Mitochondrial dysfunction as a mediator of hippocampal apoptosis in a model of hepatic encephalopathy

In this study, we describe the presence of apoptosis, associated with a mitochondrial dysfunction in the hippocampus of animals in an experimental model defined as minimal hepatic encephalopathy (MHE). This experimental model was studied after 10 days of induced portal vein calibrated stricture, leading to portal hypertension and to a moderate hyperammonemia, without the presence of other evident central nervous system changes. The molecular mechanisms here proposed indicate the presence of apoptotic intrinsic pathways that point to hippocampal mitochondria as an important mediator of apoptosis in this experimental model. In this model of MHE, the presence of DNA fragmentation is documented by 2.3-times increased number of TUNEL-positive cells. These findings together with a higher ratio of the Bcl-2 family members Bax/Bcl-xL in the outer mitochondrial membrane of the MHE animals together with 11% of cytochrome c release indicate the presence of apoptosis in this experimental model. A detailed analysis of the hippocampal mitochondrial physiology was performed after mitochondrial isolation. The determination of the respiratory rate in the presence of malate plus glutamate and ADP showed a 45% decrease in respiratory control in MHE animals as compared with the sham group. A marked decrease of cytochrome oxidase (complex IV of the electron transport chain) was also observed, showing 46% less activity in hippocampal mitochondria from MHE animals. In addition, mitochondria from these animals showed less ability to maintain membrane potential (ΔΨ _m) which was 13% lower than the sham group. Light scattering experiments showed that mitochondria from MHE animals were more sensitive to swell in the presence of increased calcium concentrations as compared with the sham group. In addition, in vitro studies performed in mitochondria from sham animals showed that mitochondrial permeability transition (MPT) could be a mitochondrial mediator of the apoptotic signaling in the presence of NH₄ ⁺ and calcium.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Effects of climate change on productivity of cereals and legumes; model evaluation of observed year-to-year variability of the CO2 response

The effect of elevated [CO₂] on the productivity of spring wheat, winter wheat and faba bean was studied in experiments in climatized crop enclosures in the Wageningen Rhizolab in 1991–93. Simulation models for crop growth were used to explore possible causes for the observed differences in the CO₂ response. Measurements of the canopy gas exchange (CO₂ and water vapour) were made continuously from emergence until harvest. At an external [CO₂] of 700 μmol mol^?1 Maximum Canopy CO₂ Exchange Rate (CCERmax) at canopy closure was stimulated by 51% for spring wheat and by 71% for faba bean. At the end of the growing season, above ground biomass increase at 700 μmol mol^?1 was 58% (faba bean), 35% (spring wheat) and 19% (winter wheat) and the harvest index did not change. For model exploration, weather data sets for the period 1975-88 and 1991–93 were used, assuming adequate water supply and [CO₂] at 350 and 700 μmol mol^?1. For spring wheat the simulated responses (35–50%) were at the upper end of the experimental results. In agreement with experiments, simulations showed smaller responses for winter wheat and larger responses for faba bean. Further model explorations showed that this differential effect in the CO₂ response may not be primarily due to fundamental physiological differences between the crops, but may be at least partly due to differences in the daily air temperatures during comparable stages of growth of these crops. Simulations also showed that variations between years in CO₂ response can be largely explained by differences in weather conditions (especially temperature) between growing seasons.     

Brain chemiluminescence and oxidative stress in hyperthyroid rats.

Newborn Wistar rats were made hyperthyroid by injection of tri-iodothyronine and assayed for survival, brain oxygen uptake, brain chemiluminescence and activity of antioxidant enzymes. Brain chemiluminescence was measured (1) by removing the parietal bones or (2) through the translucid parietal bones. Control animals showed a brain chemiluminescence of 130 +/- 12 c.p.s./cm2 and 99 +/- 10 c.p.s./cm2 for procedures (1) and (2) respectively. Hyperthyroid rats showed increases in the spontaneous brain photoemission of 46 and 70% compared with controls, measured by procedures 1 and 2 respectively. The hyperthyroid state did not modify the oxygen-dependent chemiluminescence of brain homogenates. The hyperthyroid animals showed a 30% increase in the oxygen uptake of brain slices and a dramatic shortening of life-span to about 16 weeks. Superoxide dismutase (the Cu-Zn enzyme), catalase and Se-dependent glutathione peroxidase activities of brain homogenates were increased by 18, 36 and 30% respectively in the hyperthyroid animals. Isolated brain mitochondria produced 0.18-0.20 nmol of H2O2/min per mg of protein in state 4 in the presence of succinate as substrate. No difference was observed between control and hyperthyroid animals. It is concluded that hyperthyroidism leads to hypermetabolism and oxidative stress in the brain. The increased levels of oxygen and peroxyl radicals may contribute to premature ageing in these animals.     

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Rats fed ethanol (1.74 +/- 0.12 g/day/100 g body wt for 12 weeks) showed a 45% increased microsomal production of O-2 (2.23 +/- 0.14 nmol/min/mg protein) and a 28% increased content of endoplasmic reticulum protein (26.8 +/- 1.4 mg/g liver). This could lead, at substrate saturation, to a 86% increased cytosolic production of O-2 which is not compensated by cytosolic superoxide dismutase levels that remain normal. It is claimed that this unbalance between O-2 production and superoxide dismutase leads to a peroxidative stress in agreement with the 54% increased spontaneous liver chemiluminescence (37 +/- 2 cps/cm2) measured in the ethanol-treated rats. Hydroperoxide-induced chemiluminescence was 57, 43, and 28% higher, respectively, in homogenates, mitochondria, and microsomes isolated from ethanol-treated rats as compared with controls. Vitamins E and A were more effective inhibitors of the hydroperoxide-stimulated chemiluminescence in the liver homogenates from ethanol-treated rats as compared with the effect on the homogenates from control animals. The results are consistent with a peroxidative stress in chronic alcoholism leading to increased lipoperoxidation and decreased levels of antioxidants.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.     

Systemic Oxidative Stress Associated with the Neurological Diseases of Aging

Markers of oxidative stress were measured in blood samples of 338 subjects (965 observations): Alzheimer’s, vascular dementia, diabetes (type II) superimposed to dementias, Parkinson’s disease and controls. Patients showed increased thiobarbituric acid reactive substances (+21%; P < 0.05), copper-zinc superoxide dismutase (+64%; P < 0.001) and decreased antioxidant capacity (−28%; P < 0.001); pairs of variables resulted linearly related across groups (P < 0.001). Catalase and glutathione peroxidase, involved in discrimination between diseases, resulted non-significant. When diabetes is superimposed with dementias, changes resulted less marked but significant. Also, superoxide dismutase resulted not linearly correlated with any other variable or age-related (pure Alzheimer’s peaks at 70 years, P < 0.001). Systemic oxidative stress was significantly associated (P ≪ 0.001) with all diseases indicating a disbalance in peripheral/adaptive responses to oxidative disorders through different free radical metabolic pathways. While other changes—methionine cycle, insulin correlation—are also associated with dementias, the responses presented here show a simple linear relation between prooxidants and antioxidant defenses.