Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Estimation de la fonction rénale par l'equation MDRD: intérêt et limites pour l'adaptation des doses de médicaments [Renal function estimation by MDRD equation: interest and limitations for drug dosing].

The MDRD (Modification of diet in renal disease) equation enables glomerular filtration rate (GFR) estimation from serum creatinine only. Thus, the laboratory can report an estimated GFR (eGFR) with each serum creatinine assessment, increasing therefore the recognition of renal failure. Predictive performance of MDRD equation is better for GFR < 60 ml/min/1,73 m2. A normal or near-normal renal function is often underestimated by this equation. Overall, MDRD provides more reliable estimations of renal function than the Cockcroft-Gault (C-G) formula, but both lack precision. MDRD is not superior to C-G for drug dosing. Being adjusted to 1,73 m2, MDRD eGFR has to be back adjusted to the patient's body surface area for drug dosing. Besides, C-G has the advantage of a greater simplicity and a longer use.

Post-collisional Neogene magmatism of the Mediterranean Maghreb margin: a consequence of slab breakoff

A 1 200 km-long linear magmatic belt extends along the Mediterranean coast the Maghreb from Eastern Tunisia to Morocco. This belt is mainly composed of Langan calc-alkaline metaluminous to peraluminous granitoids and associated andesites/cites Central and Eastern Algeria. In Tunisia and Oranie/Western Morocco, calc alkaline activity started later (during the Serravallian) and was followed by the placement of alkali basalts and basanites since the Tortonian to the Pliocene and, in me places, the Pleistocene. Available data on the tectonic setting, petrology, age and biochemistry of this belt show that most of its striking features, e.g. (1) very low magma production rate, subduction-related geochemical imprint, extensive crustal contamination the calc-alkaline magmatism and (2) progressive magmatic change from calc-alkaline alkaline, are consistent with magma generation during a slab breakoff process as proposed Carminati et al, in 1998. The magmatism associated with this breakoff started in Central Eastern Algeria at 16 Ma, then propagated eastwards and westwards, The upward of asthenospheric enriched plume-type mantle through the tear in the downgoing first triggered melting of the overlying lithospheric mantle which had been metasomatised during a previous subduction period. Heat supply from this uprising asthenosphere may have warmed up the continental crust and made its involvement in assimilation processes easier. As the asthenosphere ascended through the `window' in the slab, partial melting occurred at the uprising boundary between asthenosphere and lithosphere, generating basalts with transitional characteristics between those of calc-alkaline and alkaline basalts. As the asthhenospheric upwelling proceeded partial:melting then occurred in the sole asthenospheric mantle, producing alkali basalts. (C) 2000 Academie des sciences Editions scientifiques et medicales Elsevier SAS.

Identification of a key lysine residue in heat shock protein 90 required for azole and echinocandin resistance in Aspergillus fumigatus.

Heat shock protein 90 (Hsp90) is an essential chaperone involved in the fungal stress response that can be harnessed as a novel antifungal target for the treatment of invasive aspergillosis. We previously showed that genetic repression of Hsp90 reduced Aspergillus fumigatus virulence and potentiated the effect of the echinocandin caspofungin. In this study, we sought to identify sites of posttranslational modifications (phosphorylation or acetylation) that are important for Hsp90 function in A. fumigatus. Phosphopeptide enrichment and tandem mass spectrometry revealed phosphorylation of three residues in Hsp90 (S49, S288, and T681), but their mutation did not compromise Hsp90 function. Acetylation of lysine residues of Hsp90 was recovered after treatment with deacetylase inhibitors, and acetylation-mimetic mutations (K27A and K271A) resulted in reduced virulence in a murine model of invasive aspergillosis, supporting their role in Hsp90 function. A single deletion of lysine K27 or an acetylation-mimetic mutation (K27A) resulted in increased susceptibility to voriconazole and caspofungin. This effect was attenuated following a deacetylation-mimetic mutation (K27R), suggesting that this site is crucial and should be deacetylated for proper Hsp90 function in antifungal resistance pathways. In contrast to previous reports in Candida albicans, the lysine deacetylase inhibitor trichostatin A (TSA) was active alone against A. fumigatus and potentiated the effect of caspofungin against both the wild type and an echinocandin-resistant strain. Our results indicate that the Hsp90 K27 residue is required for azole and echinocandin resistance in A. fumigatus and that deacetylase inhibition may represent an adjunctive anti-Aspergillus strategy.

Evaporation from a shallow water table: Diurnal dynamics of measured and simulated water and heat regime at the vicinity of a drying sand surface

Accurate estimates of water losses by evaporation from shallow water tables are important for hydrological, agricultural, and climatic purposes. An experiment was conducted in a weighing lysimeter to characterize the diurnal dynamics of evaporation under natural conditions. Sampling revealed a completely dry surface sand layer after 5 days of evaporation. Its thickness was <1 cm early in the morning, increasing to reach 4?5 cm in the evening. This evidence points out fundamental limitations of the approaches that assume hydraulic connectivity from the water table up to the surface, as well as those that suppose monotonic drying when unsteady conditions prevail. The computed vapor phase diffusion rates from the apparent drying front based on Fick's law failed to reproduce the measured cumulative evaporation during the sampling day. We propose that two processes rule natural evaporation resulting from daily fluctuations of climatic variables: (i) evaporation of water, stored during nighttime due to redistribution and vapor condensation, directly into the atmosphere from the soil surface during the early morning hours, that could be simulated using a mass transfer approach and (ii) subsurface evaporation limited by Fickian diffusion, afterward. For the conditions prevailing during the sampling day, the amount of water stored at the vicinity of the soil surface was 0.3 mm and was depleted before 11:00. Combining evaporation from the surface before 11:00 and subsurface evaporation limited by Fickian diffusion after that time, the agreement between the estimated and measured cumulative evaporation was significantly improved.

Physical activity modulates heat shock protein-72 expression and limits oxidative damage accumulation in a healthy elderly population aged 60 90 years

BACKGROUND: Reactive oxygen species production increases during aging, whereas protective mechanisms such as heat shock proteins (HSPs) or antioxidant capacity are depressed. Physical activity has been hypothesized to provide protection against oxidative damage during aging, but results remain controversial. This study aimed to investigate the effect of different levels of physical activity during aging on Hsp72 expression and systemic oxidative stress at rest and in response to maximal exercise. METHODS: Plasma antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC), thiobarbituric acid-reactive species (TBARS), advanced oxidized proteins products (AOPP), and Hsp72 expression in leukocytes were measured before and after maximal exercise testing in 32 elderly persons (aged 73.2 years), who were assigned to two different groups depending on their level of physical activity during the past 12 months (OLow = moderate to low level; OHigh = higher level). RESULTS: The OHigh group showed higher aerobic fitness and TEAC (both representing 120% of OLow values) as well as lower oxidative damage (50% of OLow values) and Hsp72 expression. Exercise led to a lower increase in oxidative damage in the OHigh group. Aerobic fitness was positively correlated with TEAC and negatively with lipid peroxidation (TBARS). Hsp72 expression was negatively correlated with TEAC but positively correlated with TBARS levels. CONCLUSIONS: The key finding of this study is that, in people aged 60 to 90 years, long-term high level of physical activity preserved antioxidant capacity and limited oxidative damage accumulation. It also downregulated Hsp72 expression, an adaptation potentially resulting from lower levels of oxidative damage.

Further insights on the French WISC-IV factor structure through Bayesian structural equation modeling

The interpretation of the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV) is based on a 4-factor model, which is only partially compatible with the mainstream Cattell-Horn-Carroll (CHC) model of intelligence measurement. The structure of cognitive batteries is frequently analyzed via exploratory factor analysis and/or confirmatory factor analysis. With classical confirmatory factor analysis, almost all crossloadings between latent variables and measures are fixed to zero in order to allow the model to be identified. However, inappropriate zero cross-loadings can contribute to poor model fit, distorted factors, and biased factor correlations; most important, they do not necessarily faithfully reflect theory. To deal with these methodological and theoretical limitations, we used a new statistical approach, Bayesian structural equation modeling (BSEM), among a sample of 249 French-speaking Swiss children (8-12 years). With BSEM, zero-fixed cross-loadings between latent variables and measures are replaced by approximate zeros, based on informative, small-variance priors. Results indicated that a direct hierarchical CHC-based model with 5 factors plus a general intelligence factor better represented the structure of the WISC-IV than did the 4-factor structure and the higher order models. Because a direct hierarchical CHC model was more adequate, it was concluded that the general factor should be considered as a breadth rather than a superordinate factor. Because it was possible for us to estimate the influence of each of the latent variables on the 15 subtest scores, BSEM allowed improvement of the understanding of the structure of intelligence tests and the clinical interpretation of the subtest scores.

The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane.

Land plants are prone to strong thermal variations and must therefore sense early moderate temperature increments to induce appropriate cellular defenses, such as molecular chaperones, in anticipation of upcoming noxious temperatures. To investigate how plants perceive mild changes in ambient temperature, we monitored in recombinant lines of the moss Physcomitrella patens the activation of a heat-inducible promoter, the integrity of a thermolabile enzyme, and the fluctuations of cytoplasmic calcium. Mild temperature increments, or isothermal treatments with membrane fluidizers or Hsp90 inhibitors, induced a heat shock response (HSR) that critically depended on a preceding Ca(2+) transient through the plasma membrane. Electrophysiological experiments revealed the presence of a Ca(2+)-permeable channel in the plasma membrane that is transiently activated by mild temperature increments or chemical perturbations of membrane fluidity. The amplitude of the Ca(2+) influx during the first minutes of a temperature stress modulated the intensity of the HSR, and Ca(2+) channel blockers prevented HSR and the onset of thermotolerance. Our data suggest that early sensing of mild temperature increments occurs at the plasma membrane of plant cells independently from cytosolic protein unfolding. The heat signal is translated into an effective HSR by way of a specific membrane-regulated Ca(2+) influx, leading to thermotolerance.

Anti-60kDa heat shock protein antibodies in fetal serum : a biomarker for unexplained small for gestational age fetuses

Rapport de synthèseObjectifsLe retard de croissance intrautérin (RCIU) est un problème affectant 10% des grossesses et est associé à une morbidité périnatale importante. Dans environ 80% des cas, une étiologie ou un facteur de risque majeur peuvent être identifiés. Mais près de 20% des cas sont considérés comme inexpliqués. La heat shock protéine 60kDa (HSP60) est une protéine fortement immunogène dont la synthèse est considérablement augmentée lors de conditions non- physiologiques. Les HSP60 humaines et bactériennes partagent un haut degré d'homologie de séquence ce qui peut engendrer une maladie auto-immune à la suite d'une infection bactérienne. Nous avons supposé que les RCIU inexpliqués pourraient être la conséquence d'une sensibilisation à l'HSP60 humaine.MéthodesLes RCIU inexpliqués ont été identifiés par mesure échographique avec un doppler normal, sans anomalies décelables chez la mère ou le foetus. Les sera foetaux ont été obtenus par cordocentèse, effectuée lors d'analyse du caryotype en cas de RCIU inexpliqué (groupe d'étude) ou pour le dépistage d'une incompatibilité Rhésus (groupe témoin). Ils ont été testés pour l'antigène HSP60 et les IgG et IgM anti-HSP60 par ELISA ainsi que pour d'autres paramètres immunitaires et hématologiques.RésultatsLes paramètres maternels sont similaires entre les 12 cas du groupe d'étude et les 23 cas du groupe contrôle. L'âge gestationnel moyen lors de la cordocentèse est de 29 semaines. Les IgM anti-HSP60 sont détectés dans 12 cas d'étude (100%) mais dans aucun cas contrôle (p <0,00017), les IgG anti-HSP60 dans 7 cas d'étude (58%) et un seul dans le groupe contrôle (p <0,001). Trois des quatre cas avec les taux d'IgM les plus élevés sont décédés. Il n'y a pas de différences entre les deux groupes quant aux taux d'antigène HSP60 ou d'autres marqueurs immunologiques ou hématologiques.ConclusionLes foetus avec un RCIU inexpliqué expriment un taux élevé d'anticorps IgM et IgG contre l'HSP60 humaine et le taux d'IgM est un facteur prédictif de la mortalité foetale. La détection de ces anticorps indique qu'une perturbation placentaire et une réaction auto-immune foetale liée à l'HSP60 sont associées à ce retard de développement chez le foetus.

Combining heat stress and moderate hypoxia reduces cycling time to exhaustion without modifying neuromuscular fatigue characteristics.

PURPOSE: This study investigated the isolated and combined effects of heat [temperate (22 °C/30 % rH) vs. hot (35 °C/40 % rH)] and hypoxia [sea level (FiO2 0.21) vs. moderate altitude (FiO2 0.15)] on exercise capacity and neuromuscular fatigue characteristics. METHODS: Eleven physically active subjects cycled to exhaustion at constant workload (66 % of the power output associated with their maximal oxygen uptake in temperate conditions) in four different environmental conditions [temperate/sea level (control), hot/sea level (hot), temperate/moderate altitude (hypoxia) and hot/moderate altitude (hot + hypoxia)]. Torque and electromyography (EMG) responses following electrical stimulation of the tibial nerve (plantar-flexion; soleus) were recorded before and 5 min after exercise. RESULTS: Time to exhaustion was reduced (P < 0.05) in hot (-35 ± 15 %) or hypoxia (-36 ± 14 %) compared to control (61 ± 28 min), while hot + hypoxia (-51 ± 20 %) further compromised exercise capacity (P < 0.05). However, the effect of temperature or altitude on end-exercise core temperature (P = 0.089 and P = 0.070, respectively) and rating of perceived exertion (P > 0.05) did not reach significance. Maximal voluntary contraction torque, voluntary activation (twitch interpolation) and peak twitch torque decreased from pre- to post-exercise (-9 ± 1, -4 ± 1 and -6 ± 1 % all trials compounded, respectively; P < 0.05), with no effect of the temperature or altitude. M-wave amplitude and root mean square activity were reduced (P < 0.05) in hot compared to temperate conditions, while normalized maximal EMG activity did not change. Altitude had no effect on any measured parameters. CONCLUSION: Moderate hypoxia in combination with heat stress reduces cycling time to exhaustion without modifying neuromuscular fatigue characteristics. Impaired oxygen delivery or increased cardiovascular strain, increasing relative exercise intensity, may have also contributed to earlier exercise cessation.

Nuclear scaffold proteins are differently sensitive to stabilizing treatment by heat or Cu++.

The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.

Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.