Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.     

Prothrombolytic action of normobaric oxygen given alone or in combination with recombinant tissue-plasminogen activator in a rat model of thromboembolic stroke

The potential benefit of 100 vol% normobaric oxygen (NBO) for the treatment of acute ischemic stroke patients is still a matter of debate. To advance this critical question, we studied the effects of intraischemic normobaric oxygen alone or in combination with recombinant tissue-plasminogen activator (rtPA) on cerebral blood flow and ischemic brain damage and swelling in a clinically relevant rat model of thromboembolic stroke. We show that NBO provides neuroprotection by achieving cerebral blood flow restoration equivalent to 0.9 mg/kg rtPA through probable direct interaction and facilitation of the fibrinolytic properties of endogenous tPA. In contrast, combined NBO and rtPA has no neuroprotective effect on ischemic brain damage despite producing cerebral blood flow restoration. These results 1) by providing a new mechanism of action of NBO highlight together with previous findings the way by which intraischemic NBO shows beneficial action; 2) suggest that NBO could be an efficient primary care therapeutic intervention for patients eligible for rtPA therapy; 3) indicate that NBO could be an interesting alternative for patients not eligible for rtPA therapy; and 4) caution the use of NBO in combination with rtPA in acute stroke patients.     

Psycho-sensorimotor performance in divers exposed to six and seven atmospheres absolute of compressed air.

In the present study, we investigated the psycho-sensorimotor abilities of divers exposed to 6 ATA (50 m of sea water; corresponding to legal limit for occupational diving in North America, United Kingdom, and Northern Europe), or 7 ATA (60 m; the legal limit in France and Southern Europe) of compressed air (1 ATA = 100,000 Pa), using psychometric tests of manual dexterity, visual choice reaction time, and number ordination. The results of the present study showed that abilities in these tests were not significantly altered by pressure exposure to 6 ATA of compressed air. However, data obtained at 7 ATA showed slight but significant decreases in performance. Nevertheless, a few subjects presented large decreases in performance ranging from -20% to -25% of control. Finally, our results supported the ergonomic point of view that the laws limiting occupational diving to 6 ATA (50 m) are better adapted to reality and the requirements of underwater activity.     

Protein crystallography under xenon and nitrous oxide pressure: comparison with in vivo pharmacology studies and implications for the mechanism of inhaled anesthetic action

In contrast with most inhalational anesthetics, the anesthetic gases xenon (Xe) and nitrous oxide (N(2)O) act by blocking the N-methyl-d-aspartate (NMDA) receptor. Using x-ray crystallography, we examined the binding characteristics of these two gases on two soluble proteins as structural models: urate oxidase, which is a prototype of a variety of intracellular globular proteins, and annexin V, which has structural and functional characteristics that allow it to be considered as a prototype for the NMDA receptor. The structure of these proteins complexed with Xe and N(2)O were determined. One N(2)O molecule or one Xe atom binds to the same main site in both proteins. A second subsite is observed for N(2)O in each case. The gas-binding sites are always hydrophobic flexible cavities buried within the monomer. Comparison of the effects of Xe and N(2)O on urate oxidase and annexin V reveals an interesting relationship with the in vivo pharmacological effects of these gases, the ratio of the gas-binding sites' volume expansion and the ratio of the narcotic potency being similar. Given these data, we propose that alterations of cytosolic globular protein functions by general anesthetics would be responsible for the early stages of anesthesia such as amnesia and hypnosis and that additional alterations of ion-channel membrane receptor functions are required for deeper effects that progress to "surgical" anesthesia.     

Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.     

Protective Effects of Fluoxetine on Decompression Sickness in Mice

Massive bubble formation after diving can lead to decompression sickness (DCS) that can result in central nervous system disorders or even death. Bubbles alter the vascular endothelium and activate blood cells and inflammatory pathways, leading to a systemic pathophysiological process that promotes ischemic damage. Fluoxetine, a well-known antidepressant, is recognized as having anti-inflammatory properties at the systemic level, as well as in the setting of cerebral ischemia. We report a beneficial clinical effect associated with fluoxetine in experimental DCS. 91 mice were subjected to a simulated dive at 90 msw for 45 min before rapid decompression. The experimental group received 50 mg/kg of fluoxetine 18 hours before hyperbaric exposure (n = 46) while controls were not treated (n = 45). Clinical assessment took place over a period of 30 min after surfacing. At the end, blood samples were collected for blood cells counts and cytokine IL-6 detection. There were significantly fewer manifestations of DCS in the fluoxetine group than in the controls (43.5% versus 75.5%, respectively; p = 0.004). Survivors showed a better and significant neurological recovery with fluoxetine. Platelets and red cells were significantly decreased after decompression in controls but not in the treated mice. Fluoxetine reduced circulating IL-6, a relevant marker of systemic inflammation in DCS. We concluded that fluoxetine decreased the incidence of DCS and improved motor recovery, by limiting inflammation processes.     

Animal board invited review: Animal source foods in healthy,sustainable, and ethical diets – An argument against drastic limitation of livestock in the food system

Animal source foods are evolutionarily appropriate foods for humans. It is therefore remarkable that they are now presented by some as unhealthy, unsustainable, and unethical, particularly in the urban West. The benefits of consuming them are nonetheless substantial, as they offer a wide spectrum of nutrients that are needed for cell and tissue development, function, and survival. They play a role in proper physical and cognitive development of infants, children, and adolescents, and help promote maintenance of physical function with ageing. While high-red meat consumption in the West is associated with several forms of chronic disease, these associations remain uncertain in other cultural contexts or when consumption is part of wholesome diets. Besides health concerns, there is also widespread anxiety about the environmental impacts of animal source foods. Although several production methods are detrimental (intensive cropping for feed, overgrazing, deforestation, water pollution, etc.) and require substantial mitigation, damaging impacts are not intrinsic to animal husbandry. When well-managed, livestock farming contributes to ecosystem management and soil health, while delivering high-quality foodstuffs through the upcycling of resources that are otherwise non-suitable for food production, making use of marginal land and inedible materials (forage, by-products, etc.), integrating livestock and crop farming where possible has the potential to benefit plant food production through enhanced nutrient recycling, while minimising external input needs such as fertilisers and pesticides. Moreover, the impacts on land use, water wastage, and greenhouse gas emissions are highly contextual, and their estimation is often erroneous due to a reductionist use of metrics. Similarly, whether animal husbandry is ethical or not depends on practical specificities, not on the fact that animals are involved. Such discussions also need to factor in that animal husbandry plays an important role in culture, societal well-being, food security, and the provision of livelihoods. We seize this opportunity to argue for less preconceived assumptions about alleged effects of animal source foods on the health of the planet and the humans and animals involved, for less top-down planning based on isolated metrics or (Western) technocratic perspectives, and for more holistic and circumstantial approaches to the food system.     

Ex vivo and in vivo neuroprotection induced by argon when given after an excitotoxic or ischemic insult

In vitro studies have well established the neuroprotective action of the noble gas argon. However, only limited data from in vivo models are available, and particularly whether postexcitotoxic or postischemic argon can provide neuroprotection in vivo still remains to be demonstrated. Here, we investigated the possible neuroprotective effect of postexcitotoxic-postischemic argon both ex vivo in acute brain slices subjected to ischemia in the form of oxygen and glucose deprivation (OGD), and in vivo in rats subjected to an intrastriatal injection of N-methyl-D-aspartate (NMDA) or to the occlusion of middle-cerebral artery (MCAO). We show that postexcitotoxic-postischemic argon reduces OGD-induced cell injury in brain slices, and further reduces NMDA-induced brain damage and MCAO-induced cortical brain damage in rats. Contrasting with its beneficial effect at the cortical level, we show that postischemic argon increases MCAO-induced subcortical brain damage and provides no improvement of neurologic outcome as compared to control animals. These results extend previous data on the neuroprotective action of argon. Particularly, taken together with previous in vivo data that have shown that intraischemic argon has neuroprotective action at both the cortical and subcortical level, our findings on postischemic argon suggest that this noble gas could be administered during but not after ischemia, i.e. before but not after reperfusion has occurred, in order to provide cortical neuroprotection and to avoid increasing subcortical brain damage. Also, the effects of argon are discussed as regards to the oxygen-like chemical, pharmacological, and physical properties of argon.     

Quantitative study of behavioral disturbances in rats exposed to high pressure

When human divers or experimental animals are exposed to high pressure, they develop the High Pressure Neurological Syndrome (HPNS). Male Sprague-Dawley rats were exposed to high pressure in a conventional helium-oxygen breathing mixture to 80 bars. Pressure-induced behavioral motor disturbances including hyperlocomotor activity (HLA), tremor and myoclonia were monitored with a noninvasive piezoelectrical sensor device enabling a without discontinuity long-term analysis. New data were obtained on the development of the HPNS behavioral motor disturbances. Indeed, the present results suggest myoclonia would be more sensitive to constant high pressure exposure, while HLA and tremor would be more sensitive to increasing pressure. Moreover, myoclonia were found to occur significantly later in rats which developed epileptic seizures than in other. The present results constitute the quantitative basis of HPNS motor disturbances for future pharmacological pressure experiments.