Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD(+) cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.

Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD(+) or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD+/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD+/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD+/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD+/NADH redox potential may be of potential therapeutic benefit in the clinical setting.

Characterisation of the active/de-active transition of mitochondrial complex I

Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39 kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O -generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1

NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.

The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding

Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

Facile access to new C-glycosides and C-glycoside scaffolds incorporating functionalised aromatic moieties

The tandem ene/intramolecular Sakurai cyclisation (IMSC) reaction has been successfully applied to thesynthesis of a range of C-glycosides, with key intermediates offering opportunities for functionalisation ofthe glycon moiety. To demonstrate the versatility of the approach to access the 2-deoxy-C-glycoside series,we synthesised diastereomerically pure C-glucoside and galactoside derivatives incorporating functionalisedaromatic, heteroaromatic and bicyclic aromatic moieties, in addition to the C-homologue of(±)-b-2-deoxy-glucose 6-phosphate.

Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

A gestão dos resíduos hospitalares e o papel da autoridade de saúde : caso do concelho da Amad

RESUMO O Problema. A natureza, diversidade e perigosidade dos resíduos hospitalares (RH) exige procedimentos específicos na sua gestão. A sua produção depende do número de unidades de prestação de cuidados de saúde (upcs), tipo de cuidados prestados, número de doentes observados, práticas dos profissionais e dos órgãos de gestão das upcs, inovação tecnológica, entre outros. A gestão integrada de RH tem evoluído qualitativamente nos últimos anos. Existe uma carência de informação sobre os quantitativos de RH produzidos nas upcs e na prestação de cuidados domiciliários, em Portugal. Por outro lado, os Serviços de Saúde Pública, abrangendo o poder de Autoridade de Saúde, intervêm na gestão do risco para a saúde e o ambiente associado à produção de RH, necessitando de indicadores para a sua monitorização. O quadro legal de um país nesta matéria estabelece a estratégia de gestão destes resíduos, a qual é condicionada pela classificação e definição de RH por si adoptadas. Objectivos e Metodologias. O presente estudo pretende: quantificar a produção de RH resultantes da prestação de cuidados de saúde, em seres humanos e animais nas upcs, do sistema público e privado, desenvolvendo um estudo longitudinal, onde se quantifica esta produção nos Hospitais, Centros de Saúde, Clínicas Médicas e Dentárias, Lares para Idosos, Postos Médicos de Empresas, Centros de Hemodiálise e Clínicas Veterinárias do Concelho da Amadora, e se compara esta produção em dois anos consecutivos; analisar as consequências do exercício do poder de Autoridade de Saúde na gestão integrada de RH pelas upcs; quantificar a produção média de RH, por acto prestado, nos cuidados domiciliários e, com um estudo analítico transversal, relacionar essa produção média com as características dos doentes e dos tratamentos efectuados; proceder à análise comparativa das definições e classificações de RH em países da União Europeia, através de um estudo de revisão da legislação nesta matéria em quatro países, incluindo Portugal. Resultados e Conclusões. Obtém-se a produção média de RH, por Grupos I+II, III e IV: nos Hospitais, por cama.dia, considerando a taxa de ocupação; por consulta, nos Centros de Saúde, Clínicas Médicas e Dentárias e Postos Médicos de Empresas; por cama.ano, nos Lares para Idosos, considerando a sua taxa de ocupação; e por ano, nas Clínicas de Hemodiálise e Veterinárias. Verifica-se que a actuação da Autoridade de Saúde, produz nas upcs uma diferença estatisticamente significativa no aumento das contratualizações destas com os operadores de tratamento de RH. Quantifica-se o peso médio de resíduos dos Grupos III e IV produzido por acto prestado nos tratamentos domiciliários e relaciona-se esta variável dependente com as características dos doentes e dos tratamentos efectuados. Comparam-se os distintos critérios utilizados na elaboração das definições e classificações destes resíduos inscritas na legislação da Alemanha, Reino Unido, Espanha e Portugal. Recomendações. Apresentam-se linhas de investigação futura e propõe-se uma reflexão sobre eventuais alterações de aspectos específicos no quadro legal português e nos planos de gestão integrada de RH, em Portugal. ABSTRACT The problem: The nature, diversity and hazardousness of hospital wastes (HW) requires specific procedures in its management. Its production depends on the number and patterns of healthcare services, number of patients, professional and administration practices and technologic innovations, among others. Integrated management of HW has been developping, in the scope of quality, for the past few years. There is a lack of information about the amount of HW produced in healthcare units and in the domiciliary visits, in Portugal. On the other hand, the Public Health Services, embracing the Health Authority’s power, play a very important role in managing the risk of HW production to public and environmental health. They need to use some indicators in its monitorization. In a country, rules and regulations define hospital waste management policies, which are confined by the addopted classification and definition of HW. Goals and Methods: This research study aims to quantify the production of HW as a result of healthcare services in human beings and animals, public service and private one. Through a longitudinal study, this production is quantified in Hospitals, Health Centers, Medical and Dental Clinics, Residential Centers for old people, Companies Medical Centers and Veterinary and Haemodyalisis Clinics in Amadora’s Council, comparing this production in two consecutive years. This study also focus the consequences of the Health Authority’s role in the healthcare services integrated management of HW. The middle production of HW in the domiciliary treatments is also quantified and, with a transversal analytic study, its association with patients and treatments’ characteristics is enhanced. Finally, the definitions and classifications in the European Union Countries are compared through a study that revises this matter’s legislation in four countries, including Portugal. Results and Conclusions: We get the middle production of Groups I+II, III and IV: HW: in Hospitals, by bed.day, bearing the occupation rate; by consultation, in Health Centers, Medical and Dental Clinics and Companies Medical Centers; by bed.year in Residential Centers for old people, considering their occupation rate; by year, in Veterinary and Haemodyalisis Clinics. We verify that the Health Authority’s role produces a significative statistical difference in the rise of the contracts between healthcare services and HW operators. We quantify the Groups III and IV’s wastes middle weight, produced by each medical treatment in domiciliary visits and relate this dependent variable with patients and treatments’ characteristics. We compare the different criteria used in the making of definitions and classifications of these wastes registered in German, United Kingdom, Spain and Portugal’s laws. Recommendations: Lines of further investigation are explaned. We also tender a reflexion about potential changes in rules, in regulations and in the integrated plans for managing hospital wastes in Portugal. RÉSUMÉ Le Problème. La gestion des déchets d'activités hospitalières (DAH) et de soins de santé (DSS) exige des procédures spécifiques en raison de leur nature, diversité et dangerosité. Leur production dépend, parmi d’autres, du nombre d’unités de soins de santé (USS), du type de soins administrés, du nombre de malades observés, des pratiques des professionnels et des organes de gestion des USS, de l’innovation technologique. La gestion intégrée des DAH et des DSS subit une évolution qualitative dans les dernières années. Il existe un déficit d’information sur les quantitatifs de DAH et de DSS provenant des USS et de la prestation de soins domiciliaires, au Portugal. D’autre part les Services de Santé Publique, y compris le pouvoir de l’Autorité de Santé, qui interviennent dans la gestion du risque pour la santé et pour l’environnement associé à la production de DAH et de DSS, ont besoin d’indicateurs pour leur surveillance. Dans cette matière le cadre légal établit la stratégie de gestion de ces déchets, laquelle est conditionnée par la classification et par la définition des DAH et des DSS adoptées par le pays. Objectifs et Méthodologie. Cet étude prétend: quantifier la production de DAH et de DSS provenant de la prestation de soins de santé, en êtres humains et animaux dans les USS du système public et privé. À travers un étude longitudinal, on quantifie cette production dans les Hôpitaux, Centres de Santé, Cliniques Médicales et Dentaires, Maisons de Repos pour personnes âgées, Cabinets Médicaux d’ Entreprises, Centres d’Hémodialyse et Cliniques Vétérinaires du municipe d’ Amadora, en comparant cette production en deux ans consécutifs; analyser les conséquences de l’exercice du pouvoir de l’Autorité de Santé dans la gestion intégrée des DAH et des DSS par les USS; quantifier la production moyenne de DAH et de DSS dans la prestation de soins domiciliaires et, avec un étude analytique transversal, rapporter cette production moyenne avec les caractéristiques des malades et des soins administrés; procéder à l’ analyse comparative des définitions et classifications des DAH et des DSS dans des pays de l’Union Européenne, à travers un étude de révision de la législation relative à cette matière dans quatre pays, Portugal y compris. Résultats et Conclusions. On obtient la production moyenne de DAH et des DSS, par Classes I+II, III et IV: dans les hôpitaux, par lit.jour, en considérant le taux d’occupation; par consultation, dans les Centres de Santé, Cliniques Médicales et Dentaires et Cabinets Médicaux d’ Entreprises par lit.an dans les Maisons de Repos pour personnes âgées en considérant le taux d’occupation; et par an, dans les Cliniques d’Hémodialyse et Vétérinaires. On constate que l’actuation de l’Autorité de Santé produit dans les USS une différence statistiquement significative dans l’accroissement de leurs contractualisations avec les opérateurs de traitement de DAH et de DSS. On quantifie le poids moyen des déchets des Classes III et IV produit par acte de prestation de soins à domicile et on rapporte cette variable dépendante avec les caractéristiques des malades et des soins administrés. On compare les différents critères utilisés dans l’élaboration des définitions et des classifications de ces déchets inscrites dans la légis

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Exercice physique et athérosclérose : quels sont les mécanismes physiologiques et moléculaires de l'exercice qui préviennent et protègent de l'athérosclérose? : approche chez un modèle expérimental d'athérosclérose : la souris génétiquement dépourvue en apolipoprotéine E (apoE-/-)

RESUME : L'athérosclérose, pathologie inflammatoire artérielle chronique, est à l'origine de la plupart des maladies cardiovasculaires qui constituent l'une des premières causes de morbidité et mortalité en France. Les études observationnelles et expérimentales montrent que l'exercice physique prévient la mortalité cardiovasculaire. Cependant, les mécanismes précisant les bénéfices cliniques de l'exercice sur l'athérosclérose sont encore largement inconnus. Le but général de ce travail a donc été d'explorer, en utilisant un modèle expérimental d'athérosclérose, la souris hypercholestérolémique génétiquement dépourvue en apolipoprotéine E (apoE-/-), les mécanismes athéroprotecteurs de l'exercice. La dysfonction endothéliale, généralement associée aux facteurs de risque cardiovasculaire, serait l'une des étapes précoces majeures de l'athérogenèse. Elle est caractérisée par une diminution de la biodisponibilité en monoxyde d'azote (NO) avec la perte de ses propriétés vasculo-protectrices, ce qui favorise un climat pro-athérogène (stress oxydatif, adhésion et infiltration des cellules inflammatoires dans la paroi artérielle...) conduisant à la formation de la plaque athéromateuse. L'objectif de notre premier travail a donc été d'explorer les effets de l'exercice d'une part, sur le développement des plaques athéromateuses et d'autre part, sur la fonction endothéliale de la souris apoE-/-. Nos résultats montrent que l'exercice réduit significativement l'extension de l'athérosclérose et prévient la dysfonction endothéliale. L'explication pharmacologique montre que l'exercice stimule la fonction endothéliale via, notamment, une plus grande sensibilité des récepteurs endothéliaux muscariniques, ce qui active les événements signalétiques cellulaires récepteurs-dépendants à l'origine d'une bioactivité accrue de NO. Les complications cliniques graves de l'athérosclérose sont induites par la rupture de la plaque instable provoquant la formation d'un thrombus occlusif et l'ischémie du territoire tissulaire en aval. L'objectif de notre deuxième travail a été d'examiner l'effet de l'exercice sur la qualité/stabilité de la plaque. Nos résultats indiquent que l'exercice de longue durée stabilise la plaque en augmentant le nombre de cellules musculaires lisses et en diminuant le nombre de macrophages intra-plaques. Nos résultats montrent aussi que la phosphorylation de la eNOS (NO Synthase endothéliale) Akt-dépendante n'est pas le mécanisme moléculaire majeur à l'origine de ce bénéfice. Enfin, dans notre troisième travail, nous avons investigué l'effet de l'exercice sur le développement de la plaque vulnérable. Nos résultats montrent, chez un modèle murin de plaque instable (modèle d'hypertension rénovasculaire à rénine et angiotensine II élevés) que l'exercice prévient l'apparition de la plaque vulnérable indépendamment d'un effet hémodynamique. Ce bénéfice serait associé à une diminution de l'expression vasculaire des récepteurs AT1 de l'Angiotensine II. Nos résultats justifient l'importance de l'exercice comme outil préventif des maladies cardiovasculaires. ABSTRACT : Atherosclerosis, a chronic inflammatory disease, is one of the main causes of morbidity and mortality in France. Observational and experimental data indicate that regular physical exercise has a positive impact on cardiovascular mortality. However, the mechanisms by which exercise exerts clinical benefits on atherosclerosis are still unknown. The general aim of this work was to elucidate the anti-atherosclerotic effects of exercise, using a mouse model of atherosclerosis: the apolipoprotein E-deficient mice (apoE-/- mice). Endothelial dysfunction, generally associated with cardiovascular risk factors, has been recognized to be a major and early step in atherogenesis. Endothelial dysfunction is characterized by Nitric Oxide (NO) biodisponibility reduction with loss of NO-mediated vasculoprotective actions. This leads to vascular effects such as increased oxidative stress and increased adhesion of inflammatory cells into arterial wall thus playing a role in atherosclerotic plaque development. Therefore, one of the objective of our study was to explore the effects of exercise on atherosclerotic plaque extension and on endothelial function in apoE-/- mice. Results show that exercise significantly reduces plaque progression and prevents endothelial dysfunction. Pharmacological explanation indicates that exercise stimulates endothelial function by increasing muscarinic receptors sensitivity which in turn activates intracellular signalling receptor-dependent events leading to increased NO bioactivity. The clinical manifestations of atherosclerosis are the consequences of unstable plaque rupture with thrombus formation leading to tissue ischemia. The second aim of our work was to determine the effect of exercise on plaque stability. We demonstrate that long-term exercise stabilizes atherosclerotic plaques as shown by decreased macrophage and increased Smooth Muscle Cells plaque content. Our results also suggest that the Akt-dependent eNOS phosphorylation pathway is not the primary molecular mechanism mediating these beneficial effects. Finally, we assessed a putative beneficial effect of exercise on vulnerable plaque development. In a mouse model of Angiotensine II (Ang II)-mediated vulnerable atherosclerotic plaques, we provide fist evidence that exercise prevents atherosclerosis progression and plaque vulnerability. The beneficial effect of swimming was associated with decreased aortic Ang II AT1 receptor expression independently from any hemodynamic change. These findings suggest clinical benefit of exercise in terms of cardiovascular event protection.