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Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation.

Germline mutations of the tumor suppressor gene LKB1/STK11 are responsible for the Peutz-Jeghers syndrome (PJS), an autosomal-dominant disorder characterized by mucocutaneous pigmentation, hamartomatous polyps, and an increased risk of associated malignancies. In this study, we assessed the presence of pathogenic mutations in the LKB1/STK11 gene in 46 unrelated PJS families, and also carried genotype-phenotype correlation in regard of the development of cancer in 170 PJS patients belonging to these families. All LKB1/STK11 variants detected with single-strand conformational polymorphism were confirmed by direct sequencing, and those without LKB1/STK11 mutation were further submitted to Southern blot analysis for detection of deletions/rearrangements. Statistical analysis for genotype-phenotype correlation was performed. In 59% (27/46) of unrelated PJS cases, pathogenic mutations in the LKB1/STK11 gene, including 9 novel mutations, were identified. The new mutations were 2 splice site deletion-insertions, 2 missenses, 1 nonsense, and 4 abnormal splice sites. Genotype-phenotype analysis did not yield any significant differences between patients carrying mutations in LKB1/STK11 versus those without mutations, even with respect to primary biliary adenocarcinoma. This study presents the molecular characterization and cancer occurrence of a large cohort of PJS patients, increases the mutational spectrum of LKB1/STK11 allelic variants worldwide, and provides a new insight useful for clinical diagnosis and genetic counseling of PJS families

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HtrA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HtrA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HtrA2 and PINK1. HtrA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HtrA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HtrA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies.

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HtrA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HtrA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HtrA2 and PINK1. HtrA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HtrA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HtrA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies.

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HtrA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HtrA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HtrA2 and PINK1. HtrA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HtrA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HtrA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies.

The mitochondrial proteins TRAP1 and HtrA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signaling is unclear. HtrA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HtrA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HtrA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HtrA2 and PINK1. HtrA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HtrA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HtrA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies.

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestine

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene (ADE2) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER

We have developed T7 RNAi Oligo Designer (TROD), a web application for RNA interference studies. TROD greatly facilitates the design of oligodeoxynucleotide sequences for the in vitro production of siRNA duplexes with T7 RNA polymerase. Given a query cDNA sequence, the program scans for appropriate target sequences based on the constraints of the T7 RNA polymerase method and published criteria for RNA interference with siRNAs. The output is an ordered and prioritized list of ready-to-order DNA oligonucleotide sequences, with links to perform a BLAST search to ascertain target sequence specificity. The TROD web service is available at http://www.cellbio.unige.ch/RNAi.html

p23 is a small acidic protein with intrinsic molecular chaperone activity. It is best known as a co-chaperone of the major cytosolic molecular chaperone Hsp90. p23 binds the N-terminus of Hsp90 and stabilizes the ATP-bound and N-terminally closed Hsp90 dimer. It is in this configuration that many Hsp90 clients are most stably bound. Considering the important role of p23 in the Hsp90 cycle, it came as a surprise that it is not absolutely essential for viability in the budding yeast or for mouse development. Mice without p23 develop quite normally until birth and then all die perinatally because of immature lungs. The only other apparent phenotype of late stage embryos and newborns is a skin defect, which we have further characterized here. We found that skin differentiation is impaired, and that both apoptosis and cell proliferation are augmented in the absence of p23; the consequences are a severe thinning of the stratum corneum and reduced numbers of hair follicles. The altered differentiation, spontaneous apoptosis and proliferation are all mimicked by isolated primary keratinocytes indicating that they do require p23 functions in a cell-autonomous fashion. Since the phenotype of p23-null embryos is strikingly similar to that of embryos lacking the glucocorticoid receptor, a paradigmatic Hsp90-p23 client protein, we investigated glucocorticoid signaling. We discovered that it is impaired in vivo and for some aspects in isolated keratinocytes. Our results suggest that part of the phenotype of p23-null embryos can be explained by an impact on this particular Hsp90 client, but do not exclude that p23 by itself or in association with Hsp90 affects skin development and homeostasis through yet other pathways.

L'ouvrage regroupe les travaux les plus récents dans le domaine de la physiologie de la plante entière. Il porte sur les 3 grandes étapes du cycle du maïs (phase juvénile, arrêt de la phase juvénile à la fécondation, de la fécondation à la maturité) et met l'accent sur les principaux problèmes rencontrés durant ces phases. L'ouvrage s'adresse aux scientifiques et aux professionnels.

L'ouvrage regroupe les travaux les plus récents dans le domaine de la physiologie de la plante entière. Il porte sur les 3 grandes étapes du cycle du maïs (phase juvénile, arrêt de la phase juvénile à la fécondation, de la fécondation à la maturité) et met l'accent sur les principaux problèmes rencontrés durant ces phases. L'ouvrage s'adresse aux scientifiques et aux professionnels.

L'ouvrage regroupe les travaux les plus récents dans le domaine de la physiologie de la plante entière. Il porte sur les 3 grandes étapes du cycle du maïs (phase juvénile, arrêt de la phase juvénile à la fécondation, de la fécondation à la maturité) et met l'accent sur les principaux problèmes rencontrés durant ces phases. L'ouvrage s'adresse aux scientifiques et aux professionnels.

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

Previous works in our laboratory have shown that ascorbate-driven menadione-redox cycling leads to reactive oxygen species (ROS) formation and generates an oxidative stress that results in cancer cell death. This cell death is necrotic-like and the underlying mechanism involves inhibition of glycolysis (with subsequent ATP depletion) and impairment of calcium homeostasis. In this study, we wanted to determine whether additional mechanisms involved in survival signaling pathways were affected by asc/men-mediated effects. In addition to the impairment of MAPK pathways (deactivation of ERK1/2 and activation of p38), the main finding of this work is that asc/men provokes a cleavage of the hsp90 chaperone protein. This cleavage seems to be specific for cancer cells. The subsequent loss of its chaperone function leads to degradation of its client proteins including Bcr-Abl, mutated Bcr-Abl (T315I), Akt, RIP, c-Raf, and hTERT. The degradation of these proteins is accompanied by deactivation of downstream signaling pathways (i.e. MAPKinases) of these proteins. Hsp90 cleavage can be observed in living cells, in cell lysates and in both purified and recombinant proteins. The cleavage is likely provoked by a localized Fenton-type reaction, leading to a subsequent oxidation of the protein backbone, the formation of a protein radical, and a protein cleavage in a conserved sequence that is part of the N-terminal nucleotide-binding pocket of the protein. By inhibiting hsp90 function, asc/men induces the degradation of numerous oncogenic proteins, thereby impairing not only one but several signaling pathways that are important for cancer cells. Since asc/men does not rely on a classical pharmacological inhibition, which can be impaired by mutation of the target protein, its use as an adjuvant to classical cancer therapy could overcome some drug resistance. Indeed, asc/men is able to significantly delay the growth of tumors that are bearing wild-type or mutated forms of the Bcr-Abl, (SBIM 3) -- UCL, 2010

Après une réflexion approfondie sur la notion de plante-type, l'ouvrage présente les schémas d'élaboration du rendement de onze cultures regroupées en quatre classes (céréales, oléagineux, protéagineux et plantes à tubercules). En identifiant le modèle de référence pour toute nouvelle culture, la démarche suivie permet d'adapter rapidement les techniques culturales aux objectifs de rendement tout au long du cycle. Un ouvrage de référence pour ingénieurs, chercheurs et étudiants en agronomie.

Après une réflexion approfondie sur la notion de plante-type, l'ouvrage présente les schémas d'élaboration du rendement de onze cultures regroupées en quatre classes (céréales, oléagineux, protéagineux et plantes à tubercules). En identifiant le modèle de référence pour toute nouvelle culture, la démarche suivie permet d'adapter rapidement les techniques culturales aux objectifs de rendement tout au long du cycle. Un ouvrage de référence pour ingénieurs, chercheurs et étudiants en agronomie.

SYNOPSIS. In Europe, Conservation agriculture (CA) is less adopted than in the other world adopting regions and, reduced tillage is more used than no-tillage. Use of cover crop is not common. It is currently less researched than it was before the 1990s. One of the main features of CA is the reduction of the production costs which acts as a powerful driving force for the dissemination of CA technologies. In Europe, conversion from conventional agriculture to CA leads to a minor change in yields: ± 10% depending on the countries; and, this is not decisive for farmers as far as CA meets their main expectation: i.e. reduction in costs of fuel, machinery and labour saving. The adoption process responds to a step-by-step strategy and, large sized farms are the main adopters. The increase in competitiveness at the global and at European level brings the general trend of the increase of CA coverage world-wide and the current level of fuel costs together with the trend of the enlargement of the farm size in Europe will probably contribute to the adoption of CA in Europe. The process is likely already ongoing in some European countries; it has to be sustained.

Après une réflexion approfondie sur la notion de plante-type, l'ouvrage présente les schémas d'élaboration du rendement de onze cultures regroupées en quatre classes (céréales, oléagineux, protéagineux et plantes à tubercules). En identifiant le modèle de référence pour toute nouvelle culture, la démarche suivie permet d'adapter rapidement les techniques culturales aux objectifs de rendement tout au long du cycle. Un ouvrage de référence pour ingénieurs, chercheurs et étudiants en agronomie.

SYNOPSIS. In Europe, Conservation agriculture (CA) is less adopted than in the other world adopting regions and, reduced tillage is more used than no-tillage. Use of cover crop is not common. It is currently less researched than it was before the 1990s. One of the main features of CA is the reduction of the production costs which acts as a powerful driving force for the dissemination of CA technologies. In Europe, conversion from conventional agriculture to CA leads to a minor change in yields: ± 10% depending on the countries; and, this is not decisive for farmers as far as CA meets their main expectation: i.e. reduction in costs of fuel, machinery and labour saving. The adoption process responds to a step-by-step strategy and, large sized farms are the main adopters. The increase in competitiveness at the global and at European level brings the general trend of the increase of CA coverage world-wide and the current level of fuel costs together with the trend of the enlargement of the farm size in Europe will probably contribute to the adoption of CA in Europe. The process is likely already ongoing in some European countries; it has to be sustained.

L'auteur s'attache à décrire les aspects agronomiques d'une nouvelle Révolution Verte. Il met l'accent sur les productions végétales et animales. Il montre que la Révolution Doublement Verte reposera également sur l'obtention de variétés sélectionnées pour exploiter au mieux le potentiel des milieux de culture et sur la mise au point de systèmes de production capables de valoriser le potentiel de régions agricoles très variées et s'intégrant dans un aménagement global de l'espace. Elle reposera également sur la capacité des agronomes à diagnostiquer les facteurs limitant la production et/ou dégradant le milieu, à court ou moyen terme. Elle nécessitera aussi bien de nouvelles méthodes de recherche que de nouvelles technologies

L'auteur s'attache à décrire les aspects agronomiques d'une nouvelle Révolution Verte. Il met l'accent sur les productions végétales et animales. Il montre que la Révolution Doublement Verte reposera également sur l'obtention de variétés sélectionnées pour exploiter au mieux le potentiel des milieux de culture et sur la mise au point de systèmes de production capables de valoriser le potentiel de régions agricoles très variées et s'intégrant dans un aménagement global de l'espace. Elle reposera également sur la capacité des agronomes à diagnostiquer les facteurs limitant la production et/ou dégradant le milieu, à court ou moyen terme. Elle nécessitera aussi bien de nouvelles méthodes de recherche que de nouvelles technologies

Un exemple de recherche menée sur les sytèmes de culture est décrit. Le principal objectif de cette recherche est de mettre au point des systèmes de culture qui permettent la fixation et la stabilisation de l'agriculture, soient intéressants économiquement et puissent être facilement adoptés par les agriculteurs. Quand les agriculteurs font face à une situation de crise, ils n'ont pas beaucoup de temps pour trouver les solutions adéquates. La tâche des agronomes est donc de les aider à rechercher des solutions permettant de résoudre rapidement leurs problèmes. Le cas des cerrados du Brésil a été choisi pour illustrer la démarche qui a été utilisée. L'approche qui y a été développée est basée sur la mise en place d'unités de création-diffusion-formation, dans lesquelles la recherche prend en compte le système de culture

Un exemple de recherche menée sur les sytèmes de culture est décrit. Le principal objectif de cette recherche est de mettre au point des systèmes de culture qui permettent la fixation et la stabilisation de l'agriculture, soient intéressants économiquement et puissent être facilement adoptés par les agriculteurs. Quand les agriculteurs font face à une situation de crise, ils n'ont pas beaucoup de temps pour trouver les solutions adéquates. La tâche des agronomes est donc de les aider à rechercher des solutions permettant de résoudre rapidement leurs problèmes. Le cas des cerrados du Brésil a été choisi pour illustrer la démarche qui a été utilisée. L'approche qui y a été développée est basée sur la mise en place d'unités de création-diffusion-formation, dans lesquelles la recherche prend en compte le système de culture

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90α or Hsp90β, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window.

Germline mutations of the tumor suppressor gene LKB1/STK11 are responsible for the Peutz-Jeghers syndrome (PJS), an autosomal-dominant disorder characterized by mucocutaneous pigmentation, hamartomatous polyps, and an increased risk of associated malignancies. In this study, we assessed the presence of pathogenic mutations in the LKB1/STK11 gene in 46 unrelated PJS families, and also carried genotype-phenotype correlation in regard of the development of cancer in 170 PJS patients belonging to these families. All LKB1/STK11 variants detected with single-strand conformational polymorphism were confirmed by direct sequencing, and those without LKB1/STK11 mutation were further submitted to Southern blot analysis for detection of deletions/rearrangements. Statistical analysis for genotype-phenotype correlation was performed. In 59% (27/46) of unrelated PJS cases, pathogenic mutations in the LKB1/STK11 gene, including 9 novel mutations, were identified. The new mutations were 2 splice site deletion-insertions, 2 missenses, 1 nonsense, and 4 abnormal splice sites. Genotype-phenotype analysis did not yield any significant differences between patients carrying mutations in LKB1/STK11 versus those without mutations, even with respect to primary biliary adenocarcinoma. This study presents the molecular characterization and cancer occurrence of a large cohort of PJS patients, increases the mutational spectrum of LKB1/STK11 allelic variants worldwide, and provides a new insight useful for clinical diagnosis and genetic counseling of PJS families

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestine

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene (ADE2) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER

We have developed T7 RNAi Oligo Designer (TROD), a web application for RNA interference studies. TROD greatly facilitates the design of oligodeoxynucleotide sequences for the in vitro production of siRNA duplexes with T7 RNA polymerase. Given a query cDNA sequence, the program scans for appropriate target sequences based on the constraints of the T7 RNA polymerase method and published criteria for RNA interference with siRNAs. The output is an ordered and prioritized list of ready-to-order DNA oligonucleotide sequences, with links to perform a BLAST search to ascertain target sequence specificity. The TROD web service is available at http://www.cellbio.unige.ch/RNAi.html