Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Avaluació del creixement del fong ligninolític Trametes versicolor sobre diferents suports sòlids per a l'aplicació en tractaments de bioremediació

Els fongs de podridura blanca (WRF - de l'anglès: White-rot fungi) són de gran interès en l'àmbit de la bioremediació per la seva capacitat de degradar la lignina. La lacasa, un dels enzims extracel·lulars que aquests fongs excreten per degradar la lignina, pot ser utilitzada per degradar els contamiants presents en una matriu donada. Trametes versicolor ha estat estudiat per la seva capacitat de produïr aquest enzim en rersidus agrícoles com a substrats. Un primer triatge basat en la producció de CO2, la mesura de l'activitat lacasa i la quantificació de l'ergosterol han permés seleccionar els substrats on es donava un major poder oxidatiu dels cultius inoculats amb T. versicolor. Els posteriors experiments de colonització de sòls, on es monitoritzava l'activitat lacasa i l'ergosterol, han mostrat que T. vesicolor és capaç de colonitzar sòls i que té una major activitat lacasa en condicions no estèrils. També h'ha provat, mitjançant el test ND24, que T. versicolor és capaç de degradar un contaminant emergent, el naproxè, en sòls estèrils i no estèrils esmenats amb residus agrícoles.

Autosomal-Recessive Posterior Microphthalmos Is Caused by Mutations in PRSS56, a Gene Encoding a Trypsin-Like Serine Protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Bacillus thuringiensis: fermentation process and risk assessment: a short review

Several factors make the local production of Bacillus thuringiensis (Bt) highly appropriate for pest control in developing nations. Bt can be cheaply produced on a wide variety of low cost, organic substrates. Local production results in considerable savings in hard currency which otherwise would be spent on importation of chemical and biological insecticides. The use of Bt in Brazil has been limited in comparison with chemical insecticides. Although Bt is imported, some Brazilian researchers have been working on its development and production. Fermentation processes (submerged and semi-solid) were applied, using by-products from agro-industries. As the semi-solid fermentation process demonstrated to be interesting for Bt endotoxins production, it could be adopted for small scale local production. Although promising results had been achieved, national products have not been registered due to the absence of a specific legislation for biological products. Effective actions are being developed in order to solve this gap. Regardless of the biocontrol agents being considered atoxic and harmless to the environment, information related to direct and indirect effects of microbials are still insufficient in many cases. The risk analysis of the use of microbial control agents is of upmost importance nowadays, and is also discussed.

Thermal effect on the life-cycle parameters of the medically important freshwater snail species Lymnaea (Radix) luteola (Lamarck)

The snails Lymnaea (Radix) luteola exhibited marked variations in growth, longevity, and attaining sexual maturity at different temperatures and diets. At 10°C, irrespective of foods, pH and salinity of water, the snails had minimum life span, maximum death rate and lowest growth rate. At 15°C, the growth rate was comparatively higher and the snails survived for a few more days. But at these temperatures they failed to attain sexual maturity. Snails exposed to pH 5 and 9 at 20°, 25°, 30°, 35°C and room temperatures (19.6°-29.6°C); to 0.5, 1.5 and 2.5 NaCl ‰ at 20° and 35ºC; to 2.5 NaCl ‰ at 25°C and room temperatures failed to attain sexual maturity. The snails exposed to pH 7 and different salinity grades at 20°, 25°, 30°, 35°C and room temperatures became sexually mature between 25-93 days depending upon the type of foods used in the culture.

Comportamento de dípteros muscóides frente a substratos de oviposição, em laboratório, no Rio de Janeiro, RJ, Brasil

How adult females of calyptrate Diptera recognize the appropriate breeding substrate is a matter of controversy. Among holometabolic insects, the feeding opportunities of immature stages are generally determined by the adult female choice of an oviposition site. The ovipositional and larvipositional substrate preference for the synanthropic flies (Chrysomya megacephala, C. putoria, Phaenicia cuprina: Calliphoridae; Atherigona orientalis, Synthesiomyia nudiseta: Muscidae; Ravinia belforti, Parasarcophaga ruficornis, Peckia chrysostoma: Sarcophagidae) is presented in this work. The substrate used for testing were the following: bovine minced meat, fish (sardine), bovine liver, shrimp, squid, human faeces and banana. Bovine minced meat was the ovipositional and larvipositional substrate preferred by seven species. Human faeces were preferred by R. belforti.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Dibenzofuran degradation by Sphingomonas wittichii RW1 under environmental stresses

Sphingomonas wittichii is a gram-negative Alpha-proteobacterium, capable of degrading xenobiotic compounds such as dibenzofuran (DBF), dibenzo-p-dioxin, carbazole, 2-hydroxybiphenyl or nitro diphenyl ether herbicides. The metabolism of strain RW1 has been the subject of previous studies and a number of genes involved in DBF degradation have been characterized. It is known that RW1 posseses a unique initial DBF dioxygenase (encoded by the dxnAl gene) that catalyzes the first step in the degradation pathway. None of the organisms known to be able to degrade DBF have a similar dioxygenase, the closest match being the DBF dioxygenase from Rhodococcus sp. with an overall amino acid similarity of 45%. Genes participating in the conversion of the metabolite salicylate via the ortho-cleavage pathway to TCA cycle intermediates were identified as well. Apart from this scarce information, however, there is a lack of global knowledge on the genes that are involved in DBF degradation by strain RW1 and the influence of environmental stresses on DBF-dependent global gene expression. A global analysis is necessary, because it may help to better understand the behaviour of the strain under field conditions and suggest improvements for the current bioaugmentation practice. Chapter 2 describes the results of whole-genome analysis to characterize the genes involved in DBF degradation by RW1. Micro-array analysis allowed us to detect differences in gene transcription when strain RW1 was exposed to DBF. This was complemented by ultra-high throughput sequencing of mutants no longer capable of growing on salicylate and DBF. Some of the genes of the ortho-cleavage pathway were induced 2 to 4 times in the presence of DBF, as well as the initial DBF dioxygenase. However two gene clusters, named 4925 and 5102 were induced up to 19 times in response to DBF induction. The cluster 4925 is putatively participating in a meta-cleavage pathway while the cluster 5102 might be part of a gentisate pathway. The three pathways, ortho-cleavage, meta-cleavage and gentisate pathway seem to be active in parallel when strain RW1 is exposed to DBF, presenting evidence for a redundancy of genes for DBF degradation in the genome of RW1. Chapter 3 focuses on exploiting genetic tools to construct bioreporters representative for DBF degradation in RW1. A set of basic tools for genetic manipulation in Sphingomonas wittichii RW1 was tested and optimized. Both plasmids and mini-transposons were evaluated for their ability to be maintained in RW1 with or without antibiotic selection pressure, and for their ability to lead to fluorescent protein expression in strain RW1 from a constitutive promoter. Putative promoter regions of three of the previously found DBF-induced genes (Swit_4925, Swit_5102 and Swit_4897-dxnAl) were then used to construct eg/^-bioreporters in RW1. Chapter 4 describes the use of the constructed RW1-based bioreporter strains for examining the expression of the DBF degradation pathway genes under microcosm conditions. The bioreporter strains were first exposed to different carbon sources in liquid culture to calibrate the egfp induction. Contrary to our expectations from micro-array analysis only the construct with the promoter from gene cluster 4925 responded to DBF, whereas the other two constructs did not show specific induction with DBF. The response from the bioreporters was subsequently tested for sensitivity to water stress, given that this could have an important impact in soils. Exposure to liquid cultures with decreasing water potential, achieved by NaCl or PEG addition to the growth media, showed that eGFP expression in RW1 from the promoter regions 4925 and 5102 was not directly influenced by water stress, but only through an overall reduction in growth rate. In contrast, expression of eGFP from the dxnAl or an uspA promoter was also directly dependent on the extent of water stress. The RW1 with the 4925 construct was subsequently used in soil microcosms to evaluate DBF bioavailability to the cells in presence or absence of native microbiota or other contaminated material. We found that RW1 could grow on DBF added to soil, but bioreporter expression suggested that competition with native microbiota for DBF intermediates may limit its ability to proliferate to a maximum. Chapter 5 describes the results from the experiments carried out to more specifically detect genes of RW1 that might be implicated in water stress resistance. Hereto we created transposon mutagenesis libraries in RW1, either with a classical mini-Tn5 or with a variant that would express egfp when the transposon would insert in a gene induced under water stress. Classical mutant libraries were screened by replica plating under high and low water stress conditions (achieved by adding NaCl to the agar medium). In addition, we screened for smaller microcolonies formed by mutants in agarose beads that could be analized with flow cytometry. A number of mutants impaired to grow on NaCl-supplemented media were recovered and the transposon insertion sites sequenced. In a second procedure we screened by flow cytometry for mutants with a higher eGFP production after exposure to growth medium with higher NaCl concentrations. Mutants from both libraries rarely overlapped. Discovered gene functions of the transposon insertions pointed to compatible solute synthesis (glutamate and proline), cell membrane synthesis and modification of cell membrane composition. The results obtained in the present study give us a more complete picture of the mechanisms of DBF degradation by S. wittichii RW1, how it reacts to different DBF availability and how the DBF catabolic activity may be affected by the conditions found in contaminated environments. - Sphingomonas wittichii est une alpha-protéobactérie gram-négative, capable de dégrader des composés xénobiotiques tels que le dibenzofurane (DBF), la dibenzo-p-dioxine, le carbazole, le 2-hydroxybiphényle ou les herbicides dérivés du nitro-diphényléther. Le métabolisme de la souche RW1 a fait l'objet d'études antérieures et un certain nombre de gènes impliqués dans la dégradation du DBF ont été caractérisés. Il est connu que RW1 possède une unique dioxygénase DBF initiale (codée par le gène dxnAl) qui catalyse la première étape de la voie de dégradation. Aucun des organismes connus pour être capables de dégrader le DBF n'a de dioxygénase similaire. L'enzyme la plus proche étant la DBF dioxygénase de Rhodococcus sp. avec 45% d'acides aminés conservés. Les gènes qui participent à la transformation du salicylate en métabolites intermédiaires du cycle de Krebs par la voie ort/io-cleavage ont aussi été identifiés. Outre ces informations lacunaires, il y a un manque de connaissances sur l'ensemble des gènes impliqués dans la dégradation du DBF par la souche RW1 ainsi que l'effet des stress environnementaux sur l'expression génétique globale, en présence du DBF. Une analyse globale est nécessaire, car elle peut aider à mieux comprendre le comportement de la souche dans les conditions de terrain et de proposer des améliorations pour l'utilisation de la bio-augmentation comme technique de bio-remédiation. Le chapitre 2 décrit les résultats de l'analyse du génome pour caractériser les gènes impliqués dans la dégradation du DBF par RW1. Une analyse de micro-arrays nous a permis de détecter des différences dans la transcription des gènes lorsque la souche RW1 a été exposée au DBF. L'analyse a été complétée par le criblage à ultra-haut débit de mutants qui n'étaient plus capables de croître avec le salicylate ou le DBF comme seule source de carbone. Certains des gènes de la voie ortho-cleavage, dont la DBF dioxygénase initiale, ont xî été induits 2 à 4 fois, en présence du DBF. Cependant, deux groupes de gènes, nommés 4925 et 5102 ont été induits jusqu'à 19 fois en réponse au DBF. Le cluster 4925 participe probablement dans une voie de meta-cleavage tandis que le cluster 5102 pourrait faire partie d'une voie du gentisate. Les trois voies, ortho-cleavage, meta-cleavage et la voie du gentisate semblent être activées en parallèle lorsque la souche RW1 est exposée au DBF, ce qui représente une redondance de voies pour la dégradation du DBF dans le génome de RW1. Le chapitre 3 se concentre sur l'exploitation des outils génétiques pour la construction de biorapporteurs de la dégradation du DBF par RW1. Un ensemble d'outils de base pour la manipulation génétique dans Sphingomonas wittichii RW1 a été testé et optimisé. Deux plasmides et mini-transposons ont été évalués pour leur capacité à être maintenu dans RW1 avec ou sans pression de sélection par des antibiotiques, et pour leur capacité à exprimer la protéine fluorescente verte (eGFP) dans la souche RW1. Les trois promoteurs des gènes Swit_4925, Swit_5102 et Swit_4897 (dxnAl), induits en réponse au DBF, ont ensuite été utilisés pour construire des biorapporteurs dans RW1. Le chapitre 4 décrit l'utilisation des souches biorapportrices construites pour l'analyse de l'expression des gènes de la voie de dégradation du DBF dans des microcosmes avec différents types de sols. Les souches biorapportrices ont d'abord été exposées à différentes sources de carbone en cultures liquides afin de calibrer l'induction de la eGFP. La construction avec le promoteur du gène 4925 a permis une réponse au DBF. Mais contrairement à nos attentes, basées sur les résultats de l'analyse des micro-arrays, les deux autres constructions n'ont pas montré d'induction spécifique au DBF. La réponse des biorapporteurs a ensuite été testée pour la sensibilité au stress hydrique, étant donné que cela pourrait avoir un impact important dans les microcosmes. La diminution du potentiel hydrique en culture liquide est obtenue par addition de NaCl ou de PEG au milieu de croissance. Nous avons montré que l'expression de la eGFP contrôlée par les promoteurs 4925 et 5102 n'était pas directement influencée par le stress hydrique, mais seulement par une réduction globale des taux de croissance. En revanche, l'expression de la eGFP dépendante des promoteurs dxnAl et uspA était aussi directement dépendante de l'ampleur du stress hydrique. La souche avec la construction 4925 a été utilisée par la suite dans des microcosmes avec différents types de sols pour évaluer la biodisponibilité du DBF en présence ou absence des microbes indigènes et d'autres composés contaminants. Nous avons constaté que RW1 pouvait se développer si le DBF a été ajouté au sol, mais l'expression de la eGFP par le biorapporteur suggère que la compétition avec la microbiota indigène pour les métabolites intermédiaires du DBF peut limiter sa capacité à proliférer de manière optimale. Le chapitre 5 décrit les résultats des expériences réalisées afin de détecter spécifiquement les gènes de RW1 qui pourraient être impliquées dans la résistance au stress hydrique. Ici on a crée des bibliothèques de mutants de RW1 par transposon, soit avec un mini-Tn5 classique ou avec une variante qui exprime la eGFP lorsque le transposon s'insère dans un gène induit par le stress hydrique. Les bibliothèques de mutants ont été criblées par la méthode classique de repiquage sur boîtes, dans des conditions de stress hydrique élevé (obtenu par l'addition de NaCl dans les boîtes). En outre, nous avons criblé des micro¬colonies dans des billes d'agarose qui ont pu être analysées par cytométrie de flux. Un certain nombre de mutants déficients à croître sur des milieux supplémentés avec du NaCl ont été isolés et les sites d'insertion du transposon séquencés. Dans une deuxième procédure nous avons criblé par cytométrie de flux des mutants avec une production de eGFP supérieure, après exposition à un milieu de croissance avec une concentration élevée de NaCl. Les mutants obtenus dans les deux bibliothèques n'étaient pas similaires. Les fonctions des gènes où se trouvent les insertions de transposons sont impliqués dans la synthèse de solutés compatibles (glutamate et de la proline), dans la synthèse de la membrane cellulaire et dans la modification de la composition de la membrane cellulaire. Les résultats obtenus dans la présente étude nous donnent une image plus complète des mécanismes de dégradation du DBF par S. wittichii RW1, comment cette souche réagit à la disponibilité du DBF et comment l'activité catabolique peut être affectée par les conditions rencontrées dans des environnements contaminés.

A study of the aging of fingerprints by GC/MS analysis.

Determining the time since deposition of fingermarks may prove necessary in order to assess their relevance to criminal investigations. The crucial factor is the initial composition of fingermarks because it represents the starting point of any ageing model. This study mainly aimed to characterize the initial composition of fingerprints, which show a high variability between donors (inter-variability), but also to investigate the variations among fingerprints from the same donor (intra-variability). Solutions to reduce this initial variability using squalene and cholesterol as target compounds are proposed and should be further investigated. The influence of substrates was also evaluated and the initial composition was observed to be larger on porous surface than non-porous surfaces. Preliminary aging of fingerprints over 30 days was finally studied on a porous and a non-porous substrate to evaluate the potential for dating of fingermarks. Squalene was observed to decrease in a faster rate on a non-porous substrate.

Local heating of human skin causes hyperemia without mediation by muscarinic cholinergic receptors or prostanoids.

RESUME Les changements locaux de la température à la surface de la peau humaine ont une influence importante sur sa perfusion. La chaleur augmente localement le flux sanguin cutané, mais les mécanismes et les médiateurs de cette réponse (réponse thermique d'hyperémie) sont incomplètement élucidés. Dans la présente étude, nous avons examiné la relation possible entre la réponse thermique d'hyperémie, les récepteurs cholinergiques muscariniques et la production des prostaglandines vasodilatatrices. Chez 13 sujets de sexe masculin en bonne santé âgés entre 20 et 30 ans, une chambre métallique (contenant de l'eau) dont la température peut être contrôlée, a été placée sur la face palmaire de leur avant-bras et utilisée pour augmenter la température de surface de 34 à 41°C. L'hyperémie cutanée consécutive a été enregistrée par l'intermédiaire d'un scanner laser-Doppler. Dans une expérience, chacun des 8 sujets a reçu un bolus i.v. de glycopyrolate (agent antimuscarinique) (4 µg/kg) lors d'une visite et de NaCl 0,9% lors de l'autre visite. La réponse thermique d'hyperémie a été déterminée dans l'heure suivant les injections. Les glycopyrolate a efficacement empêché la vasodilation des micro-vaisseaux cutanés induite par iontophorèse d'acétylcholine mais n'a pas influencé la réponse thermique d'hyperémie. Dans une deuxième expérience entreprise avec 5 autres sujets 1 g d'aspirine (inhibiteur de la cyclooxygénase) administrée oralement a totalement supprimé la vasodilatation induite dans la peau par le courant anodique, sans modifier la réponse thermique d'hyperémie. La présente étude confirme l'absence de stimulation des récepteurs muscariniques et la production de prostaglandines vaso-dilatatrices dans la vasodilatation induite chez l'homme par réchauffement local de la peau de l'avant-bras. ABSTRACT Local changes in surface temperature have a powerful influence on the perfusion of human skin. Heating increases local skin blood flow (SkBF), but the mechanisms and mediators of this response (thermal hyperemia response) are incompletely elucidated. In the present study, we examined the possible dependence of the thermal hyperemia response on stimulation of muscarinic cholinergic receptors and on production of vasodilator prostanoids. In 13 male healthy subjects aged 20 - 30 years, a temperature- controlled chamber was positioned on the volar face of one forearm and used to raise surface temperature from 34to41°C. The time-course of the resulting thermal hyperemia response was recorded with a laser-Doppler imager. In one experiment, each of 8 subjects received an i.v. bolus of the antimuscarinic agent glycopyrrolate (4µg/kg) on one visit and saline on the other. The thermal hyperemia response was determined within the hour following the injections. Glycopyrrolate effectively inhibited the skin vasodilation induced by iontophoresis of acetylcholine, but did not influence the thermal hyperemia response. In a second experiment conducted in 5 other subjects, 1 gram of the cyclooxygenase inhibitor aspirin administered orally totally abolished the vasodilation induced in the skin by anodal current, but also failed to modify the thermal hyperemia response. The present study excludes the stimulation of muscarinic receptors and the production of vasodilator prostaglandins as essential and nonredundant mechanisms for the vasodilation induced by local heating in human forearm skin.

Detection of extracellular proteases from microorganisms on agar plates

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate.

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5°C or at room temperature for one hour, or by storage at -80°C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k(obs) varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13) during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km) of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.