Effect of cAMP on macromolecule synthesis in the pathogenic protozoa Trypanosoma cruzi

Macromolecule synthesis of Trypanosoma cruzi in culture was monitored using radioactive tracers. Cells of different days in culture displayed a preferential incorporation of precursors as follows: 1 day for (³H)-thymidine cells; 3 days for (³H)-uridine cells, and 4 days for (³H)-leucine cells. Autoradiographic studies showed that (³H)-thymidine was incorporated in the DNA of both kinetoplast and nucleus in this order. Shifts in the intracellular content of cAMP either by addition of dibutyryl-cAMP or by stimulation of the adenylcyclase by isoproterenol, caused an inhibition in the synthesis of DNA, RNA and proteins. Addition to the T. cruzi cultures of these agents which elevate the intracellular content ofcAMP provoked an interruption of cell proliferation as a result of the impairment of macromolecule synthesis. A discrimination was observed among the stereoisomers of isoproterenol, the L configuration showing to be most active.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Stereoselective total synthesis of some insect pheromones: (±)-serricornine and (±)-invictolide

An efficient (12 steps, 12% overallyield) and stereoselective total synthesis of (±)-serricornine (1) the sex pheromone of the cigarette beetle (Lasioderma serricornine F) is described. The preparation of intermediate 5, which encompasses the proper relative configuration of three contiguous chiral centers of (±)-invictolide, (3), is discussed.

Methodology in structural determination and synthesis of insect pheromone

By means of ethereal washing of insect pheromone glands of female moths, GC-MS detection along with microchemical reactions and electroantennogram (EAG) survey, six economically important insect species were targeted for pheromone identification. The discovery of a natural pheromone inhibitor, chemo-selectivity and species isolation by pheromone will be described. The modified triple bond migration and triethylamine liganded vinyl cuprate were applied for achiral pheromone synthesis in double bond formation. Some optically active pheromones and their stereoisomers were synthesized through chiral pool or asymmetric synthesis. Some examples of chiral recognition of insects towards their chiral pheromones will be discussed. A CaH2 and silica gel catalyzed Sharpless Expoxidation Reaction was found in shortening the reaction time.

Parallel spinors on pseudo-riemannian spin(q) manifolds

We study simply-connected irreducible non-locally symmetric pseudo-Riemannian Spin(q) manifolds admitting parallel quaternionic spinors.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

Developmental critical period in gentic redox dysregulation: animal and human studies in schizophrenia

Converging evidence favors an abnormal susceptibility to oxidative stress in schizophrenia. Decreased levels of glutathione (GSH), the major cellular antioxidant and redox regulator, was observed in cerebrospinal-fluid and prefrontal cortex of patients. Importantly, abnormal GSH synthesis of genetic origin was observed: Two case-control studies showed an association with a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease associated genotypes (35% of patients) correlated with decreased GCLC protein, GCL activity and GSH content. Similar GSH system anomalies were observed in early psychosis patients. Such redox dysregulation combined with environmental stressors at specific developmental stages could underlie structural and functional connectivity anomalies. In pharmacological and knock-out (KO) models, GSH deficit induces anomalies analogous to those reported in patients. (a) morphology: spine density and GABA-parvalbumine immunoreactivity (PV-I) were decreased in anterior cingulate cortex. KO mice showed delayed cortical PV-I at PD10. This effect is exacerbated in mice with increased DA from PD5-10. KO mice exhibit cortical impairment in myelin and perineuronal net known to modulate PV connectivity. (b) physiology: In cultured neurons, NMDA response are depressed by D2 activation. In hippocampus, NMDA-dependent synaptic plasticity is impaired and kainate induced g-oscillations are reduced in parallel to PV-I. (c) cognition: low GSH models show increased sensitivity to stress, hyperactivity, abnormal object recognition, olfactory integration and social behavior. In a clinical study, GSH precursor N-acetyl cysteine (NAC) as add on therapy, improves the negative symptoms and decreases the side effects of antipsychotics. In an auditory oddball paradigm, NAC improves the mismatched negativity, an evoked potential related to pre-attention and to NMDA receptors function. In summary, clinical and experimental evidence converge to demonstrate that a genetically induced dysregulation of GSH synthesis combined with environmental insults in early development represent a major risk factor contributing to the development of schizophrenia Conclusion Based on these data, we proposed a model for PSIP1 promoter activity involving a complex interplay between yet undefined regulatory elements to modulate gene expression.

Induction of synthesis of the rat cystatin S protein by the submandibular gland during the acute phase of experimental Chagas disease

Rats experimentally infected with Trypanosoma cruzi Y strain exhibited hypertrophy of the submandibular gland at 18 days after infection.SDS-PAGE of infected rats saliva revealed the presence of an additional band with an apparent molecular weight of about 13KDa. Electrophoresis of protein salivaand immunochemical analysis with antibody against rat cystatin S confirmed that the protein was identical to that induced by beta adrenergic stimulation.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores, and indirect defences in ant-attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high and low elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high elevation ecotypes. We observed an elevation-driven trade-off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade-offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.

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.