Tumor sensitizing function and mechanism of action of a RasGAP derived peptide

Cancer is the second cause of death after cardio-vascular diseases in economically developed countries. Two of the most commonly used anti-cancer therapies are chemo and radiotherapy. Despite the remarkable advances made in term of delivery and specificity of these two anti-tumor regimens, their toxicity towards healthy tissue remains a limitation. A promising approach to overcome this obstacle would be the utilization of therapeutic peptides that specifically augment the sensitivity of tumoral cells to treatments. Lower therapeutical doses would then be required to kill malignant cells, limiting toxic effects on healthy tissues. It was previously shown in our laboratory that the caspase-3 generated fragment N2 of RasGAP is able to potentiate the genotoxin-induced apoptosis selectively in cancer cells. In this work we show that fragment N2 strictly requires a cytoplasmic localization to deliver its pro-apoptotic effect in genotoxin-treated cancer cells. The tumor sensitizing capacity of fragment N2 was found to reside within the 10 amino acid sequence 317-326. Our laboratory earlier demonstrated that a peptide corresponding to amino acids 317 to 326 of RasGAP fused to the TAT cell permeable moiety, called TAT-RasGAP317.326, is able to sensitize cancer cells, but not normal cells, to genotoxin-induced apoptosis. In the present study we describe the capacity of TAT-RasGAP 317.326 to sensitize tumors to both chemo and radiotherapy in an in vivo mouse model. The molecular mechanism underlying the TAT-RasGAP 317.326-mediated sensitization starts now to be elucidated. We demonstrate that G3BP1, an endoribonuclease binding to amino acids 317-326 of RasGAP, is not involved in the sensitization mechanism. We also provide evidence showing that TAT-RasGAP3 17-326 potentiates the genotoxin-mediated activation of Bax in a tBid-dependent manner. Altogether our results show that TAT-RasGAP 317.326 could be potentially used in cancer therapy as sensitizer, in order to improve the efficacy of chemo and radiotherapy and prolong the life expectancy of cancer patients. Moreover, the understanding of the TAT-RasGAP317.326 mode of action might help to unravel the mechanisms by which cancer cells resist to chemo and radiotherapy and therefore to design more targeted and efficient anti-tumoral strategies.

Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen synthesis and breakdown.

The purpose of this study was to verify in man the relationships of muscle glycogen synthase and phosphorylase activities with glycogen concentration that were reported in animal studies. The upper level of glycogen concentration in muscle is known to be tightly controlled, and glycogen concentration was reported to have an inhibitory effect on synthase activity and a stimulatory effect on phosphorylase activity. Glycogen synthase and phosphorylase activity and glycogen concentration were measured in muscle biopsies in a group of nine normal subjects after stimulating an increase of their muscle glycogen concentration through either an intravenous glucose-insulin infusion to stimulate glycogen synthesis, or an Intralipid (Vitrum, Stockholm, Sweden) infusion in the basal state to inhibit glycogen mobilization by favoring lipid oxidation at the expense of glucose oxidation. Phosphorylase activity increased from 71.3 +/- 21.0 to 152.8 +/- 20.0 nmol/min/mg protein (P < .005) after the glucose-insulin infusion. Phosphorylase activity was positively correlated with glycogen concentration (P = .005 and P = .0001) after the glucose-insulin and Intralipid infusions, respectively. Insulin-stimulated glycogen synthase activity was significantly negatively correlated with glycogen concentration at the end of the Intralipid infusion (P < .005). In conclusion, by demonstrating a negative correlation of glycogen concentration with glycogen synthase and a positive correlation with phosphorylase, this study might confirm in man the double-feedback mechanism by which changes in glycogen concentration regulate glycogen synthase and phosphorylase activities. It suggests that this mechanism might play an important role in the regulation of glucose storage.

Physico-chemical characterization and oxidative reactivity evaluation of aged brake wear particles

Brake wear dust is a significant component of traffic emissions and has been linked to adverse health effects. Previous research found a strong oxidative stress response in cells exposed to freshly generated brake wear dust. We characterized aged dust collected from passenger vehicles, using microscopy and elemental analyses. Reactive oxygen species (ROS) generation was measured with acellular and cellular assays using 2′7-dichlorodihydrofluorescein dye. Microscopy analyses revealed samples to be heterogeneous particle mixtures with few nanoparticles detected. Several metals, primarily iron and copper, were identified. High oxygen concentrations suggested that the elements were oxidized. ROS were detected in the cell-free fluorescent test, while exposed cells were not dramatically activated by the concentrations used. The fact that aged brake wear samples have lower oxidative stress potential than fresh ones may relate to the highly oxidized or aged state of these particles, as well as their larger size and smaller reactive surface area.

The monocarboxylate transporter MCT4 : mechanism of regulation in astrocytes and its putative role in cerebral ischemia

Despite its small fraction of the total body weight (2%), the brain contributes for 20% and 25% respectively of the total oxygen and glucose consumption of the whole body. Indeed, glucose has been considered the energy substrate par excellence for the brain. However, evidence accumulated over the last half century revealed an important role for the monocarboxylate lactate in fulfilling the energy needs of neurons. This is particularly true during physiological neuronal activation and in pathological conditions. Lactate transport into and out of the cell is mediated by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, only three of them have been well characterized: MCT2 is the predominant neuronal isoform, while the other non¬neuronal cell types of the brain express the ubiquitous isoform MCT1. Quite recently, the MCT4 isoform has been described in astrocytes. Due to its high transport capacity compared to the other two isoforms, MCT4 is particularly adapted for glycolytic cells. Because of its recent discovery in the brain, nothing was known about its regulation in the central nervous system. Here we show that MCT4 is regulated by oxygen levels in primary cultures of astrocytes in a time- and concentration-dependent manner via the hypoxia inducible factor-la (HIF-la). Moreover, we showed that MCT4 expression is essential for astrocyte survival under low oxygen conditions. In parallel, we investigated the possible implication of the pyruvate kinase isoform Pkm2, a strong enhancer of glycolysis, in its regulation. Then we showed that MCT4 expression, as well as the expression of the other two MCT isoforms, is altered in a murine model of stroke. Surprisingly, neurons started to express MCT4, as well as MCT1, under such conditions. Altogether, these data suggest that MCT4, due to its high transport capacity for lactate, may be the isoform that enables cells to operate a major metabolic adaptation in response to pathological situations that alter metabolic homeostasis of the brain. -- Le cerveau représente 2% du poids corporel total, mais il contribue pour 20% de la consommation totale d'oxygène et 25% de celle de glucose au repos. Le glucose est considéré comme le substrat énergétique par excellence pour le cerveau. Néanmoins, depuis un demi- siècle maintenant, de plus en plus de travaux ont démontré que le lactate joue un rôle majeur dans le métabolisme cérébral et est capable du subvenir aux besoins énergétiques des neurones. Le lactate est tout particulièrement nécessaire pendant l'activation neuronale ainsi qu'en situation pathologique. Le transport du lactate à travers la barrière hématoencéphalique ainsi qu'à travers les membranes cellulaires est assuré par la famille des transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, uniquement trois d'entre eux ont été décrits: MCT2 est considéré comme le transporteur neuronal, alors que les autres types cellulaires qui constituent le cerveau expriment l'isoforme ubiquitaire MCT1. Récemment, l'isoforme MCT4 a été rapportée sur les astrocytes. Dû à sa grande capacité de transport pour le lactate, MCT4 est tout particulièrement adapté pour soutenir le métabolisme des cellules hautement glycolytiques, comme les astrocytes. En raison de sa toute récente découverte, les aspects comprenant sa régulation et son rôle dans le cerveau sont pour l'instant méconnus. Les résultats exposés dans ce travail démontrent dans un premier temps que l'expression de MCT4 est régulée par les niveaux d'oxygène dans les cultures d'astrocytes corticaux par le biais du facteur de transcription HIF-la. De plus, nous avons démontré que l'expression de MCT4 est essentielle à la survie des astrocytes quand le niveau d'oxygénation baisse. En parallèle, des résultats préliminaires suggèrent que l'isoforme 2 de la pyruvate kinase, un puissant régulateur de la glycolyse, pourrait jouer un rôle dans la régulation de MCT4. Dans la deuxième partie du travail nous avons démontré que l'expression de MCT4, ainsi que celle de MCT1 et MCT2, est altérée dans un modèle murin d'ischémie cérébrale. De façon surprenante, les neurones expriment MCT4 dans cette condition, alors que ce n'est pas le cas en condition physiologique. En tenant compte de ces résultats, nous suggérons que MCT4, dû à sa particulièrement grande capacité de transport pour le lactate, représente le MCT qui permet aux cellules du système nerveux central, notamment les astrocytes et les neurones, de s'adapter à de très fortes perturbations de l'homéostasie métabolique du cerveau qui surviennent en condition pathologique.

Diploid males support a two-step mechanism of endosymbiont-induced thelytoky in a parasitoid wasp.

BACKGROUND: Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. RESULTS: We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7-1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. CONCLUSIONS: Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts' sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.