Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.     

Elicitor activity of a fungal endopolygalacturonase in tobacco requires a functional catalytic site and cell wall localization

CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared: CLPG1, with or without its signal peptide (SP; PG1, PG1deltaSP); CLPG1 with the tobacco expansin1 SP instead of its own SP (Exp::PG1deltaSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and beta-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco.     

Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.     

Synthesis of glycocluster-tumor antigenic peptide conjugates for dendritic cell targeting

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.     

Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase

Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the pi-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.     

Re-distribution of phospholipase C gamma 2 in macrophage precursors is mediated by the actin cytoskeleton under the control of the Src kinases

Macrophage colony-stimulating factor (M-CSF) is a growth factor that is known to trigger several signalling pathways through receptor tyrosine kinase activation. We investigated the specific requirements for the activation of phospholipase C gamma 2 (PLC-γ2) during the differentiation of mouse bone marrow-derived macrophage precursors. M-CSF stimulation induced rapid PLC-γ2 translocation and phosphorylation from the cytosolic compartment to the cell periphery. Both events were dependent on cytoskeleton integrity and Src kinase activity, but only PLC-γ2 phosphorylation did not require PI3-kinase activity. Biochemical experiments as well as confocal microscopy analyses indicate that the translocation of PLC-γ2 is mediated by the direct association of this protein with the actin cytoskeleton. Using GST-fusion proteins containing various deletions of the PLC-γ2 Src homology region, it was found that PLC-γ2 binds to F-actin via its SH2 domains, a feature that has equally been found in a co-sedimentation assay. This association, which is increased during actin reorganisation and disrupted by cytoskeleton inhibitors, seems to be a primary means to recruit this enzyme to the cell periphery. These results indicate that, upon M-CSF stimulation, PLC-γ2 cellular localisation and phosphorylation are strongly dependent on cytoskeleton architecture of the macrophage precursor as well as the PI3-kinase and the Src kinases.     

Water-soluble dendrigrafts bearing saccharidic moieties: elaboration and application to enzyme linked OligoSorbent Assay (ELOSA) diagnostic tests

The synthesis of a series of water-soluble galactopyranose-functionalized polystyrene-polyvinyl ether dendrigrafts and their characterization (in solution and thin solid deposits) have been achieved. The presence of external galactopyranose groups on dendritic polymers has been exploited to prepare dendrigraft-oligonucleotide conjugates using a simple one-step coupling procedure with amino-ended oligonucleotides (ODNs). Several parameters such as the peripherical density of hydrophilic branches, the polymerization degree of polystyrene or poly(hydroxyethyl vinyl ether) blocks, and the number of galactopyranose groups were tuned. A capture test with short labeled complementary ODNs (25 bases) confirmed the presence of covalently bound ODNs on various kinds of dendrigrafts. The ability of the dendritic polymers to enhance the sensitivity of enzyme-linked oligosorbent assay (ELOSA) diagnostic tests (detection of hepatitis B virus, DNA target of 2400 bases) was then evaluated, especially the influence of the macromolecular architecture and the impact of the structural parameters. The dendrigraft-ODN conjugate with the lower saccharide external density was found to lead to a very significant amplification of the fluorescence signal, corresponding to a limit of sensitivity of 10(9) DNA copies per milliliter (instead of 10(11) DNA copies per milliliter without using dendrigrafts). Conversely, the dendrigrafts exhibiting a very high number of branches and galactopyranose groups at their periphery were not able to induce a better sensitivity due to steric hindrance generated by the peripheral congestion on these polymers.     

Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients

Background

While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression.

Methodology/Principal findings

Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While α-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis.

Conclusion/Significance

We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients.