Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.     

Endotoxemia impairs heart mitochondrial function by decreasing electron transfer, ATP synthesis and ATP content without affecting membrane potential

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O₂ consumption of rat heart (1 mm³ tissue cubes) by 33% (from 4.69 to 3.11 μmol O₂/min. g tissue). Mitochondrial O₂ consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O₂^●− and H₂O₂ due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO⁻ generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O₂ uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia.     

Content of liver and brain ubiquinol-9 and ubiquinol-10 after chronic ethanol intake in rats subjected to two levels of dietary alpha-tocopherol

To assess the effect of chronic ethanol ingestion in the content of the reduced forms of coenzymes Q9 (ubiquinol-9) and Q10 (ubiquinol-10) as a factor contributing to oxidative stress in liver and brain, male Wistar rats were fed ad libitum a basal diet containing either 10 or 2.5 mg alpha-tocopherol/100 g diet (controls), or the same basal diet plus a 32% ethanol-25% sucrose solution. After three months treatment, ethanol chronically-treated rats showed identical growth rates to the isocalorically pair-fed controls, irrespectively of alpha-tocopherol dietary level. Lowering dietary alpha-tocopherol led to a decreased content of this vitamin in the liver and brain of control rats, without changes in that of ubiquinol-9, and increased levels of hepatic ubiquinol-10 and total glutathione (tGSH), accompanied by a decrease in brain tGSH. At the two levels of dietary alpha-tocopherol, ethanol treatment significantly decreased the content of hepatic alpha-tocopherol and ubiquinols 9 and 10. This effect was significantly greater at 10 mg alpha-tocopherol/100 g diet than at 2.5, whereas those of tGSH were significantly elevated by 43% and 9%, respectively. Chronic ethanol intake did not alter the content of brain alpha-tocopherol and tGSH, whereas those of ubiquinol-9 were significantly lowered by 20% and 14% in rats subjected to 10 and 2.5 mg alpha-tocopherol/100 g diet, respectively. It is concluded that chronic ethanol intake at two levels of dietary alpha-tocopherol induces a depletion of hepatic alpha-tocopherol and ubiquinols 9 and 10, thus contributing to ethanol-induced oxidative stress in the liver tissue. This effect of ethanol is dependent upon the dietary level of alpha-tocopherol, involves a compensatory enhancement in hepatic tGSH availability, and is not observed in the brain tissue, probably due to its limited capacity for ethanol biotransformation and glutathione synthesis.     

Kinetic analysis of thapsigargin-induced thymocyte apoptosis

Thapsigargin addition to thymocytes increased cytosolic Ca2+ by a factor of 8.5 with a time for half maximal effect (t1/2) of 2.5 min. Calcium signaling increased mitocondrial and endoplasmic reticulum nitric oxide synthase (NOS) activities by five and six times, with t1/2 of 16 and 48 min, respectively, followed by increases of 140% in intracellular [H2O2], 73% in hydroperoxide content, and 250% in thiobarbituric reactive substance content, with t1/2 of 13, 27, and 30 min, respectively. Mitochondrial dysfunction followed, and was characterized by decreased respiratory control, membrane depolarization, and decrease cytochrome c content release, processes with t1/2 of 101, 129, and 133 min, respectively. Increased UDP-GT gene expression, observed by mRNA synthesis, and the enzymatic activity of this protein had t1/2 of 52 and 187 min, respectively. These events were followed by caspase-3 activation (t1/2 = 210 min) and DNA laddering (t1/2 = 260 min) at the completion of the cell death program. Preincubation of thymocytes with NOS inhibitors (NG-methyl-L-arginine and L-Nomega-nitro-L-arginine methylester) halted the whole process through inhibition of mitochondrial and endoplasmic reticulum NOS activities and of DNA laddering.     

Improving ability to identify malaria and correctly use chloroquine in children at household level in Nakonde District, Northern Province of Zambia

BACKGROUND: This study investigated causes of malaria and how cases were managed at household level, in order to improve the ability to identify malaria and ensure correct use of chloroquine. It was conducted in Nakonde District, Northern Province of Zambia, between 2000 and 2001. Nakonde district is in a hyperendemic malaria province, where Plasmodium falciparum is predominant. The district has a total population of 153, 548 people, the majority of whom are peasant farmers. The main aim of the post intervention survey was to establish the proportion of caretakers of children five years and below, who were able to identify simple and severe malaria and treat it correctly using chloroquine in the home. METHODS: A baseline survey was conducted in five wards divided into intervention and control.Intervention and control wards were compared. Village health motivators and vendors were identified and trained in three intervention wards, as a channel through which information on correct chloroquine dose could be transmitted. A total of 575 carers, who were 15 years old and above and had a child who had suffered from malaria 14 days before the survey commenced, were interviewed. The two control wards received no intervention. 345 caretakers were from the intervention wards, while 230 came from the control wards. Identification of malaria and correct use of anti-malarial drugs was assessed in terms of household diagnosis of malaria in children under five years, type and dose of anti-malarial drugs used, self medication and the source of these anti-malarials. RESULTS: The majority of respondents in the study were females (81%). Chloroquine was the most frequently used anti-malarial (48.5%) in both the intervention and control wards. There was no difference between the intervention and control wards at pre-intervention (P = 0.266 and P = 0.956), in the way mothers and other caretakers identified simple and severe malaria. At baseline, knowledge on correct chloroquine dosage in the under five children was comparable between intervention and control wards. Post-intervention revealed that mothers and other caretakers were 32% and 51%, respectively, more likely to identify simple and severe malaria. There was a 60% increase on correct chloroquine dosage in all age groups among carers living in post-intervention wards. CONCLUSION: Compliance with standard therapeutic doses and correct identification of malaria was poorest in control wards, where no motivators and vendors were trained.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats

Portal hypertension is a major complication of human cirrhosis that frequently leads to central nervous system dysfunction. In our study, rats with prehepatic portal hypertension developed hippocampal mitochondrial dysfunction as indicated by decreased respiratory rates, respiratory control and mitochondrial nitric oxide synthase (mtNOS) activity in mitochondria isolated from the whole hippocampus. Succinate-dependent respiratory rates decreased by 29% in controlled state 4 and by 42% in active state 3, and respiratory control diminished by 20%. Portal hypertensive rats showed a decreased mtNOS activity of 46%. Hippocampal mitochondrial dysfunction was associated with ultrastructural damage in the mitochondria of hippocampal astrocytes and endothelial cells. Swollen mitochondria, loss of cristae and rupture of outer and inner membrane was observed in astrocytes and endothelial cells of the blood-brain barrier in parallel with the ammonia gradient. It is concluded that the moderate increase in plasma ammonia that followed portal hypertension was the potential primary cause of the observed alterations.