laser flash in dark adapted leaves are virtually the same in wild type (WT) and mutants with diminished (about 50%) monogalactosyldiacylglycerol (MGDG) content (mgd1 mutant); (ii) significant changes of the F(t)/F(0) curves are observed in mutants with a severely reduced DGDG content; (iii) in mutants dgd1 and dgd1 dgd2-1 with DGDG contents of 1/15 of the control and below the detection limit, respectively, the probability of the dissipative recombination reaction between P680(+)(*) and Q(A)(-) increases by factors of about two and four, respectively; (iv) the acceptor side reactions are only slightly affected; (v) excitation with actinic laser flash energies above the saturation level of photosynthesis gives rise to elevated carotenoid triplet formation in mutants dgd1 and dgd1 dgd2-1; and (vi) the relationship between DGDG content and functional effect(s) on PS II is strikingly nonlinear. A small fraction of DGDG molecules of the total pool is inferred to be specifically bound to PS II as an essential constituent for its functional competence.