Synchronization of the function of respiratory chain enzymes and ATP-synthetase in energized mitochondria

The present study revealed that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of the respiratory chain functioning could be observed under different absolute values of delta phi on the mitochondrial membrane. This points out that the membrane potential is not a unique regulator in the coupling of the ATP-synthetase and respiratory chain activities. At the same time, we succeeded in obtaining some evidence testifying that under conditions of ATP-synthetase inhibition the amount of functioning respiratory chains has to be proportional the functioning of the ATP-synthetases units. The osmolarity of the incubation medium was shown to control the state of the oxidative phosphorylation system. The respiratory chain and ATP-synthetase should be considered as an enzymatic supercomplex only when the osmolarity is close to 150-300 mOsm (within the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. It is concluded that the respiratory chain and ATP-synthetase are tightly bound from the kinetic point of view. The ATP-synthetase inhibition induces proportional inhibition of the respiratory chain enzymes and vice versa, the respiratory chain inhibition induces proportional inhibition of ATP-synthetase.     

Growth and buoyant density of Escherichia coli at very low osmolarities.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM. With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.     

Heterogeneous transmural proteoglycan distribution provides a mechanism for regulating residual stresses in the aorta

The arterial wall contains a significant amount of charged proteoglycans, which are inhomogeneously distributed, with the greatest concentrations in the intimal and medial layers. The hypothesis of this study is that the transmural distribution of proteoglycans plays a significant role in regulating residual stresses in the arterial wall. This hypothesis was first tested theoretically, using the framework of mixture theory for charged hydrated tissues, and then verified experimentally by measuring the opening angle of rat aorta in NaCl solutions of various ionic strengths. A three-dimensional finite element model of aortic ring, using realistic values of the solid matrix shear modulus and proteoglycan fixed-charge density, yielded opening angles and changes with osmolarity comparable to values reported in the literature. Experimentally, the mean opening angle in isotonic saline (300 mosM) was 15 +/- 17 degrees and changed to 4 +/- 19 degrees and 73 +/- 18 degrees under hypertonic (2,000 mosM) and hypotonic (0 mosM) conditions, respectively (n = 16). In addition, the opening angle in isotonic (300 mosM) sucrose, an uncharged molecule, was 60 +/- 16 degrees (n = 11), suggesting that the charge effect, not cellular swelling, was the major underlying mechanism for these observations. The extent of changes in opening angle under osmotic challenges suggests that transmural heterogeneity of fixed-charge density plays a crucial role in governing the zero-stress configuration of the aorta. A significant implication of this finding is that arterial wall remodeling in response to altered wall stresses may occur via altered deposition of proteoglycans across the wall thickness, providing a novel mechanism for regulating mechanical homeostasis in vascular tissue.     

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.     

Effect of dehydrocorrine-cobalt complex on mitochondria

The effects of the decamethyloctadehydrocorrine-cobalt complex (Co-C) on respiration and the ATP-synthetase activity of rat liver mitochondria were investigated. The Co-C complex was found to be an effective shunt of the respiratory chain. It accepts electrons from ubiquinone and donates them directly to O2. The Co-C complex inhibits the ATPase and ATP-synthetase activities of mitochondria.     

Metabolite transport in mitochondria as a function of osmolarity

The effect of increasing sucrose concentrations on some mitochondrial functions was studied. The results showed that high osmolarity inhibits oxidative phosphorylation as well as ATPase activity and ATP-dependent delta phi formation as a consequence of adenine nucleotide translocase inhibition. It is also shown that high osmolarity does not affect delta phi formation and energy-dependent Ca2+ uptake as driven by succinate oxidation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of membrane proteins showed a different reactivity to o-phenantroline/Cu2+ as function of osmolarity. It is proposed that high sucrose concentrations induce a collapse of the matrix compartment that results in a restricted diffusion of some metabolites.     

The elasticity of uniform, unilamellar vesicles of acidic phospholipids during osmotic swelling is dominated by the ionic strength of the media

Uniform, unilamellar vesicles have been prepared by the pH-modification technique. The initial sizes of the vesicles were from 200 to 700 nm and were measured to within 1-3% by photo correlation spectroscopy. Vesicles were made of the dioleoyl esters of phosphatidic acid, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, the diphytanyl ethers of phosphatidylglycerol, Escherichia coli lipids, and lac permease reconstituted into E. coli lipids. The vesicle suspensions were prepared and then diluted with electrolyte (KCl) and/or nonelectrolyte (sucrose, trehalose, pentaerythritol) impermeants. The amplitude of the swelling is linearly proportional to the osmotic pressure difference across the bilayer. We have determined the elastic modulus, the elastic limit (percent surface expansion at bursting), and the transbilayer pressure difference at bursting for each of these vesicles at constant osmolarity but at different ionic strengths. We find that the elastic properties of the bilayer vary by a factor of 10 in electrolyte media as compared to isosmolal nonelectrolyte media and that this variation appears to be related to both the charge density at the surface and the ionic strength of the media. Anionic lipid vesicles in 150 mM KCl have a significantly higher modulus (50 X 10(7) dyn/cm2) and transbilayer pressure difference (40 mosM) at bursting with a small capacity to stretch (3-4% surface expansion) compared to the same vesicles suspended in nonelectrolyte impermeants. The latter vesicles undergo a significant surface expansion (8-10%), display a low modulus (3 X 10(7) dyn/cm2), and burst at 3-4 mosM bilayer pressure difference. Vesicles suspended in media of constant osmolarity at various ionic strengths display properties with proportional values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks (Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C. leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642 +/- 7 mosM) was significantly reduced compared to SW-captured animals (1067 +/- 21 mosM). In FW animals, sodium, chloride and urea were 208 +/- 3, 203 +/- 3 and 192 +/- 2 mmol l(-1), respectively. Plasma sodium, chloride and urea in SW-captured C. leucas were 289 +/- 3, 296 +/- 6 and 370 +/- 10 mmol l(-1), respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24 per thousand SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1 per thousand rise in salinity. Between 24 per thousand and 33 per thousand, plasma osmolarity increased by 33% or 4.7% per 1 per thousand rise in salinity, largely due to a sharp increase in plasma urea between 28 per thousand and 33 per thousand. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0-10 per thousand, 11-20 per thousand and 21-33 per thousand. A comparison between C. leucas captured in FW and estuarine environments (20-28 per thousand ) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C. leucas moving between FW and SW, as well as the ecological implications of these data are discussed.     

Energetics of swelling in isolated hepatocytes: A comprehensive study

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.     

Ion transport in rat liver mitochondria: the effect of the incubation medium osmolarity

A decrease in the incubation medium osmolarity from 320 to 120 mosM reverses the pH dependence of K+ efflux from rat liver mitochondria. The K+ efflux is no longer inhibited by oligomycin and a free radical scavenger butylhydroxytoluene. At 320 mosM, the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) accelerates the K+ efflux, while EGTA inhibits it. At 120 mosM these CCCP and EGTA effects are reversed. In either case the K+ efflux is inhibited by Mg2+. The decrease in osmolarity changes the ruthenium red-insensitive Ca2+ efflux in the same manner. It has thus been shown that the modification of the mitochondrial structure by changing the incubation medium osmolarity results in a qualitative alteration of the systems regulating the K+ and Ca2+ effluxes.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

Effects of changes in osmolarity on isolated human airways

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.     

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin,ERK, and p38 MAPK

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.     

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri.

In Shigella flexneri, the ompB locus (containing the ompR and envZ genes) was found to modulate expression of the vir genes, which are responsible for invasion of epithelial cells. vir gene expression was markedly enhanced under conditions of high osmolarity (300 mosM), similar to that encountered in tissues both extra- and intracellularly. Two ompB mutants were constructed and tested for virulence and for osmotic regulation of vir genes. An envZ::Tn10 mutant remained invasive, although its virulence was significantly decreased as a result of its inability to survive intracellularly. By using a vir::lac operon fusion, this mutation was shown to decrease beta-galactosidase expression both in low- and high-osmolarity conditions but did not affect vir expression in response to changes in osmolarity. A delta ompB deletion mutant was also constructed via allelic exchange with an in vitro-mutagenized ompB locus of Escherichia coli. This mutation severely impaired virulence and abolished expression of the vir::lac fusion in both low- and high-osmolarity conditions. Therefore, a two-component regulatory system modulates virulence according to environmental conditions. In addition, the mutation affecting a spontaneous avirulent variant of S. flexneri serotype 5, M90T, has been mapped at the ompB locus and was complemented by the cloned E. coli ompB locus. Introduction of the vir::lac fusion into this mutant did not result in the expression of beta-galactosidase (Lac-).     

Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells

Glutamate is an excitotoxin responsible for causing neuronal damage associated with mitochondria dysfunction. We have analyzed the relationship between the mitochondrial respiratory rate, the membrane potential (delta psi) and the activity of mitochondrial complexes in retinal cells in culture, used as neuronal models. Glutamate (10 microM-10 mM) dose-dependently decreased the O2 consumption and the membrane potential. A linear correlation was found between these parameters, suggesting that the mitochondrial respiratory function was affected. Exposure to glutamate (100 microM) for 10 min, in the absence of Mg2+, inhibited the activity of complex I (26.3%), complexes II/III (22.2%) and complex IV (26.7%). MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), a non-competitive antagonist of the NMDA (N-methyl-D-aspartate) receptors, completely reversed the effect exerted by 100 microM glutamate at the level of complexes I, II/III and IV. These results suggest that NMDA receptor-mediated inhibition of mitochondrial respiratory chain complexes may be responsible for the alteration in the respiratory rate of chick retinal cells submitted to glutamate.