MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

Tailed Duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing.

Purpose of the Study: To elucidate the mechanism of homologous recombination and double-strand break repair mediated by the eukaryotic recombination pin, Rad51.

Effect of diets high or low in unavailable and slowly digestible carbohydrates on the pattern of 24-h substrate oxidation and feelings of hunger in humans.

BACKGROUND: The pattern of substrate utilization with diets containing a high or a low proportion of unavailable and slowly digestible carbohydrates may constitute an important factor in the control, time course, and onset of hunger in humans. OBJECTIVE: We tested the hypothesis that isoenergetic diets differing only in their content of unavailable carbohydrates would result in different time courses of total, endogenous, and exogenous carbohydrate oxidation rates. DESIGN: Two diets with either a high (H diet) or a low (L diet) content of unavailable carbohydrates were fed to 14 healthy subjects studied during two 24-h periods in a metabolic chamber. Substrate utilization was assessed by whole-body indirect calorimetry. In a subgroup of 8 subjects, endogenous and exogenous carbohydrate oxidation were assessed by prelabeling the body glycogen stores with [(13)C]carbohydrate. Subjective feelings of hunger were estimated with use of visual analogue scales. RESULTS: Total energy expenditure and substrate oxidation did not differ significantly between the 2 diets. However, there was a significant effect of diet (P: = 0.03) on the carbohydrate oxidation pattern: the H diet elicited a lower and delayed rise of postprandial carbohydrate oxidation and was associated with lower hunger feelings than was the L diet. The differences in hunger scores between the 2 diets were significantly associated with the differences in the pattern of carbohydrate oxidation among diets (r = -0.67, P: = 0. 006). Exogenous and endogenous carbohydrate oxidation were not significantly influenced by diet. CONCLUSIONS: The pattern of carbohydrate utilization is involved in the modulation of hunger feelings. The greater suppression of hunger after the H diet than after the L diet may be helpful, at least over the short term, in individuals attempting to better control their food intake.

On problems of calculating energy expenditure and substrate utilization from respiratory exchange data.

Indirect calorimetry based on respiratory exchange measurement has been successfully used from the beginning of the century to obtain an estimate of heat production (energy expenditure) in human subjects and animals. The errors inherent to this classical technique can stem from various sources: 1) model of calculation and assumptions, 2) calorimetric factors used, 3) technical factors and 4) human factors. The physiological and biochemical factors influencing the interpretation of calorimetric data include a change in the size of the bicarbonate and urea pools and the accumulation or loss (via breath, urine or sweat) of intermediary metabolites (gluconeogenesis, ketogenesis). More recently, respiratory gas exchange data have been used to estimate substrate utilization rates in various physiological and metabolic situations (fasting, post-prandial state, etc.). It should be recalled that indirect calorimetry provides an index of overall substrate disappearance rates. This is incorrectly assumed to be equivalent to substrate "oxidation" rates. Unfortunately, there is no adequate golden standard to validate whole body substrate "oxidation" rates, and this contrasts to the "validation" of heat production by indirect calorimetry, through use of direct calorimetry under strict thermal equilibrium conditions. Tracer techniques using stable (or radioactive) isotopes, represent an independent way of assessing substrate utilization rates. When carbohydrate metabolism is measured with both techniques, indirect calorimetry generally provides consistent glucose "oxidation" rates as compared to isotopic tracers, but only when certain metabolic processes (such as gluconeogenesis and lipogenesis) are minimal or / and when the respiratory quotients are not at the extreme of the physiological range. However, it is believed that the tracer techniques underestimate true glucose "oxidation" rates due to the failure to account for glycogenolysis in the tissue storing glucose, since this escapes the systemic circulation. A major advantage of isotopic techniques is that they are able to estimate (given certain assumptions) various metabolic processes (such as gluconeogenesis) in a noninvasive way. Furthermore when, in addition to the 3 macronutrients, a fourth substrate is administered (such as ethanol), isotopic quantification of substrate "oxidation" allows one to eliminate the inherent assumptions made by indirect calorimetry. In conclusion, isotopic tracers techniques and indirect calorimetry should be considered as complementary techniques, in particular since the tracer techniques require the measurement of carbon dioxide production obtained by indirect calorimetry. However, it should be kept in mind that the assessment of substrate oxidation by indirect calorimetry may involve large errors in particular over a short period of time. By indirect calorimetry, energy expenditure (heat production) is calculated with substantially less error than substrate oxidation rates.

Use of combined suspension laryngoscopy, flexible bronchoscopy and high frequency jet ventilation for Y-shaped airway stents delivery

La trachée et les bronches proximales sont de fins conduits subtils, ingénieusement structurés par une partie cartilagineuse antérieure résistante aux variations de pression et une partie membraneuse postérieure souple. Par leurs faibles volumes (espace mort) ils délivrent un grand pourcentage de l'air inspiré aux voies distales, puis au parenchyme pulmonaire, permettant les échanges de gaz. Cette belle harmonie respiratoire peut être rapidement mise à mal dès qu'un processus atteint ces voies respiratoires proximales, soit en les comprimant, processus sténosant, soit en affaiblissant leur structure, trachéo-bronchomalacie, soit en ouvrant leur paroi sur les structures médiastinales, fistule trachéo/broncho-médiastinales, pleurales ou autres. Le pronostic vital est alors rapidement engagé au vu de l'absolue nécessité du bon fonctionnement de ces fins conduits, une petite diminution du calibre de leurs fines lumières provoquant une baisse importante de leurs surfaces. Dans ces situations à haut potentiel de complication majeure les interventions endoscopiques pour restaurer l'intégrité de ces conduits sont alors fort risquées, et il est primordial de pouvoir les effectuer dans un cadre sécurisé au maximum. La réalisation de ces gestes par la technique décrite dans notre article « Use of combined suspension laryngoscopy, flexible bronchoscopy and high frequency jet ventilation forY-shaped airway stents delivery" permet la sécurité nécessaire à ces situations instable, en effet -la laryngoscopie en suspension expose les voies proximales en offrant un accès le plus large possible à l'arbre trachéobronchique ce qui permet l insertion de multiples instruments parfois volumineux, -la Jet ventilation assure une oxygénation et une ventilation adéquate par un fin cathéter placé soit dans le poumon sain, soit en distalité de la lésion -la bronchoscopie souple, passant au travers d'endroits exigus et courbes permet le déploiement sous vision direct, au millimètre près, de divers dispositifs. Cette association remplace avantageusement la technique traditionnelle qui insère les stents à l'aveugle, et en apnée, ce qui représente de haut risque de mauvais positionnement des stents avec des conséquences immédiates sur l'oxygénation et la ventilation souvent déjà bien altérées. Perspective et conclusion : cette technique est utile pour l'insertion des stents en Y, centraux, comme décrit dans notre article, et les indications peuvent être étendues aux stents distaux pour lesquels l'accès n'est parfois pas aisé avec le bronchoscope rigide, et pour d'autres interventions endoscopiques, laser, cryothérapie, radiofréquence ou l'insertion de nouveaux dispositifs.

Efficiency of flexible derotator in walking cerebral palsy children

Introduction The flexible derotator is one of the therapeutic resources used to combat primary and secondary abnormalities in walking cerebral palsy children. It was developed to reduce abnormal femoral and tibial torsions and lessen the latter's negative functional impact. Objective To determine the effect of wearing a flexible derotator on anatomic and functional parameters in walking cerebral palsy children. Methods We performed a retrospective study of walking cerebral palsy children by gathering data on bone-related parameters (femoral and tibial torsion) and functional parameters (distance and speed gait, and the energy expenditure index (EEI)). Fifteen walking cerebral palsy children were treated with the flexible derotator for one year and 15 untreated walking cerebral palsy children were included as controls. The two groups were compared in terms of the various parameters' change over time between the initial examination (the last examination prior to the start of the study or prior to use of the flexible derotator) and the final examination (after one year of follow-up). Results Right femoral anteversion and right and left external tibial torsion improved. There was a significant increase in distance and speed gait and a decrease in the EEI in walking cerebral palsy children. Conclusion Our retrospective study revealed a significant improvement in functional parameters in children with cerebral palsy, as a result of wearing the flexible derotator for at least 6 hours a day for a year. Bone parameters only improved slightly. Use of the flexible derotator could improve these children's quality of life.

Femur fracture in preschool children : experience with flexible intramedullary nailing in 72 children

RESUME Fractures du fémur chez les enfants d'âge préscolaire. Expérience avec l'enclouage centromédullaire élastique stable chez 72 enfants Introduction L'immobilisation plâtrée est le traitement le plus fréquemment utilisé pour traiter les fractures du fémur chez les enfants d'âge préscolaire de moins de 6 ans. L'enclouage centromédullaire élastique stable (ECMES), qui a remplacé les immobilisations plâtrées chez les enfants d'âge scolaire, est une alternative qui n'a jamais été étudiée spécifiquement dans la tranche d'âge préscolaire. Matériel et Méthode Nous avons réalisé une étude rétrospective de tous les cas de fractures du fémur chez l'enfant de moins de 6 ans traitées par ECMES dans le service de chirurgie pédiatrique du Centre Hospitalier Universitaire Vaudois et de l'Hôpital de l'Enfance de Lausanne sur une période de 15 ans. Résultats Parmi les 210 fractures du fémur traitées par ECMES entre le 1.1.1988 et le 31.12.2003, 74 fractures du fémur ont été identifiées chez 73 enfants âgés de 1.5 à 5.9 ans. Ces fractures étaient sous-trochantériennes (n=5), diaphysaires (n=64, dont 5 ouvertes), ou métaphysaires discales (n=4). Le type de fracture était transverse (n=35, dont 2 ouvertes), oblique (n=28, dont 3 ouvertes) au spiroïde (n=11). Quatre fractures étaient comminutives. Le temps opératoire moyen était de 56,9 minutes (limites entre 20 et 155 min.) pour les enfants ne présentant pas d'autre pathologie chirurgicale. Le séjour hospitalier moyen était de 9.1 jours (limites entre 1 et 46 jours) pour tous les enfants n'ayant pas de pathologie associée. Chez les enfants sans lésion ou pathologie associée, la première mise en charge s'est effectuée en moyenne au 14,1 ème jour post-opératoire (limites entre 1 et 42ème jour) alors que la première mobilisation a eu lieu en moyenne dès le 2,7ème jour post-opératoire (limites entre le 1 et le 14ème jour). 64 enfants ont été suivis à long terme avec un recul moyen de. 36,8 mois (limites entre 4 et 124 mois). Nous avons relevés 6 enfants avec une inégalité de longueur de plus d'un centimètre, alors que nous n'avons jamais constaté de défaut de rotation. Durant le 11 premières années de l'étude, 9 enfants ont dû être réopérés pour raccourcissement secondaire de broches extériorisées ou douloureuses sous la peau. Aucun problème de broche n'a été observé après introduction d'une nouvelle pince à couper. 2 réductions de fracture se sont faites à foyer ouvert. Une infection localisée transitoire du point de ponction d'une broche a été notée, sans ostéite associée. Discussion L' ECMES chez le petit enfant est techniquement réalisable sans véritable limite inférieure d'âge. Il favorise la mobilisation et la charge précoces. Les complications sont avant tout en rapport avec la technique et peuvent être évitées. Les résultats sont au moins aussi bons et meilleurs sur certains points que ceux publiés en utilisant les immobilisations. En outre ce traitement évite une longue hospitalisation. Conclusions L'ECMES peut être appliqué aux enfants de moins de 6 ans avec les mêmes bénéfices que ceux observés pour les plus grands, sans en augmenter la morbidité. La limite inférieure d'âge reste à déterminer. Un suivi à long terme s'impose pour vérifier l'absence d'inégalité de longueur des membres inférieurs.

Influences of body weight, body composition, and substrate oxidation rate on resting postabsorptive glucose production and gluconeogenesis.

OBJECTIVE: To determine the influence of body weight, fat mass, and fat distribution on resting endogenous glucose production in healthy lean and overweight individuals. DESIGN: measurements were performed in the resting postabsorptive state in individuals receiving an unrestricted diet. SETTING: Institute of Physiology of Lausanne University. MEASUREMENTS: resting post absorptive glucose production, glycogenolysis and gluconeogenesis; resting energy expenditure and net substrate oxidation. RESULTS: Endogenous glucose production was positively correlated with body weight, lean body mass, energy expenditure and carbohydrate oxidation. Gluconeogenesis was positively correlated with net lipid oxidation and energy expenditure, and negatively correlated with net carbohydrate oxidation. No correlation with body fat or fat distribution was observed. CONCLUSIONS: Gluconeogenesis shows a large interindividual variability. Net lipid oxidation and not body fat appears to be a major determinant of gluconeogenesis.

Un partenariat public-privé rigide ou flexible ? Théorie et application aux concessions routières à péage

In this article, we explore the contractual design of toll road concession contracts. We highlight the fact that contracting parties try to sign not only complete rigid contracts in order to avoid renegotiations but also flexible contracts in order to adapt contractual framework to contingencies and to create incentives for cooperative behavior. This gives rise to multiple toll adjustment provisions and to a tradeoff between rigid and flexible contracts. Such a tradeoff is formalized using an incomplete contract framework - including ex post maladaptation and renegotiation costs - and propositions are tested using an original database of 71 concession contracts. Our results suggest an important role for economic efficiency concerns, as well as politics, in designing such public-private contracts. Codes JEL : D23, H11, H54, L14, L9.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate.

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.