Subcellular heterogeneity of mitochondrial membrane potential in anther tapetum

Studies on animal material have revealed that changes in the mitochondrial permeability transition pore (PTP), which cause a reduction in the mitochondrial transmembrane potential (_m) followed by release of cytochrome c, belong to the earliest manifestations of some types of apoptosis. We have attempted to monitor the _m of mitochondria during programmed cell death (PCD) of the secretory tapetum using JC-1, a fluorochrome dye that detects mitochondrial membrane potential and to relate changes in this potential to mitochondrial ultrastructure. Analysis of tapetal cells isolated from Ornithogalum virens anthers revealed that the _m of mitochondria in the tapetal cells alters during development; the change, however, is not uniform in the mitochondrial population within a single tapetal cell. In young tapetal cells, at the tetrad stage, we detected only the red fluorescence of JC-1 aggregates in all tapetal mitochondria, which indicates highly negative _m. In an advanced stage of PCD at the late microspore stage, in each tapetal cell we detected both mitochondria with red (as formerly) and mitochondria with green fluorescence. The green fluorescence of JC-1 monomers indicates mitochondria with depolarised membranes. These changes in _m are related to observed changes in mitochondria ultrastructure. This is the first documentation of intracellular heterogeneity of _m during anther tapetum development. Alteration in _m suggests a relationship between mitochondrial function and PCD processes in tapetal cells.     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Protonmotive force in Brevibacterium flavum: the lack of intracellular pH homeostasis

Brevibacterium flavum 22LD-P cells were shown to maintain a transmembrane pH gradient (pH) from 0.6 to 1.8–2 units and a transmembrane electric potential difference () from 0 to 200 mV depending on the pH and ionic composition of the incubation medium, grwoth substrate and concentration of cells. decreased from 120–140 mV to 0 when medium pH was lowered from neutral to 5.0–5.5 and increased to 180–200 mV when medium pH was raised to 8–9 in cells utilizing acetate or endogenous substrate. Cells growing on sucrose, kept around 100–120 mV at neutral as well as acidic medium pH. Intracellular pH in the acetate utilizing or endogenously respiring cells was maintained with the range of 8.9 to 5.5 at medium pH ranging from 9.1 to 4.0, respectively. Sucrose grown cells were able to maintain a more stable intracellular pH. Endogenously respiring cells in potassium phosphate buffer at high biomass concentrations maintained larger pH and relatively smaller , than the same cells in diluted suspensions. Cells in sodium phosphate buffer possessed larger and almost no pH, but was still dependent on biomass concentration.The lack of intracellular pH homeostasis and the collapse of at acid medium pH are discussed in the context of cell membrane proton permeability.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Modulation of a plant mitochondrial K+ATP channel and its involvement in cytochrome c release

Pea stem mitochondria, resuspended in a KCl medium (de-energized mitochondria), underwent a swelling, as a consequence of K⁺ entry, that was inhibited by ATP. This inhibition was partially restored by GTP and diazoxide (K⁺ _ATP channel openers). In addition, glyburide and 5-hydroxydecanate (K⁺ _ATP channel blockers) induced an inhibition of the GTP-stimulated swelling. Mitochondrial swelling was inhibited by H₂O₂, but stimulated by NO. The same type of responses was also obtained in succinate-energized mitochondria. When the succinate-dependent transmembrane electrical potential () had reached a steady state, the addition of KCl induced a dissipation that was inhibited by H₂O₂ and stimulated by NO. The latter stimulation was prevented by carboxy-PTIO, a NO scavenger. Phenylarsine oxide (a thiol oxidant) and NEM (a thiol blocker) stimulated the KCl-induced dissipation of , while DTE prevented this effect in both cases. In addition, DTE transiently inhibited the NO-induced dissipation of , but then it caused a more rapid collapse. These results, therefore, show that the plant mitochondrial K⁺ _ATP channel resembles that present in mammalian mitochondria and that it appears to be modulated by dithiol–disulfide interconversion, NO and H₂O₂. The aperture of this channel was linked to the partial rupture of the outer membrane. The latter effect led to a release of cytochrome c, thus suggesting that this release may be involved in the manifestation of programmed cell death.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 g dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (_m) and the plasma membrane (_p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K⁺:Na⁺:Ca²⁺ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.Abbreviations RP-HPLC Reversed-phase high-performance liquid chromatography - BLM Black lipid membrane - DAD Diode-array detector - _m Mitochondrial membrane potential - _p Plasma membrane potential - JC-1 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenz-imidazolo carbocyanine iodide - AM Calcein acetoxymethyl ester - PI Propidium iodide - MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - ESI-IT-MS Electrospray ionization ion trap mass spectrometry - EC₅₀ Endpoint concentration which caused 50% change in the viability parameters - FCCP Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone     

Transmembrane signal defect and absence of cancer extract induced leukocyte adherence inhibition (LAI) for leukocytes from patients with advanced cancer

Summary Leukocytes from patients with early cancer exhibit leukocyte adherence inhibition (LAI) when incubated with extracts of cancer of the same organ and histogenesis, whereas leukocytes from patients with advanced cancer seldom do. To understand the reason for this refractory state, tumor antigen-induced LAI and transmembrane signalling were measured in the same leukocytes. Transmembrane signalling was measured by changes in membrane potential () by the [³H]tetraphenylphosphonium equilibration technique. When leukocytes from patients with early breast cancer were incubated with extracts of breast cancer and malignant melanoma they showed changes consisting of depolarization and hyperpolarization beginning within 0.5 min after addition of the breast cancer extract and finishing 15 min later. Moreover, they showed no changes when incubated with extracts of normal breast tissue. Leukocytes from subjects without cancer seldom showed changes. In criss-cross experiments, leukocytes from patients with melanoma only exhibited changes when incubated with the melanoma extract. There was a strong correlation between cancer extract-induced change and LAI. The change was triggered by leukotriene-like mediators from antibody-dependent monocytes. Authentic leukotrienes triggered changes in all subpopulation of leukocytes. Leukocytes from patients with advanced breast cancer when incubated with breast cancer extract did not transmit a signal or show LAI. Brief elevation of intracellular cyclic AMP restored both change and LAI induced by breast cancer extracts, indicating that reactive leukocytes are present but in a refractory state. We conclude that leukocytes from patients with advanced cancer do not react in LAI because tumor antigen does not trigger a transmembrane signal to initiate the cascade of biochemical reactions and physiological changes for LAI.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - cyclic AMP cyclic adenosine monophosphate - ETYA eicosatetraynoic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethansulfonate - LAI leukocyte adherence inhibition - NAI nonadherence index - OSN organ-specific cancer neoantigen - PBL peripheral blood leukocytes - PGE₂ prostaglandin E₂ - [³H]TPP⁺ [phenyl³H]tetraphenylphosphonium bromide - transmembrane potential     

Plant Uncoupling Mitochondrial Protein Activity in Mitochondria Isolated from Tomatoes at Different Stages of Ripening

In the present study we have observed a higher state of coupling in respiring mitochondriaisolated from green as compared to red tomatoes (Lycopersicon esculentum, Mill.). Greentomato mitochondria produced a membrane potential () high enough to phosphorylate ADP,whereas in red tomato mitochondria, BSA and ATP were required to restore to the levelof that obtained with green tomato mitochondria. This supports the notion that such uncouplingin red tomato mitochondria is mediated by a plant uncoupling mitochondrial protein (PUMP;cf. Vercesi et al., 1995). Nevertheless, mitochondria from both green and red tomatoes exhibitedan ATP-sensitive linoleic acid (LA)-induced decrease providing evidence that PUMP isalso present in green tomatoes. Indeed, proteoliposomes containing reconstituted green or redtomato PUMP showed LA uniport and LA-induced H⁺ transport. It is suggested that the higherconcentration of free fatty acids (PUMP substrates) in red tomatoes could explain the lowercoupling state in mitochondria isolated from these fruits.     

Phloem turgor and the regulation of sucrose loading in Ricinus communis L.

The influence of plant water relations on phloem loading was studied in Ricinus communis L. Phloem transport was maintained in response to bark incisions even at severe water deficits. Water stress was associated with a net increase in the solute content of the sieve tubes, which resulted in maintenance of a positive phloem turgor pressure _p. There was a significant increase in solute flux through the phloem with decreasing xylem water potential (). In addition, sugar uptake by leaf discs was examined in media adjusted to different water potentials with either sorbitol (a relatively impermeant solute) or ethylene glycol (a relatively permeant solute). The limitations in this experimental system are discussed. The results nevertheless indicated that sucrose uptake can be stimulated by a reduction in cell _p, but that it is little affected by cell or solute potential _s. On the basis of these data we suggest that sucrose loading is turgor-pressure dependent. This may provide the mechanism by which transport responds to changes in sink demand in the whole plant.Abbreviations water potential - _s solute potential - _p pressure potential     

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport

An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg²⁺-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (pH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess of mannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent K _m, of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K⁺, which indicated an electrogenic sucrose-H⁺ symport. Membrane potentials () were imposed across the vesicle membrane using valinomycin and K⁺. A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a , positive inside, inhibited uptake. Conditions that produce a negative in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H⁺ symport as the mechanism of sucrose transport across the PM in Beta leaves.Abbreviations and symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - cyt cytochrome - PM plasma-membrane(s) - electrical potential difference     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.     

An investigation of the role of solutes in the xylem sap and in the xylem parenchyma as the source of root pressure

Summary Solute osmotic potentials (_x) in the vessels of hydroponically grown maize roots were measured to assess the osmotic-xylem-sap mechanism for generating root pressure (indicated by guttation). Solutes in vessels were measured in situ by X-ray microanalysis of plants frozen intact while guttating. Osmotic potentials outside the roots (_o) were changed by adding polyethylene glycol to the nutrient solution. Guttation rate fell when _o was decreased, but recovered towards the control value during 3–5 days when _o was greater than or equal to –0.3 MPa, but not when _o was equal to –0.4 MPa. In roots stressed to _o = –0.3 MPa, _x, was always more positive than _o, and _x changed only slightly (ca. 0.05 MPa). Thus the adjustment in the roots which increased root pressure cannot be ascribed to _x, contradicting the osmotic-xylem-sap mechanism. An alternative driving force was sought in the osmotic potentials of the vacuoles of the living cells (_v), which were analysed by microanalysis and estimated by plasmolysis. _v showed larger responses to osmotic stress (0.1 MPa). Some plants were pretreated with abundant KNO₃ in the nutrient solution. These plants showed very large adjustments in _v (0.4 MPa) but little change in _x (0.08 MPa). They guttated by 4 h after _o was lowered to –0.4 MPa. It is argued that turgor pressure of the living cells is a likely alternative source of root pressure. Published evidence for high solute concentrations in the xylem sap is critically assessed.Abbreviations _o external water potential - _x osmotic potential of xylem sap - _v osmotic potential of vacuolar sap - EDX energy dispersive X-ray microanalysis - CSEM cryo-scanning electron microscope - LN₂ liquid nitrogen - PEG polyethylene glycol     

Coupling Chemical Energy by the hsp70/tim44 Complex to Drive Protein Translocation into Mitochondria

A dynamic complex between the mitochondrial cognate of hsp70 (mthsp70) and the inner membrane protein tim44 couples energy derived from ATP hydrolysis to drive multiple steps in the mitochondrial protein import pathway: (1) The dependent import step and the mthsp70/tim44 complex cooperate to facilitate the unidirectional transfer of the mitochondrial targeting signal across the inner membrane. (2) The mthsp70/tim44 complex helps to unfold domains on precursors proteins that arrive at the import apparatus in a folded conformation on the cis side of the outer membrane. (3) Completion of import is then driven by the mthsp70/tim44 complex in a manner that is independent of . Mechanisms proposed to explain how the mthsp70/tim44 complex harvests chemical energy to drive these aspects of the import process are discussed.     

Asbestos-induced alveolar epithelial cell apoptosis: Role of mitochondrial dysfunction caused by iron-derived free radicals

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-induced AEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (_m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces _m after a 4 h exposure period. Further, the _m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell _m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell _m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.