The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO₂) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag⁺-induced ΔQO₂ was associated with H₂O₂ production and both effects depended on the presence of external Ca²⁺; (b) Ca²⁺ influx was markedly increased by treatment with NEM; (c) the Ca²⁺ channel blocker LaCl₃ inhibited ΔQO₂, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl₃ also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H₂O₂. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca²⁺ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.     

Root Effect Haemoglobins in Fish May Greatly Enhance General Oxygen Delivery Relative to Other Vertebrates

The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O₂) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O₂ affinity (Bohr effect) and carrying capacity (Root effect). This, combined with a large arterial-venous pH change (ΔpH_a-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO₂ tensions for rainbow trout and determined that, when Hb-O₂ saturation is 50% or greater, the change in oxygen partial pressure (ΔPO₂) associated with ΔpH_a-v can exceed that of the mammalian Bohr effect by at least 3-fold, but as much as 21-fold. Using known ΔpH_a-v and assuming a constant arterial-venous PO₂ difference (P_a-vO₂), Root effect Hbs can enhance O₂ release to the tissues by 73.5% in trout; whereas, the Bohr effect alone is responsible for enhancing O₂ release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpH_a-v, and thus enhancement of O₂ delivery, could be even larger. Modeling with known P_a-vO₂ in fish during exercise and hypoxia indicates that O₂ release from the Hb and therefore potentially tissue O₂ delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.     

Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag⁺, HgCl₂, etc.) can induce a marked, transitory stimulation of O₂ uptake (QO₂) in Egeria densa leaves, insensitive to CN⁻ and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O₂ consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H⁺-ATPase. In this investigation we compared the FC-induced increase in O₂ consumption with those induced by NEM and Ag⁺, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO₂. The results show (a) the different nature of the FC- and NEM- or Ag⁺-induced increases of QO₂; (b) that FC counteracts the NEM- (and Ag⁺)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN⁻-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.     

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.     

Studies of Root Function in Zea mays: III. Xylem Sap Composition at Maximum Root Pressure Provides Evidence of Active Transport into the Xylem and a Measurement of the Reflection Coefficient of the Root

The cut ends of excised Zea mays roots were sealed to a pressure transducer and their root pressures recorded. These rose approximately hyperbolically to a maximum value of 4.21 ± 0.34 bar after 30 to 40 minutes. Xylem exudate could not be collected at this pressure since the flow rate was zero. Samples of exudate were collected at lower applied pressures (ΔP), however, and Δπ, the osmotic pressure difference between them and the solution bathing the root, was measured by freezing point depression. A plot of ΔP/Δπ against J_v/Δπ, where J_v is the volume flux, proved to be a straight line whose intercept, equal to σ, the reflection coefficient, was 0.853 ± 0.016. The maximum xylem concentrations of various chemical species were found by a similar extrapolative method and compared with those in the cell sap. This indicated that (a) Ca²⁺, Mg²⁺, NO₃²⁻, SO₄²⁻, and most amino acids move from the cells to the xylem down an electrochemical potential gradient; (b) relative to these ions H⁺, NH₄⁺, glutamine and asparagine are actively transported into the xylem; and (c) H₂PO₄⁻, and K⁺ are actively retained in the symplasm.     

Influence of Transepithelial Potential Difference on the Sodium Uptake at the Outer Surface of the Isolated Frog Skin

The unidirectional uptake of sodium across the outer surface of the isolated frog skin (J₁₂^Na) was measured in the presence of transepithelial potential difference (Δ_ψ) ranging from +100 to -100 mV. With a sodium concentration of 115 mM in the bathing solutions J₁₂^Na increases significantly when the spontaneous Δ_ψ is reduced to zero by short-circuiting the skin. With an Na concentration of 6 mM a progressive increase J₁₂^Na can be observed when Δ_ψ is decreased in several steps from +100 to -100 mV (serosal side positive and negative, respectively). The observed change J₁₂^Na amounts to a fraction only of that predicted from the shift in Δ_ψ. The results suggest that under open circuit conditions the potential step across the outside surface is at most one half of Δ_ψ and that the resistance across the outside and inside barrier of the skin is ohmic. This is in agreement with measurements of intracellular potentials in the frog skin and with resistance measurements carried out in the toad skin. The data strongly support the view that the saturating component of J_ψ proceeds via a charged carrier system. Exposure to negative values of Δ_ψ of 50 mV or more for times of 24 min or more result in a marked reduction of J₁₂^Na which shows only partial or no reversibility.     

Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants

The absence of Tsa1, a key peroxiredoxin that scavenges H₂O₂ in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can^R mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can^R mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.     

Silver ion (Ag+)-Induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: Reversal by thiol reagents

Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag⁺) on K⁺ transport and respiration with extracellular K⁺ and O₂ electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO₃) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K⁺ efflux (K _m =10^–4 m,V _max=379 nmol K⁺/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO₂) (13.9±1.1 to 25.7±4.4 nmol O₂/min/mg,P<0.001), was observed 19 sec after the K⁺ efflux induced by AgNO₃ (10^–4 m), suggesting a delayed increase in Na⁺ entry into the cell. Ouabain-insensitive QO₂, nystatin-stimulated QO₂, and CCCP-uncoupled QO₂ were not significantly affected, indicating preserved function of the Na⁺, K⁺-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K⁺ transport and QO₂. We conclude that Ag⁺ causes early changes in the permeability of the cell membrane to K⁺ and then to Na⁺ at concentrations that do not limit Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag⁺ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.     

Biosurfactant Production and Surface Translocation Are Regulated by PlcR in Bacillus cereus ATCC 14579 under Low-Nutrient Conditions

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 ΔplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K₂HPO₄, 3.0 g KH₂PO₄, 0.1 g MgSO₄·7H₂O, 0.1 g (NH₄)₂SO₄, 0.01 g CaCl₂, 0.001 g FeSO₄, 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the ΔplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and ΔplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the ΔplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the ΔplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.     

Iodoacetate inhibits the biosynthesis of alanine in glial cells and its utilization in photoreceptors of the honeybee drone (Apis mellifera) retina

The aim of this work was to study the effects of iodoacetate on the metabolism of the honeybee drone retina. In the superfused retina, iodoacetate only at high concentration (3 mmol·1^-1) causes a 77% decrease in the O₂ consumption induced by a flash of light. Chromatographic analysis showed that 3 mmol·1^-1 iodoacetate strongly inhibited glycolysis in the retinal glial cells and consequently suppressed the biosynthesis of alanine, which is the fuel transferred from the glia to the photoreceptors. However, the synthesis of ¹⁴C-alanine from [1-¹⁴C]-pyruvate was not affected by iodoacetate. It was therefore surprising to find that superfusion of the retina with 10 mmol·1^-1 pyruvate had no protective effect on the decrease in O₂ consumption, and that the ¹⁴CO₂ production from [1-¹⁴C]-pyruvate was inhibited 60% by iodoacetate. Also, no protection from the effect of iodoacetate was obtained by adding 10 and 20 mmol·1^-1 alanine in the superfusate, even though the transport of ¹⁴C-alanine in the photoreceptor cells was not significantly affected by 3 mmol·1^-1 iodoacetate. However, exposure to iodoacetate strongly inhibited the production of ¹⁴C-glutamate from ¹⁴C-alanine. In contrast, the transformation of ¹⁴C-proline to ¹⁴C-glutamate was not affected by iodoacetate. Indeed, in the presence of iodoacetate, photostimulation caused a decrease in the total concentration of proline and glutamate. It appears therefore that 3 mmol·1^-1 iodoacetate inhibits not only glycolysis and, consecutively, the formation of alanine, but also its use in the photoreceptors. Possibly a large intracellular store of proline, whose mitochondrial use was not affected, contributed in slowing down the inhibition of O₂-consumption by iodoacetate.Abbreviations DNP dinitrophenol - HPLC high pressure liquid chromatography - IAA iodoacetate - QO ₂ change in oxygen consumption - QO ₂ oxygen consumption - PO ₂ partial pressure of O₂     

Hydrogen Photoproduction by Immobilized N2-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process

We have investigated two approaches to enhance and extend H₂ photoproduction yields in heterocystous, N₂-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO₂ supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H₂ production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N₂ by periodic treatments of air, or air plus CO₂. These treatments restored the photosynthetic activity of the cells and led to a high level of H₂ production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO₂) but not in the Anabaena PCC 7120 strain (for which H₂ yields did not change after air treatments). The highest H₂ yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO₂ under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O₂ from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.     

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K⁺ Channel (PmitoK_ATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K⁺ uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoK_ATP and ⁺/H⁺ antiporter allows such a great operation of a K⁺ cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoK_ATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics. [BMB Reports 2013; 46(8): 391-397]     

Glucose-dependent respiration in suspensions of rabbit cortical tubules

Summary The effects of glucose on cellular respiration were examined in suspensions of rabbit cortical tubules. When glucose was removed from the bathing fluid, oxygen consumption (QO₂) decreased from 18.6±0.8 to 15.7±0.5 nmol O₂/mg protein·min (P<0.01). The transported but nonmetabolized analogue of glucose, -methyl-d-glucoside (MG), was found to support QO₂ to the same extent as glucose. These observations were also evident in the presence of butyrate, a readily oxidized substrate of the renal cortex. Additional studies with nystatin and ouabain indicated that glucose-related changes in QO₂ were the result of changes in Na, K-ATPase associated respiration. The effect of glucose was localized to the luminal membrane since phlorizin (10^–5 m), a specific inhibitor of liminalk glucose-sodium cotransport, also significantly reduced QO₂ by 10±1%. Phlorizin inhibition of QO₂ was also evident in the presence of MG but was abolished when glucose was removed from the bathing medium. Finally, measurement of NADH fluorescence showed that addition of glucose (5mm) to a tubule suspension causes an oxidation of NAD. These data are all consistent with glucose acting to increase respiration by stimulating sodium entry at the luminal membrane (via glucose-sodium cotransport) followed by increased sodium pump activity and its associated increase in mitochondrial respiration.     

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Ca_v1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Ca_v1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Ca_v1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca²⁺ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca²⁺ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca²⁺ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.     

Bacillus subtilis polynucleotide phosphorylase 3′-to-5′ DNase activity is involved in DNA repair

In the presence of Mn²⁺, an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3′ → 5′ polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn²⁺ and low-level inorganic phosphate (P_i), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg²⁺ or high-level P_i. In contrast, the RNase activity of PNPase requires Mg²⁺ and P_i, suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (ΔpnpA) is not epistatic with ΔrecA, but is epistatic with ΔrecN and Δku, which by themselves are non-epistatic. The addA5, ΔrecO, ΔrecQ (ΔrecJ), ΔrecU and ΔrecG mutations (representative of different epistatic groups), in the context of ΔpnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.     

Transmembrane Proton Electrochemical Gradients in Dark Aerobic and Anaerobic Cells of the Cyanobacterium (Blue-Green Alga) Anacystis nidulans: Evidence for Respiratory Energy Transduction in the Plasma Membrane

The transmembrane proton electrochemical potential gradient Δμ_H+ in whole cells of Anacystis nidulans was measured in aerobic and anaerobic dark conditions using the distribution, between external medium and cell interior, of radioactively labeled weak acids (acetylsalicyclic acid, 5,5-dimethyloxazolidine-2,4-dione) or bases (imidazole, methylamine), and permeant ions (tetraphenylphosphonium cation, thiocyanate anion), as determined by flow dialysis. Alternatively, the movements across the plasma membrane of ΔpH-indicating atebrin or 9-aminoacridine, and of ΔΨ-indicating 8-anilino-l-naphthalenesulfonate were qualitatively followed by fluorescence measurements. Attempts were made to discriminate between the individual chemiosmotic gradients across the cytoplasmic (plasmalemma) and the intracytoplasmic (thylakoid) membranes. By use of the ionophores nigericin, monensin, and valinomycin, the components of the proton motive force, namely the proton concentration gradient ΔpH and the electric membrane potential ΔΨ were shown to be mutually exchangeable within the range of external pH values tested (3.2-11.0). Both components were depressed by the uncoupler carbonylcyanide m-chlorophenylhydrazone, though inhibition of ΔpH was much more pronounced than that of ΔΨ, notably in the alkaline pH₀ range. The total proton electrochemical gradient across the plasma membrane was significantly higher in aerobic than in anaerobic cells and increased markedly (i.e. became more negative) towards lower pH₀ values. This increase was paralleled by a similar increase in the rate of endogenous respiration of the cells. At the same time the ATPase inhibitor dicyclohexylcarbodiimide only slightly affected the proton motive force across the plasma membrane of aerobic cells. The results will be discussed in terms of a respiratorily competent plasma membrane in Anacystis nidulans.     

Identification of C18:1-Phytoceramide as the Candidate Lipid Mediator for Hydroxyurea Resistance in Yeast

Recent studies showed that deletion of ISC1, the yeast homologue of the mammalian neutral sphingomyelinase, resulted in an increased sensitivity to hydroxyurea (HU). This raised an intriguing question as to whether sphingolipids are involved in pathways initiated by HU. In this study, we show that HU treatment led to a significant increase in Isc1 activity. Analysis of sphingolipid deletion mutants and pharmacological analysis pointed to a role for ceramide in mediating HU resistance. Lipid analysis revealed that HU induced increases in phytoceramides in WT cells but not in isc1Δ cells. To probe functions of specific ceramides, we developed an approach to supplement the medium with fatty acids. Oleate (C_18:1) was the only fatty acid protecting isc1Δ cells from HU toxicity in a ceramide-dependent manner. Because phytoceramide activates protein phosphatases in yeast, we evaluated the role of CDC55, the regulatory subunit of ceramide-activated protein phosphatase PP2A. Overexpression of CDC55 overcame the sensitivity to HU in isc1Δ cells. However, addition of oleate did not protect the isc1Δ,cdc55Δ double mutant from HU toxicity. These results demonstrate that HU launches a lipid pathway mediated by a specific sphingolipid, C_18:1-phytoceramide, produced by Isc1, which provides protection from HU by modulating Swe1 levels through the PP2A subunit Cdc55.     

The role of the diffusion of oxygen in the respiration of rat diaphragm at different temperatures

Summary 1. Data are presented to support the hypothesis that the respiration rate of hemidiaphragms and tissue slices is restrictedin vitro because oxygen fails to reach the innermost layers of the tissue.2. Calculation of limiting thickness fromWarburg's formula (Equation 1) requires use of a value for QO₂, but the true value is unknown since it is in turn dependent upon thickness. The dilemma is not avoided by making thinner slices because this damages tissues and reduces the QO₂.3. We found that for rat diaphragm a plot of log QO₂ versus 1/T yields a straight line between -6° and 13.5° C, and a line of half that slope from 18° to 38° C.4. Equations are presented for calculating QO₂ at these temperatures, assuming that oxygenation is incomplete above about 18° C. The calculated values agree well with the observed values. Further, QO₂ of diaphragm at 33° C was higher at an oxygen pressure of 2 atmospheres than 1 atmosphere in agreement with theoretical considerations.

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Die Bedeutung der Sauerstoffdiffusion für die Atmung des Rattendiaphragmas bei verschiedenen Temperaturen

Kurzfassung Es werden Ergebnisse vorgelegt, welche die Hypothese untermauern, daß die Respirationsrate von Hemidiaphragmen und Gewebescheiben in vitro eingeschränkt ist, weil der Sauerstoff die innersten Gewebeschichten nicht zu erreichen vermag. Die Errechnung der kritischen Gewebedicke nachWarburgs Formel erfordert die Einsetzung des Wertes für QO₂, der ist aber unbekannt, da er ja selbst wiederum von der Dicke abhängt. Man kann diesem Dilemma nicht dadurch ausweichen, daß man dünnere Scheiben verwendet; denn das würde die Gewebe beschädigen und so den QO₂ herabsetzen. Wir haben festgestellt, daß die Auftragung von log QO₂ gegen 1/T für das Rattendiaphragma eine gerade Linie ergibt zwischen -6° und 13,5° C und eine Linie mit dem halben Anstiegswinkel zwischen 18° und 38° C. Unter der Annahme, daß das Sauerstoffangebot über etwa 18° C unzureichend wird, werden Gleichungen gegeben für die Errechnung des QO₂ bei den oben genannten Temperaturen. Die errechneten Daten stimmen mit den beobachteten Werten gut überein. Bei 33° C war der QO₂-Wert höher bei einem Sauerstoffdruck von 2 Atmosphären als bei einem solchen von 1 Atmosphäre; dieser Sachverhalt steht in Übereinstimmung mit den theoretischen Erörterungen.

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This work was supported in part by a grant from the National Science Foundation.     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.     

Isolation and separation of toad bladder epithelial cells

Summary The epithelium of the urinary bladder ofBufo marinus is composed of 5 cell types, i.e., granular (Gr), mitochondria-rich (MR) and goblet (G) cells which face the urinary lumen, microfilament-rich (MFR) and undifferentiated cells (Un) located basally. The epithelium was dissociated by collagenase and EGTA treatment. Fractionation of dispersed cells by isopycnic centrifugation on dense serum albumin solutions yielded 4 fractions: (i) a very light fraction ( ) enriched in MR and MFR cells; (ii) a light fraction ( ) enriched in vacuolated Gr cells; (iii) a heavy fraction ( ) composed essentially of aggregated Gr cells, and (iv) a pellet ( ) enriched in G and undifferentiated cells. Recoveries were based on cell counts and DNA measurements. DNA content per cell was 13.2 pg±0.9 (n=37). From 1 g fresh tissue, 62±5×10⁶ (n=10) cells were recovered before isopycnic centrifugation of which about 70% excluded Trypan blue. After centrifugation, 90 to 95% of the cells excluded the vital dye and 3⁹×10⁶ cells were recovered from the gradient. Cell metabolism in each fraction was estimated by oxygen consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption was threefold higher in the very light and light fractions when compared to the heavy and pellet fractions. Ouabain sensitive oxygen consumption (QO₂) represented 12 to 35% of the total O₂ consumption depending on the cell fraction, and acetazolamide sensitive QO₂ varied from –0.8% in the heavy fractions to 20% in the lighter fractions. DNP increased QO₂ in all fractions by 20 to 50%. Finally, the cells were able to reaggregate and form junctional complexes upon addition of calcium to the medium.