The GTPase RalA regulates different steps of the secretory process in pancreatic beta-cells.

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway.

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.

Introducing systematic dispatcher-assisted cardiopulmonary resuscitation (telephone-CPR) in a non-Advanced Medical Priority Dispatch System (AMPDS): implementation process and costs.

OBJECTIVE: In order to improve the quality of our Emergency Medical Services (EMS), to raise bystander cardiopulmonary resuscitation rates and thereby meet what is becoming a universal standard in terms of quality of emergency services, we decided to implement systematic dispatcher-assisted or telephone-CPR (T-CPR) in our medical dispatch center, a non-Advanced Medical Priority Dispatch System. The aim of this article is to describe the implementation process, costs and results following the introduction of this new "quality" procedure. METHODS: This was a prospective study. Over an 8-week period, our EMS dispatchers were given new procedures to provide T-CPR. We then collected data on all non-traumatic cardiac arrests within our state (Vaud, Switzerland) for the following 12months. For each event, the dispatchers had to record in writing the reason they either ruled out cardiac arrest (CA) or did not propose T-CPR in the event they did suspect CA. All emergency call recordings were reviewed by the medical director of the EMS. The analysis of the recordings and the dispatchers' written explanations were then compared. RESULTS: During the 12-month study period, a total of 497 patients (both adults and children) were identified as having a non-traumatic cardiac arrest. Out of this total, 203 cases were excluded and 294 cases were eligible for T-CPR. Out of these eligible cases, dispatchers proposed T-CPR on 202 occasions (or 69% of eligible cases). They also erroneously proposed T-CPR on 17 occasions when a CA was wrongly identified (false positive). This represents 7.8% of all T-CPR. No costs were incurred to implement our study protocol and procedures. CONCLUSIONS: This study demonstrates it is possible, using a brief campaign of sensitization but without any specific training, to implement systematic dispatcher-assisted cardiopulmonary resuscitation in a non-Advanced Medical Priority Dispatch System such as our EMS that had no prior experience with systematic T-CPR. The results in terms of T-CPR delivery rate and false positive are similar to those found in previous studies. We found our results satisfying the given short time frame of this study. Our results demonstrate that it is possible to improve the quality of emergency services at moderate or even no additional costs and this should be of interest to all EMS that do not presently benefit from using T-CPR procedures. EMS that currently do not offer T-CPR should consider implementing this technique as soon as possible, and we expect our experience may provide answers to those planning to incorporate T-CPR in their daily practice.

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.

Two-stage, extreme albitization of A-type granites from Rajasthan, NW India

Albitization is a common process during which hydrothermal fluids convert plagioclase and/or K-feldspar into nearly pure albite; however, its specific mechanism in granitoids is not well understood. The c. 1700 Ma A-type metaluminous ferroan granites in the Khetri complex of Rajasthan, NW India, have been albitized to a large extent by two metasomatic fronts, an initial transformation of oligoclase to nearly pure albite and a subsequent replacement of microcline by albite, with sharp contacts between the microcline-bearing and microcline-free zones. Albitization has bleached the original pinkish grey granite and turned it white. The mineralogical changes include transformation of oligoclase (similar to An(12)) and microcline (similar to Or(95)) to almost pure albite (similar to An(0 center dot 5-2)), amphibole from potassian ferropargasite (X-Fe 0 center dot 84-0 center dot 86) to potassic hastingsite (X-Fe 0 center dot 88-0 center dot 97) and actinolite (X-Fe 0 center dot 32-0 center dot 67), and biotite from annite (X-Fe 0 center dot 71-0 center dot 74) to annite (X-Fe 0 center dot 90-0 center dot 91). Whole-rock isocon diagrams show that, during albitization, the granites experienced major hydration, slight gain in Si and major gain in Na, whereas K, Mg, Fe and Ca were lost along with Rb, Ba, Sr, Zn, light rare earth elements and U. Whole-rock Sm-Nd isotope data plot on an apparent isochron of 1419 +/- 98 Ma and reveal significant disturbance and at least partial resetting of the intrusion age. Severe scatter in the whole-rock Rb-Sr isochron plot reflects the extreme Rb loss in the completely albitized samples, effectively freezing Sr-87/Sr-86 ratios in the albite granites at very high values (0 center dot 725-0 center dot 735). This indicates either infiltration of highly radiogenic Sr from the country rock or, more likely, radiogenic ingrowth during a considerable time lag (estimated to be at least 300 Myr) between original intrusion and albitization. The albitization took place at similar to 350-400 degrees C. It was caused by the infiltration of an ascending hydrothermal fluid that had acquired high Na/K and Na/Ca ratios during migration through metamorphic rocks at even lower temperatures in the periphery of the plutons. Oxygen isotope ratios increase from delta O-18 = 7 parts per thousand in the original granite to values of 9-10 parts per thousand in completely albitized samples, suggesting that the fluid had equilibrated with surrounding metamorphosed crust. A metasomatic model, using chromatographic theory of fluid infiltration, explains the process for generating the observed zonation in terms of a leading metasomatic front where oligoclase of the original granite is converted to albite, and a second, trailing front where microcline is also converted to albite. The temperature gradients driving the fluid infiltration may have been produced by the high heat production of the granites themselves. The confinement of the albitized granites along the NE-SW-trending Khetri lineament and the pervasive nature of the albitization suggest that the albitizing fluids possibly originated during reactivation of the lineament. More generally, steady-state temperature gradients induced by the high internal heat production of A-type granites may provide the driving force for similar metasomatic and ore-forming processes in other highly enriched granitoid bodies.

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.

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

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.