"In vitro" study of the molecular mechanism of the "E.Coli" Hsp 70/Hsp40/NEF chaperone system and its interactions with ?-sinuclein

SUMMARY Under stressful conditions, mutant or post-translationally modified proteins may spontaneously misfold and form toxie species, which may further assemble into a continuum of increasingly large and insoluble toxic oligomers that may further condense into less toxic, compact amyloids in the cell Intracellular accumulation of aggregated proteins is a common denominator of several neurodegenerative diseases. To cope with the cytotoxicity induced by abnormal, aggregated proteins, cells have evolved various defence mechanisms among which, the molecular chaperones Hsp70. Hsp70 (DnaK in E. coii) is an ATPase chaperone involved in many physiological processes in the cell, such as assisting de novo protein folding, dissociating native protein oligomers and serving as pulling motors in the import of polypeptides into organelles. In addition, Hsp70 chaperones can actively solubilize and reactivate stable protein aggregates, such as heat- or mutation-induced aggregates. Hsp70 requires the cooperation of two other co-chaperones: Hsp40 and NEF (Nucleotide exchange factor) to fulfil its unfolding activity. In the first experimental section of this thesis (Chapter II), we studied by biochemical analysis the in vitro interaction between recombinant human aggregated α-synuclein (a-Syn oligomers) mimicking toxic a-Syn oligomers species in PD brains, with a model Hsp70/Hsp40 chaperone system (the E. coii DnaK/DnaJ/GrpE). We found that chaperone-mediated unfolding of two denatured model enzymes were strongly affected by α-Syn oligomers but, remarkably, not by monomers. This in vitro observed dysfunction of the Hsp70 chaperone system resulted from the sequestration of the Hsp40 proteins by the oligomeric α-synuclein species. In the second experimental part (Chapter III), we performed in vitro biochemical analysis of the co-chaperone function of three E. coii Hsp40s proteins (DnaJ, CbpA and DjlA) in the ATP-fuelled DnaK-mediated refolding of a model DnaK chaperone substrate into its native state. Hsp40s activities were compared using dose-response approaches in two types of in vitro assays: refolding of heat-denatured G6PDH and DnaK-mediated ATPase activity. We also observed that the disaggregation efficiency of Hsp70 does not directly correlate with Hsp40 binding affinity. Besides, we found that these E. coii Hsp40s confer substrate specificity to DnaK, CbpA being more effective in the DnaK-mediated disaggregation of large G6PDH aggregates than DnaJ under certain conditions. Sensibilisées par différents stress ou mutations, certaines protéines fonctionnelles de la cellule peuvent spontanément se convertir en formes inactives, mal pliées, enrichies en feuillets bêta, et exposant des surfaces hydrophobes favorisant l'agrégation. Cherchant à se stabiliser, les surfaces hydrophobes peuvent s'associer aux régions hydrophobes d'autres protéines mal pliées, formant des agrégats protéiques stables: les amyloïdes. Le dépôt intracellulaire de protéines agrégées est un dénominateur commun à de nombreuses maladies neurodégénératives. Afin de contrer la cytotoxicité induite par les protéines agrégées, les cellules ont développé plusieurs mécanismes de défense, parmi lesquels, les chaperonnes moléculaires Hsp70. Hsp70 nécessite la collaboration de deux autres co-chaperonnes : Hsp40 et NEF pour accomplir son activité de désagrégation. Hsp70 (DnaK, chez E. coli) est impliquée par ailleurs dans d'autres fonctions physiologiques telles que l'assistanat de protéines néosynthétisées à la sortie du ribosome, ou le transport transmembranaire de polypeptides. Par ailleurs, les chaperonnes Hsp70 peuvent également solubiliser et réactiver des protéines agrégées à la suite d'un stress ou d'une mutation. Dans la première partie expérimentale de cette thèse (Chapter II), nous avons étudié in vitro l'interaction entre les oligomères d'a-synucleine, responsables entre autres, de la maladie de Parkinson, et le système chaperon Hsp70/Hsp40 (système Escherichia coli DnaK/DnaJ/GrpE). Nous avons démontré que contrairement aux monomères, les oligomères d'a-synucleine inhibaient le système chaperon lors du repliement de protéines agrégées. Cette dysfonction du système chaperon résulte de la séquestration des chaperonnes Hsp40 par les oligomères d'a-synucleine. La deuxième partie expérimentale (Chapitre III) est consacrée à une étude in vitro de la fonction co-chaperonne de trois Hsp40 d'is. coli (DnaJ, CbpA, et DjlA) lors de la désagrégation par DnaK d'une protéine pré-agrégée. Leurs activités ont été comparées par le biais d'une approche dose-réponse au niveau de deux analyses enzymatiques: le repliement de la protéine agrégée et l'activité ATPase de DnaK. Par ailleurs, nous avons mis en évidence que l'efficacité de désagrégation d'Hsp70 et l'affinité des chaperonnes Hsp40 vis-à-vis de leur substrat n'étaient pas corrélées positivement. Nous avons également montré que ces trois chaperonnes Hsp40 étaient directement impliquées dans la spécificité des fonctions accomplies par les chaperonnes Hsp70. En effet, DnaK en présence de CbpA assure la désagrégation de large agrégats protéiques avec une efficacité nettement plus accrue qu'en présence de DnaJ.

The daily energy expenditure in stable chronic obstructive pulmonary disease.

Resting energy expenditure is frequently increased in chronic obstructive pulmonary disease (COPD), but it is unknown if this hypermetabolism holds true over 24 h. The aim of this study was to measure the actual 24-h energy expenditure (24-h EE) in patients with stable COPD. Energy expenditure was measured by indirect calorimetry, using a metabolic chamber for 24-h EE and a canopy for basal metabolic rate (BMR). Physical activity was detected in the chamber by a radar system, and its duration was quantified. Two groups matched for age and height were studied: 16 male ambulatory patients with stable COPD and 12 male normal subjects. Body weight was 92 +/- 12% of ideal body weight in the group with COPD and 108 +/- 11% in the control group (p = 0.01). BMR was 120 +/- 7% of predicted in the group with COPD and 108 +/- 12% in the control group (p < 0.01). However, 24-h EE was similar in the two groups, amounting to 1,935 +/- 259 kcal in patients with COPD and 2,046 +/- 253 kcal in the control group (NS). This corresponded to 145% and 137% of predicted BMR, and to 121% and 126% of measured BMR in patients with COPD and the control group, respectively (NS). Patients were allowed to pursue their usual treatment within the chamber, and a positive correlation existed between 24-h EE and the daily dose of inhaled beta 2-agonists (p < 0.03). During daytime, physical activity was lower in patients with COPD. This study shows that patients with stable COPD are characterized by a normal daily energy expenditure in controlled conditions in spite of an increased basal metabolic rate. They appear to save energy by reducing their spontaneous level of physical activity.

Neural regulation of pancreatic islet cell mass and function.

Intracellular glucose signalling pathways control the secretion of glucagon and insulin by pancreatic islet α- and β-cells, respectively. However, glucose also indirectly controls the secretion of these hormones through regulation of the autonomic nervous system that richly innervates this endocrine organ. Both parasympathetic and sympathetic nervous systems also impact endocrine pancreas postnatal development and plasticity in adult animals. Defects in these autonomic regulations impair β-cell mass expansion during the weaning period and β-cell mass adaptation in adult life. Both branches of the autonomic nervous system also regulate glucagon secretion. In type 2 diabetes, impaired glucose-dependent autonomic activity causes the loss of cephalic and first phases of insulin secretion, and impaired suppression of glucagon secretion in the postabsorptive phase; in diabetic patients treated with insulin, it causes a progressive failure of hypoglycaemia to trigger the secretion of glucagon and other counterregulatory hormones. Therefore, identification of the glucose-sensing cells that control the autonomic innervation of the endocrine pancreatic and insulin and glucagon secretion is an important goal of research. This is required for a better understanding of the physiological control of glucose homeostasis and its deregulation in diabetes. This review will discuss recent advances in this field of investigation.

Entropy of a correlated system of nucleons

Realistic nucleon-nucleon interactions induce correlations to the nuclear many-body system, which lead to a fragmentation of the single-particle strength over a wide range of energies and momenta. We address the question of how this fragmentation affects the thermodynamical properties of nuclear matter. In particular, we show that the entropy can be computed with the help of a spectral function, which can be evaluated in terms of the self-energy obtained in the self-consistent Green's function approach. Results for the density and temperature dependences of the entropy per particle for symmetric nuclear matter are presented and compared to the results of lowest order finite-temperature Brueckner-Hartree-Fock calculations. The effects of correlations on the calculated entropy are small, if the appropriate quasiparticle approximation is used. The results demonstrate the thermodynamical consistency of the self-consistent T-matrix approximation for the evaluation of the Green's functions.

Relaxation times in a bistable system with parametric, white noise: Theory and experiment

We consider the effects of external, multiplicative white noise on the relaxation time of a general representation of a bistable system from the points of view provided by two, quite different, theoretical approaches: the classical Stratonovich decoupling of correlations and the new method due to Jung and Risken. Experimental results, obtained from a bistable electronic circuit, are compared to the theoretical predictions. We show that the phenomenon of critical slowing down appears as a function of the noise parameters, thereby providing a correct characterization of a noise-induced transition.

Unilateral urinary flow impairment at the pelviureteral junction: outcome of renal function with respect to therapeutic strategy.

OBJECTIVES: To evaluate the renal function outcome in children with unilateral hydronephrosis and urinary flow impairment at the pelviureteral junction with respect to the therapeutic strategy. METHODS: We retrospectively selected 45 children with iodine-123-hippuran renography performed at diagnosis and after 3 or more years of follow-up. All children had bilateral nonobstructive pattern findings on diuretic renography at follow-up. Eleven children were treated conservatively, and 34 underwent unilateral pyeloplasty. Split and individual renal function, measured by an accumulation index, was computed from background-corrected renograms for the affected and contralateral kidneys at diagnosis and the follow-up examination. RESULTS: Of 11 children treated conservatively, 9 had normal bilateral function at diagnosis, all had reached normal function at follow-up. Of the 34 operated kidneys, 12 (38%) had initially normal function that remained normal at the follow-up examination, and 22 had impaired function that had normalized at the follow-up examination in 15 (68%). The function of the contralateral kidneys was increased in 5 of 8 children with persistently abnormal affected kidneys. Pyeloplasty was performed in 23 children (68%) and 11 children (32%) younger and older than 1 year, respectively. The function of the affected kidneys increased in both groups, but normalization occurred only in the younger children. CONCLUSIONS: Of the children selected for conservative treatment, 82% had normal bilateral renal function at diagnosis that was normal in all at the follow-up examination. Of the children treated surgically, 65% had initially impaired function of the affected kidney that improved in 87% after pyeloplasty. Normalization of function was observed only in children who were younger than 1 year old at surgery. Persistently low function of the affected kidney was compensated for by the contralateral one regardless of the age at surgery.

Current status of a model system: the gene Gp-9 and its association with social organization in fire ants.

The Gp-9 gene in fire ants represents an important model system for studying the evolution of social organization in insects as well as a rich source of information relevant to other major evolutionary topics. An important feature of this system is that polymorphism in social organization is completely associated with allelic variation at Gp-9, such that single-queen colonies (monogyne form) include only inhabitants bearing B-like alleles while multiple-queen colonies (polygyne form) additionally include inhabitants bearing b-like alleles. A recent study of this system by Leal and Ishida (2008) made two major claims, the validity and significance of which we examine here. After reviewing existing literature, analyzing the methods and results of Leal and Ishida (2008), and generating new data from one of their study sites, we conclude that their claim that polygyny can occur in Solenopsis invicta in the U.S.A. in the absence of expression of the b-like allele Gp-9(b) is unfounded. Moreover, we argue that available information on insect OBPs (the family of proteins to which GP-9 belongs), on the evolutionary/population genetics of Gp-9, and on pheromonal/behavioral control of fire ant colony queen number fails to support their view that GP-9 plays no role in the chemosensory-mediated communication that underpins regulation of social organization. Our analyses lead us to conclude that there are no new reasons to question the existing consensus view of the Gp-9 system outlined in Gotzek and Ross (2007).

High-precision satellite positioning system as a new tool to study the biomechanics of human locomotion.

New Global Positioning System (GPS) receivers allow now to measure a location on earth at high frequency (5Hz) with a centimetric precision using phase differential positioning method. We studied whether such technique was accurate enough to retrieve basic parameters of human locomotion. Eight subjects walked on an athletics track at four different imposed step frequencies (70-130steps/min) plus a run at free pace. Differential carrier phase localization between a fixed base station and the mobile antenna mounted on the walking person was calculated. In parallel, a triaxial accelerometer, attached to the low back, recorded body accelerations. The different parameters were averaged for 150 consecutive steps of each run for each subject (total of 6000 steps analyzed). We observed a perfect correlation between average step duration measured by accelerometer and by GPS (r=0.9998, N=40). Two important parameters for the calculation of the external work of walking were also analyzed, namely the vertical lift of the trunk and the velocity variation per step. For an average walking speed of 4.0km/h, average vertical lift and velocity variation were, respectively, 4.8cm and 0.60km/h. The average intra-individual step-to-step variability at a constant speed, which includes GPS errors and the biological gait style variation, were found to be 24. 5% (coefficient of variation) for vertical lift and 44.5% for velocity variation. It is concluded that GPS technique can provide useful biomechanical parameters for the analysis of an unlimited number of strides in an unconstrained free-living environment.

Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system. Experience in 45 consecutive patients.

BACKGROUND: This study is a single-institution validation of video-assisted thoracoscopic (VATS) resection of a small solitary pulmonary nodule (SPN) previously localized by a CT-guided hook-wire system in a consecutive series of 45 patients. METHODS: The records of all patients undergoing VATS resection for SPN preoperatively localized by CT-guided a hook-wire system from January 2002 to December 2004 were assessed with respect to failure to localize the lesion by the hook-wire system, conversion thoracotomy rate, duration of operation, postoperative complications, and histology of SPN. RESULTS: Forty-five patients underwent 49 VATS resections, with simultaneous bilateral SPN resection performed in 4. Preoperative CT-guided hook-wire localization failed in two patients (4%). Conversion thoracotomy was necessary in two patients (4%) because it was not possible to resect the lesion by a VATS approach. The average operative time was 50 min. Postoperative complications occurred in 3 patients (6%), one hemothorax and two pneumonia. The mean hospital stay was 5 days (range: 2-18 days). Histological assessment revealed inflammatory disease in 17 patients (38%), metastasis in 17 (38%), non-small-cell lung cancer (NSCLC) in 4 (9%), lymphoma in 3 (6%), interstitial fibrosis in 2 (4%), histiocytoma in one (2%), and hamartoma in one (2%). CONCLUSIONS: Histological analysis of resected SPN revealed unexpected malignant disease in more than 50% of the patients indicating that histological clarification of SPN seems warranted. Video-assisted thoracoscopic resection of SPN previously localized by a CT-guided hook-wire system is related to a low conversion thoracotomy rate, a short operation time, and few postoperative complications, and it is well suited for the clarification of SPN.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Elastic constants of the beta-Cu-Zn alloy system: A Monte Carlo study

Monte Carlo calculations of the isothermal elastic constants of the beta-CuxZn1-x alloy system as a function of the composition have been carried out. We assume the atoms interact via a two-body Morse potential function and use numerical values for the potential parameters evaluated taking into account experimental data. We find a quite good agreement between our results and the expected experimental behavior.

The scarf osteotomy for the treatment of hallux valgus deformity: a review of 84 cases.

This study reviewed the subjective, clinical and radiological outcome of 71 patients (84 feet) treated by scarf osteotomy for hallux valgus deformity at our institution from 1995 to 1998 with an average follow-up time of 22 months (range, 17 to 48 months). At the time of follow-up, 39% of the patients were very satisfied, 50% were satisfied and 11% were not satisfied. The mean AOFAS score raised significantly from 43 points (14-68) preoperatively to 82 points (39 to 100) at follow-up (p &lt; 0.001). The radiological angles including M1-M2, M1-P1, M1-M5 and DMAA improved significantly (p &lt; 0.001). Among the 16 complications recorded, seven (8%) were minor and nine (11%) required an additional procedure. The scarf osteotomy of the first metatarsal coupled with a lateral soft-tissue release and, in three-quarters of our cases, with a basal closing wedge varisation osteotomy of the first phalanx, resulted in overall high satisfaction rate as well as significant clinical and radiological improvements in our series. Nevertheless, the range of motion of the first MP joint remained low: 30 degrees to 74 degrees in 52 patients (62%) and &lt;30 degrees in four patients (5%). Furthermore, the mobility of the first ray as well as the consequences of the procedure in the sagittal plane need to be assessed more accurately, and this may be achieved by incorporating measurement of the plantar pressures in the forefoot area into the global rating system.

Sleep function: current questions and new approaches.

The mammalian brain oscillates through three distinct global activity states: wakefulness, non-rapid eye movement (NREM) sleep and REM sleep. The regulation and function of these 'vigilance' or 'behavioural' states can be investigated over a broad range of temporal and spatial scales and at different levels of functional organization, i.e. from gene expression to memory, in single neurons, cortical columns or the whole brain and organism. We summarize some basic questions that have arisen from recent approaches in the quest for the functions of sleep. Whereas traditionally sleep was viewed to be regulated through top-down control mechanisms, recent approaches have emphasized that sleep is emerging locally and regulated in a use-dependent (homeostatic) manner. Traditional markers of sleep homeostasis, such as the electroencephalogram slow-wave activity, have been linked to changes in connectivity and plasticity in local neuronal networks. Thus waking experience-induced local network changes may be sensed by the sleep homeostatic process and used to mediate sleep-dependent events, benefiting network stabilization and memory consolidation. Although many questions remain unanswered, the available data suggest that sleep function will best be understood by an analysis which integrates sleep's many functional levels with its local homeostatic regulation.

Stabilization of soil hydraulic properties under a long term no-till system

The area under the no-tillage system (NT) has been increasing over the last few years. Some authors indicate that stabilization of soil physical properties is reached after some years under NT while other authors debate this. The objective of this study was to determine the effect of the last crop in the rotation sequence (1st year: maize, 2nd year: soybean, 3rd year: wheat/soybean) on soil pore configuration and hydraulic properties in two different soils (site 1: loam, site 2: sandy loam) from the Argentinean Pampas region under long-term NT treatments in order to determine if stabilization of soil physical properties is reached apart from a specific time in the crop sequence. In addition, we compared two procedures for evaluating water-conducting macroporosities, and evaluated the efficiency of the pedotransfer function ROSETTA in estimating the parameters of the van Genuchten-Mualem (VGM) model in these soils. Soil pore configuration and hydraulic properties were not stable and changed according to the crop sequence and the last crop grown in both sites. For both sites, saturated hydraulic conductivity, K0, water-conducting macroporosity, εma, and flow-weighted mean pore radius, R0ma, increased from the 1st to the 2nd year of the crop sequence, and this was attributed to the creation of water-conducting macropores by the maize roots. The VGM model adequately described the water retention curve (WRC) for these soils, but not the hydraulic conductivity (K) vs tension (h) curve. The ROSETTA function failed in the estimation of these parameters. In summary, mean values of K0 ranged from 0.74 to 3.88 cm h-1. In studies on NT effects on soil physical properties, the crop effect must be considered.

MODELING OF SOIL LOAD-BEARING CAPACITY AS A FUNCTION OF SOIL MECHANICAL RESISTANCE TO PENETRATION

Estimation of soil load-bearing capacity from mathematical models that relate preconsolidation pressure (σp) to mechanical resistance to penetration (PR) and gravimetric soil water content (U) is important for defining strategies to prevent compaction of agricultural soils. Our objective was therefore to model the σp and compression index (CI) according to the PR (with an impact penetrometer in the field and a static penetrometer inserted at a constant rate in the laboratory) and U in a Rhodic Eutrudox. The experiment consisted of six treatments: no-tillage system (NT); NT with chiseling; and NT with additional compaction by combine traffic (passing 4, 8, 10, and 20 times). Soil bulk density, total porosity, PR (in field and laboratory measurements), U, σp, and CI values were determined in the 5.5-10.5 cm and 13.5-18.5 cm layers. Preconsolidation pressure (σp) and CI were modeled according to PR in different U. The σp increased and the CI decreased linearly with increases in the PR values. The correlations between σp and PR and PR and CI are influenced by U. From these correlations, the soil load-bearing capacity and compaction susceptibility can be estimated by PR readings evaluated in different U.