Retinal dehydrogenase 12 (RDH12) mutations in leber congenital amaurosis

Leber congenital amaurosis (LCA), the most early-onset and severe form of all inherited retinal dystrophies, is responsible for congenital blindness. Ten LCA genes have been mapped, and seven of these have been identified. Because some of these genes are involved in the visual cycle, we regarded the retinal pigment epithelium and photoreceptor-specific retinal dehydrogenase (RDH) genes as candidate genes in LCA. Studying a series of 110 unrelated patients with LCA, we found mutations in the photoreceptor-specific RDH12 gene in a significant subset of patients (4.1%). Interestingly, all patients harboring RDH12 mutations had a severe yet progressive rod-cone dystrophy with severe macular atrophy but no or mild hyperopia.     

Phylogenetic analysis of bacterial populations in waters of the former Texcoco Lake, Mexico

Molecular techniques were used to compare the compositions of the bacterial communities of the 2 following lagoons from the former soda Texcoco Lake, Mexico: the restored Facultativa lagoon and the Nabor Carrillo lagoon. Ribosomal intergenic spacer analysis (RISA) revealed that bacterial communities of the 2 lagoons were different and presented a relatively low diversity. Clone libraries of 16S rDNA genes were constructed, and significant phylotypes were distinguished by restriction fragment length polymorphism (RFLP). A representative clone from each phylotype was partially sequenced. Molecular identification and phylogenetic analyses based on ribosomal sequences revealed that the Facultativa lagoon harbored mainly gamma- and beta-Proteobacteria, low G+C Gram-positive bacteria, and several members of the Halobacteriaceae family of archaea. The Nabor Carrillo lagoon mainly included typical halophilic and alkaliphilic low G+C Gram-positive bacteria, gamma-Proteobacteria, and beta-Proteobacteria similar to those found in other soda lakes. Several probably noncultured new bacterial species were detected. Three strains were isolated from the Nabor Carrillo lagoon, their partial 16S rDNA sequences were obtained. On this basis, they were identified as Halomonas magadiensis (H1), Halomonas eurihalina (H2), and Staphylococcus sciuri (H3). This is the first study that uses molecular techniques to investigate potential genetic diversity in the Texcoco lakes. In this preliminary evaluation, we infer the presence of alkalophilic, halophilic, or haloalkaliphilic bacteria potentially useful for biotechnology.     

Autologous cell transplantation and cardiac tissue engineering: potential applications in heart failure

The promising concept of cell transplantation and cardiac tissue engineering has been developed in the last few years and focused on strategies attempting to replace dysfunctional, necrotic, and/or apoptotic cardiomyocytes with new cells of mesodermal origin. Transplantation of autologous cells minimizes the risk of neoplasia and avoids immune rejection associated with allogenic or xenogenic cells and recent data hold enormous hopes for short term clinical practices. Tissue engineering represents another promising approach that makes possible the creation of new functional tissues to replace the lost or failing one. Three-dimensional polymeric scaffolds provide the mechanical support for the candidate cells until the formation of cardiac-like tissue prior to surgical repair of the infarcted myocardium. For ultimate clinical applications, further investigations have to select the appropriate cell types, to determine the sufficient number of grafted cells and to provide the long term evaluation of these strategies in the global improvements of cardiac function (neoangiogenesis, synchronous contraction and extracellular matrix remodelling).     

Recombinant (2-->3)-alpha-sialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl Lewis(x) and Lewis(a) precursor oligosaccharides

The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni(2+)-NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [alpha-Neu5Ac-(2-->3)-D-Galp-(1-->4)-beta-D-GlcpNAc-O-CH(2)](2)-C-(CH(2)OBn)(2) (12); (ii) the dansylated derivative, alpha-Neu5Ac-(2-->3)-D-Galp-(1-->3)-beta-D-GlcpNAc-O-(CH(2))(6)-NH-dansyl and; (iii) the tetrasacharide alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O-CH(3). Compound 12 was itself prepared from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.     

In vitro induction of microglial and endothelial cell apoptosis by cerebrospinal fluids from patients with human African trypanosomiasis

In human African trypanosomiasis, trypanosomes first develop in the blood and lymph (Stage 1), then spread to the central nervous system (CNS) (Stage 2). Disruption of the blood-brain barrier of unknown mechanism occurs in Stage 2 disease. The hypothesis that cerebrospinal fluids (CSF) from African trypanosomiasis patients might contain factor(s) able to induce apoptosis in endothelial cells led us to evaluate this effect by two methods, the TdT-mediated dUTP nick end labelling (TUNEL) method and the measurement of soluble nucleosomes released by apoptotic cells in culture supernatant by ELISA. Apoptosis induction by CSF was also studied with microglial cells, the resident macrophages in the brain, which participate in the blood-brain barrier in the perivascular area. In contrast with control CSF, African trypanosomiasis patients' CSF induced apoptosis in both microglial and endothelial cells. The results obtained with the two methods correlated well, and showed that Stage 2 CSF induced apoptosis at higher levels in microglial cells, whereas the disease stage was not decisive for apoptosis induction in endothelial cells. We measured soluble Fas ligand (sFasL) and anti-Fas antibodies levels, two potent inducers of the Fas signalling pathway leading to apoptosis, in CSF from African trypanosomiasis patients and controls. CSF from African trypanosomiasis patients contained sFasL, and anti-Fas antibodies at higher levels than in controls. Stage 2 CSF contained more sFasL than Stage 1 CSF, and anti-Fas antibodies were detected only in Stage 2 CSF. Caspase-8 inhibitor effect and statistical data suggest that other pro-apoptotic factors may be involved in some CSF-induced apoptosis. Apoptosis induction may participate in the pathogenesis during African trypanosomiasis, and the presence of sFasL and anti-Fas antibodies may provide new tools for diagnosis and prognosis of the disease.     

Mapping the paratope of anti-CD4 recombinant Fab 13B8.2 by combining parallel peptide synthesis and site-directed mutagenesis

We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a beta-sheet secondary structure similar to that of wild-type Fab. Compared with the CD4-binding capacity of wild-type Fab 13B8.2, 11 light (Y32-L, W35-L, Y36-L, H91-L, and Y92-L) and heavy chain (H35-H, R38-H, W52-H, R53-H, F100K-H, and W103-H) Fab single mutants showed a decrease in CD4 recognition as demonstrated by enzyme-linked immunosorbent assay, BIAcore, and flow cytometry analyses. The five remaining Fab mutants showed antigen-binding properties similar to those of wild-type Fab. Recombinant Fab mutants that showed decreased CD4 binding also lost their capacity to inhibit human immunodeficiency virus promoter activation and the antigen-presenting ability that wild-type Fab displays. Molecular modeling of the 13B8.2 antibody paratope indicated that most of these critical residues are appropriately positioned inside the putative CD4-binding pocket, whereas the five SCR that were not confirmed by mutagenesis show an unfavorable positioning. Taken together, these results indicate that most of the residues defined by the Spot method as critical matched with important residues defined by mutagenesis in the whole protein context. The identification of critical residues for CD4 binding in the paratope of anti-CD4 recombinant Fab 13B8.2 provides the opportunity for the generation of improved anti-CD4 molecules with more efficient pharmacological properties.     

Regeneration of peroxiredoxins during recovery after oxidative stress: only some overoxidized peroxiredoxins can be reduced during recovery after oxidative stress

Peroxiredoxins (prx) are redox enzymes using an activated cysteine as their active site. This activated cysteine can be easily overoxidized to cysteine sulfinic acid or cysteine sulfonic acid, especially under oxidative stress conditions. The regeneration of peroxiredoxins after a short, intense oxidative stress was studied, using a proteomics approach. Important differences in regeneration speed were found, prx2 being the fastest regenerated protein, followed by prx1, whereas prx3 and prx6 were regenerated very slowly. Further study of the mechanism of this regeneration by pulse-chase experiments using stable isotope labeling and cycloheximide demonstrated that the fast-regenerating peroxiredoxins are regenerated at least in part by a retroreduction mechanism. This demonstrates that the overoxidation can be reversible under certain conditions. The pathway of this retroreduction and the reasons explaining the various regeneration speeds of the peroxiredoxins remain to be elucidated.     

Concomitant recruitment of ERK1/2 and p38 MAPK signalling pathway is required for activation of cytoplasmic phospholipase A2 via ATP in articular chondrocytes

Extracellular ATP is a pro-inflammatory mediator involved in the release of prostaglandin from articular chondrocytes, but little is known about its effects on intracellular signaling. ATP triggered the rapid release of prostaglandin E(2) (PGE(2)) by acting on P2Y(2) receptors in rabbit articular chondrocytes. We have explored the signaling events involved in this synthesis. ATP significantly increased arachidonic acid production, which involved the activation of the 85-kDa cytosolic phospholipase A(2) (cPLA(2)) but not a secreted form of PLA(2), as demonstrated by various PLA(2) inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activated-protein kinases (MAPKs). Both PD98059, an inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK, completely inhibited the ATP-induced release of PGE(2). Finally, dominant-negative plasmids encoding p38 and ERK transfected alone into the cells impaired the ATP-induced release of PGE(2) to about the same extent as both plasmids transfected together. These results suggest that PGE(2) production induced by ATP requires the activation of both ERK1/2 and p38 MAPKs. Thus, ATP acts via P2Y(2)-purine receptors to recruit cPLA(2) by activating both ERK1/2 and p38 MAPKs and stimulates the release of PGE(2) from articular chondrocytes.     

Effect of S 17092, a novel prolyl endopeptidase inhibitor,on substance P and alpha-melanocyte-stimulating hormone breakdown in the rat brain

In the present study, we have investigated the effects of a novel prolyl endopeptidase (EC 3.4.21.26, PEP) inhibitor, compound S 17092, on substance P (SP) and alpha-melanocyte-stimulating hormone (alpha-MSH) metabolism in the rat brain. In vitro experiments revealed that S 17092 inhibits in a dose-dependent manner PEP activity in rat cortical extracts (IC50 = 8.3 nm). In addition, S 17092 totally abolished the degradation of SP and alpha-MSH induced by bacterial PEP. In vivo, a significant decrease in PEP activity was observed in the medulla oblongata after a single oral administration of S 17092 at doses of 10 and 30 mg/kg (-78% and -82%, respectively) and after chronic oral treatment with S 17092 at doses of 10 and 30 mg/kg per day (-75% and -88%, respectively). Concurrently, a single administration of S 17092 (30 mg/kg) caused a significant increase in SP- and alpha-MSH-like immunoreactivity (LI) in the frontal cortex (+41% and +122%, respectively) and hypothalamus (+84% and +49%, respectively). In contrast, chronic treatment with S 17092 did not significantly modify SP- and alpha-MSH-LI in the frontal cortex and hypothalamus. Collectively, the present results show that S 17092 elevates SP and alpha-MSH concentrations in the rat brain by inhibiting PEP activity. These data suggest that the effect of S 17092 on memory impairment can be accounted for, at least in part, by inhibition of catabolism of promnesic neuropeptides such as SP and alpha-MSH.