Concentrations Of DNP Research Articles

Продукция активных форм кислорода при действии ультрафиолетового-В излучения на клетки дрожжей

The present investigation is devoted to the study of the effect of ultraviolet-B (UV-B) radiation on the survival and production of reactive oxygen species in yeast cells. It has been determined that under the action of UV-B radiation on cells, depending on its dose, the rate of oxidation of the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCF•DA) increases and a high intensity of DCF fluorescence is observed. When cells are modified with ascorbic acid before irradiation, the rate of H2DCF oxidation and the intensity of DCF fluorescence decreases. The survival rate of modified cells is increased. Ascorbic acid reduces the amount of ROS in the suspension of irradiated cells. At high doses (4.5-10 erg/mm2) of irradiation, the antioxidant effect of ascorbic acid in yeast cells is negligible. The concentration of 2-4 dinitrophenol (DNF) 10-7 M was determined as a protector, in which the substance potentially contributes to the manifestation of the effect of "soft uncoupling" in cells and, under the influence of UV-B radiation on yeast cells, helps to reduce the production of reactive oxygen species and an increase in survival. High concentrations (10–3M–10–5 M) of DNP adversely affect the survival of yeast cells and ROS production. So, it can be concluded that under the action of UV-B rays on yeast cells, antioxidants can possibly participate in the regulation of redox homeostasis.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 μM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e− ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.

Enhancing the Performance of Sequencing Batch Reactors by Adding Crushed Date Seeds to Remove High Concentrations of 2,4‐Dinitrophenol

Wastewater treatment systems using simultaneous adsorption and biodegradation processes have been successful in treating toxic pollutants present in industrial wastewater. The goal of this investigation was to assess the effectiveness of date seeds in reducing the toxic effects of 2,4-dinitrophenol (DNP) on activated sludge microorganisms. Two identical sequencing batch reactors (SBRs) (4-L glass vessel), each with a 3.5-L working volume, were used. The initial DNP concentrations in the reactor were 50, 75, 100, 250, and 500 mg/L. The reactor amended with date seeds was capable of degrading DNP at significantly greater rates (11 +/- 2.5 mg/L x h) than the control SBR (4 +/- 1.2 mg/L x h) at a 95% confidence level. Date seeds can be added to the mixed liquor of activated sludge treatment plants to remove high concentrations of DNP from wastewater, to protect the treatment plant against toxic components in the influent and enhance the settling characteristics of the mixed liquor.

Expression Profile of Rat Hippocampal Neurons Treated with the Neuroprotective Compound 2,4-Dinitrophenol: Up-Regulation of cAMP Signaling Genes

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

Dendroaspis Natriuretic Peptide Induces the Apoptosis of Cardiac Muscle Cells

Early heart failure is characterized by elevated plasma Dendroaspis natriuretic peptide-like immunoreactivity (DNP-LI). However, the direct effects of DNP on heart or the heart-associated cell system are not well known. Therefore, we investigated whether DNP induces the apoptosis of H9c2 cardiac muscle cells. H9c2 cardiac muscle cells and rat neonatal cardiomyocytes were treated with various concentrations of DNP. Cell viability and nuclear morphology change were determined by trypan blue staining and Hoechst 33258 staining, respectively. Caspase-3-like activity was measured using specific fluorogenic substrates. Pro-and antiapoptotic proteins were assayed by Western blotting. DNP induced the apoptosis of H9c2 cardiac muscle cells in a dose-dependent manner. Maximum effects occurred at 100 nM concentration of DNP, with a 7–8-fold increase in apoptotic cells, to reach a maximum apoptotic index of 17%. We also identified that H9c2 cardiac muscle cells expressed Natriuretic peptide reactor -A and -B, which respond to DNP to generate cGMP. The treatment with DNP also markedly reduced levels of Bcl-2, inhibitor of apoptosis protein-1, and inhibitor of apoptosis protein-2 and increased the level of Bax and cytochrome c release into cytoplasm and subsequent caspase-3 activation, which co-occurred with increased apoptosis. DNP-induced apoptosis was mediated by cyclic GMP, and this effect was mimicked by dibutylyl-cGMP (30 µM), a membrane permeable analog of cGMP. Furthermore, DNP-induced apoptosis was observed in rat neonatal cardiomyocytes. These results suggestthat DNP induces the apoptosis of H9c2 cardiac muscle cells and of cardiomyocytes via cGMP and demonstrate that the operative mechanism includes the regulation of Bcl-2 family proteins.

Dendroaspis Natriuretic Peptide Induces the Apoptosis of Cardiac Muscle Cells

Early heart failure is characterized by elevated plasma Dendroaspis natriuretic peptide-like immunoreactivity (DNP-LI). However, the direct effects of DNP on heart or the heart-associated cell system are not well known. Therefore, we investigated whether DNP induces the apoptosis of H9c2 cardiac muscle cells. H9c2 cardiac muscle cells and rat neonatal cardiomyocytes were treated with various concentrations of DNP. Cell viability and nuclear morphology change were determined by trypan blue staining and Hoechst 33258 staining, respectively. Caspase-3-like activity was measured using specific fluorogenic substrates. Pro-and antiapoptotic proteins were assayed by Western blotting. DNP induced the apoptosis of H9c2 cardiac muscle cells in a dose-dependent manner. Maximum effects occurred at 100 nM concentration of DNP, with a 7–8-fold increase in apoptotic cells, to reach a maximum apoptotic index of 17%. We also identified that H9c2 cardiac muscle cells expressed Natriuretic peptide reactor -A and -B, which respond to DNP to generate cGMP. The treatment with DNP also markedly reduced levels of Bcl-2, inhibitor of apoptosis protein-1, and inhibitor of apoptosis protein-2 and increased the level of Bax and cytochrome c release into cytoplasm and subsequent caspase-3 activation, which co-occurred with increased apoptosis. DNP-induced apoptosis was mediated by cyclic GMP, and this effect was mimicked by dibutylyl-cGMP (30 µM), a membrane permeable analog of cGMP. Furthermore, DNP-induced apoptosis was observed in rat neonatal cardiomyocytes. These results suggestthat DNP induces the apoptosis of H9c2 cardiac muscle cells and of cardiomyocytes via cGMP and demonstrate that the operative mechanism includes the regulation of Bcl-2 family proteins.

Substrate inhibition of degradation of 2,4-dinitrophenol in activated sludge

Substrate inhibition of 2,4-dinitrophenol (2,4-DNP) degradation was investigated in activated sludge previously adapted to mineralize low concentrations of DNP. Typically, the time required for 95% degradation of 10 mg/L DNP under aerobic conditions and with mixed liquor suspended solids (MLSS) of 600 to 1000 mg/L was less than 4 hours without any lag. However, when the initial DNP concentration increased to 75 mg/L, lags and even complete inhibition of DNP degradation were observed. The length of the lag was found to increase proportionally with decreasing MLSS. When dilute activated sludge was incubated for extended periods (192 hours), degradation of 75 mg/L DNP did eventually occur after lag periods of 37 to 144 hours, depending on MLSS concentration. Assays for most probable number of DNP-degrading bacteria were made to ascertain the effect of high DNP concentrations on bacterial populations in the activated sludge.

The nature of uncoupling by n-hexane, 1-hexanethiol and 1-hexanol in rat liver mitochondria

We have analyzed the effects of n-hexane, 1-hexanethiol, and 1-hexanol on the coupled respiration of rat liver mitochondria. Incubation of mitochondria with n-hexane, 1-hexanethiol and 1-hexanol resulted in a stimulation, at low concentrations, and an inhibition, at high concentrations, of the state 4 mitochondrial respiration. Three criteria, all based on the comparison with the effect of DNP, have been used to establish whether the stimulation of respiration, at low concentrations of n-hexane, 1-hexanethiol, and 1-hexanol, depends on protonophoric mechanisms. First, the quantitative relationship between the extents of respiratory stimulation and membrane potential depression: a strong decrease of membrane potential was induced by increasing concentrations of DNP and a negligible depression by increasing concentrations of n-hexane or 1-hexanethiol. Only a slight decrease was induced by 1-hexanol. Second, the quantitative relationship between the extents of respiratory stimulation and of proton conductance increase: at equivalent rates of respiration, the enhancement of the proton conductance induced by DNP was very marked, by n-hexane and 1-hexanethiol practically negligible, and by 1-hexanol much smaller than that induced by DNP. Third, in titrations with redox inhibitors of the proton pumps, the pattern of the relationship between proton pump conductance and membrane potential was markedly different for protonophoric and non-protonophoric uncouplers: almost linear in the case of DNP, highly non-linear in the case of n-hexane, 1-hexanethiol and 1-hexanol. These three criteria support the view that n-hexane, 1-hexanethiol, and partially 1-hexanol, uncouple mitochondrial respiration by a non-protonophoric mechanism.

PH-dependent effects of 2,4-dinitrophenol (DNP) on proliferation, endocytosis, fine structure and DNP resistance in Tetrahymena.

In view of the importance of external pH on cytotoxic effects of ionizable agents, the pH-dependent effects of 2,4-dinitrophenol (DNP) were investigated. As uncoupler of oxidative phosphorylation, DNP interferes with the proton gradient across the inner mitochondrial membrane. DNP was added to proliferating Tetrahymena pyriformis in media of different initial pH. Effects studied were rates of cell proliferation and endocytosis, and fine structure. Findings correlated with the calculated concentration of undissociated DNP, taking into account that pH changes with time and cell density in Tetrahymena cultures. A linear relationship thus emerged between initial concentrations of undissociated DNP and lengths of the lag preceding cell proliferation. Once resumed, the rate of proliferation corresponded to that of control cells, even in different concentrations of undissociated DNP, presumably indicating an adaptation mechanism. Endocytosis was elevated throughout a wide range of undissociated DNP concentrations with a sharp transition towards inhibition at high DNP concentrations causing lethality with time. Changes in fine structure of DNP-treated cells (mitochondria, peroxisomes, nucleoli) also depended on the concentration of undissociated DNP.

Relative Effect of 2,4-Dinitrophenol on the Respiratory Chain of Heterotrophic Euglena gracilis Adapted and Non-adapted to Antimycin A

A study of the action of DNP on the respiration of Euglena gracilis (Z Klebs strain) treated or untreated with antimycin shows that the generation time of cells increases proportionally to DNP concentration from 10-6 to 10-5 M, with a further increase beyond. The intensity of respiration also increases but reverts after 24 hours. Cyanide resistance, related to the concentration of DNP, appears only 24 hours after addition of the latter, indicating a block towards the end of the respiratory chain. The photoconsumption of oxygen, due to the action of UV on the ubiquinone complex, diminishes and becomes negligible at high concentrations of DNP, implying that DNP acts on ubiquinone.

A Ferro-Heme Protein Senses Oxygen Levels, Which Modulate the Glucagon-Dependent Activation of the Phosphoenolpyruvate Carboxykinase Gene in Rat Hepatocyte Cultures

Oxygen modulates the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene. The respiratory chain or heme proteins have been proposed to function as O 2-sensors. The functions of the respiratory chain are impaired by uncouplers such as 2,4-dinitrophenol (DNP); those of ferro-heme proteins are affected by carbon monoxide (CO), which locks heme in the oxy conformation. Therefore, the effects of different concentrations of CO and DNP on the glucagon-dependent induction of PCK mRNA and PCK activity were investigated at different physiological oxygen tensions in primary rat hepatocyte cultures. The cells were cultured under standard conditions from 4-24 h. After addition of fresh media PCK was induced with 1 nM glucagon. PCK mRNA and PCK activity were elevated after 2h and 3h, respectively, to 100% at 16% O 2 (mimicking arterial oxygen tensions) and to about 60% at 8% O 2 (mimicking venous oxygen tensions). CO counteracted the reduced induction at lower oxygen tensions: Under 8% O 2 + 2% CO PCK mRNA could be elevated again to about 90% and PCK activity to about 80%. CO did not impair the induction by insulin of omithine decarboxylase (ODC) and the incorporation of 14C-leucine into total protein. CO did not cause lactate dehydrogenase (LDH) to leak from the cells or influence the cell structures at the microscopical level. DNP (50 μM) unspecifically lowered PCK gene expression without affecting its modulation by oxygen. These results are in line with the proposal that a ferro-heme protein rather than the respiratory chain acted as an O 2 sensor in the activation of the PCK gene.

Coexisting Bacterial Populations Responsible for Multiphasic Mineralization Kinetics in Soil

Experiments were conducted to study populations of indigenous microorganisms capable of mineralizing 2,4-dinitrophenol (DNP) in two soils. Previous kinetic analyses indicated the presence of two coexisting populations of DNP-mineralizing microorganisms in a forest soil (soil 1). Studies in which eucaryotic and procaryotic inhibitors were added to this soil indicated that both populations were bacterial. Most-probable-number counts with media containing different concentrations of DNP indicated that more bacteria could mineralize low concentrations of DNP than could metabolize high concentrations of it. Enrichments with varying concentrations of DNP and various combinations of inhibitors consistently resulted in the isolation of the same two species of bacteria from soil 1. This soil contained a large number and variety of fungi, but no fungi capable of mineralizing DNP were isolated. The two bacterial isolates were identified as a Janthinobacterium sp. and a Rhodococcus sp. The Janthinobacterium sp. had a low mu(max) and a low K(m) for DNP mineralization, whereas the Rhodococcus sp. had much higher values for both parameters. These differences between the two species of bacteria were similar to differences seen when soil was incubated with different concentrations of DNP. Values for mu(max) from soil incubations were similar to mu(max) values obtained in pure culture studies. In contrast, K(s) and K(m) values showed greater variation between soil and pure culture studies. The results of this study help to confirm predictions that two physiologically distinct bacterial populations are responsible for the multiphasic mineralization kinetics observed in the soil studied.

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre

The adsorption of 1,8-dinitropyrene (DNP) to α-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the α-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP had been dissolved, and (iii) the relative concentrations of DNP and α-cellulose. We suggest that this model system could be applied to other mutagens, and that α-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.

Carboxyatractylate inhibits the uncoupling effect of free fatty acids

The ATP/ADP-antiporter inhibitors and ADP decrease the palmitate-induced stimulation of the mitochondrial respiration in the controlled state. The degree of inhibition decreases in the order: carboxyatractylate > bongkrekic acid, palmitoyl-CoA, ADP > atractylate. GDP is ineffective. The inhibiting concentration of carboxyatractylate coincides with this arresting the state 3 respiration. Carboxyatractylate inhibition decreases when the palmitate concentration increases. Stimulation of controlled respiration by FCCP or gramicidin D at any concentration of these uncouplers is carboxyatractylate-resistant, whereas that by low concentrations of DNP is partially suppressed by carboxyatractylate. These data together with observations that palmitate does not increase H + conductance in bilayer phospholipid membranes and in cytochrome oxidase-asolectin proteoliposomes indicate that the ATP/ADP-antiporter is somehow involved in the uncoupling by low concentrations of fatty acids (or DNP), whereas that by FCCP and gramicidin D is due to their effect on the phospholipid bilayer. It is suggested that the antiporter facilitates translocation of palmitate anion across the mitochondrial membrane.

2,4-Dinitrophenol, muscle biopsy, and McArdle's disease.

Exercise is simulated in muscle biopsy preparations by using low concentrations of 2,4-dinitrophenol (DNP), which do not produce contracture or anatomic damage. The validity of this simulation is supported by (1) the biochemical effects of simultaneous muscle contraction and DNP are not additive, suggesting that exercise and DNP stress the same pathways; (2) the effects of increasing concentrations of DNP and increasing levels of stimulation are similar with an early drop in phosphocreatine, increasing lactate and inosine monophosphate (IMP), and a late fall in ATP levels; and (3) DNP provocation in a patient with McArdle's disease demonstrated an absence of lactate and high levels of IMP correlating with clinical findings. DNP provocation may be a simple way of studying metabolic pathways in neuromuscular diseases.

The effect of 2,4-Dinitrophenol on steroid transport in membrane vesicles of Pseudomonas testosteroni

Steroid transport in Pseudomonas testosteroni membrane vesicles was significantly inhibited by the uncoupled 2,4-dinitrophenol (DNP). Inhibition of steroid transport was not due to inhibition of the 3β- and 17β-hydroxysteroid dehydrogenase by concentrations of up to 1 mM DNP. However, inhibition of this membrane-bound enzyme was measured at 10 mM DNP. The solubilized 3β- and 17β-hydroxysteroid dehydrogenase was more sensitive, being inhibited at both 1 and 10mM DNP indicating a specific inhibition of this enzyme by DNP. Testosterone-dependent oxygen consumption was stimulated slightly at low concentrations of DNP and inhibited at high concentrations. The inhibition of testosterone-dependent oxygen consumption correlated with the inhibition of transport. This indicated that the inhibition of transport by DNP was due to a direct inhibition of metabolism. The existence of an electrochemical gradient is used to explain these results.

Decreased insulin binding and degradation associated with depressed intracellular ATP content.

The effect of metabolic inhibitors, 2,4-dinitrophenol (DNP) and NaF, on insulin binding and degradation has been studied in cultured Buffalo rat liver (BRL) cells. In control studies, 1.8 fmol of 125I-insulin binds to 1.2 x 10(6) cells, possessing approximately 40,000 receptor sites per cell with binding affinity of 5.52 x 10(-8) M. When the cells were preincubated with increasing concentrations of either DNP or NaF, a dose- and time-dependent decrease in both insulin binding and degradation was observed. The total amount of 125I-insulin bound to BRL cells preincubated with metabolic inhibitors was reduced to 1.2 fmol per 1.2 x 10(6) cells. The point of 1/2 B max was achieved in the presence of 50 ng/ml of native insulin, 1.7 times that of the control level. The number of receptor sites was unaffected by either DNP or NaF, but an average affinity profile revealed a decrease in the affinity of the ATP-depleted cells for insulin (KD: 7.31 x 10(-8) M and 7.06 x 10(-8) M in DNP- and NaF-treated cells, respectively). The decrease in insulin binding and degradation following the exposure of the BRL cells to the metabolic inhibitors was associated with a 20% reduction in intracellular ATP and adenylate energy charge. DNP and NaF did not affect the equilibrium constant for the myokinase catalyzed reaction and the intracellular concentration of hypoxanthine was stable, confirming the integrity of the cells during the experiments. It is suggested that ATP levels must remain intact to maintain normal insulin receptor affinity. Furthermore, the rate of insulin degradation by ATP-depleted cells is slower than that of intact cells. It is conceivable that the depression of insulin degradation by partially ATP-depleted cells results from either diminished binding or decreased endocytosis and lysosomal activity, all of which appear to be energy dependent.

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and α-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide- p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ∼10 nmol/10 8 cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ( I 50 ⋍ 7 μM ). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-(14)C]IAA, 0-2.0 μM) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101-121, 1974) are also found in a tissue capable of polar auxin transport. - 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. - 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. - 4. Non-radioactive IAA (10 μM) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0-10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. - 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. - 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.

Human liver mitochondria: III. ATPase activity as an index of mitochondrial damage

Optimal conditions for the determination of human liver mitochondrial ATPase are described. The reaction medium containing EDTA (0.5 m m), KCl (optimal concentration 75 m m), and sucrose (100 m m) was found suitable for the assay. The optimal concentration of DNP in order to obtain maximal stimulation was higher at the alkaline pH and in the presence of albumin; the interactions between mitochondria, albumin, and DNP are discussed. The pH curve of activity (measured in optimal conditions with Tris-acetate buffer) showed minimal values of basal and Mg-ATPase at neutral pH and higher values at lower and higher pH; the DNP-ATPase presented small values in the acid range of pH and a broad maximum in the alkaline range of pH. Human liver mitochondria were more sensitive to aging at 37°C than rat liver mitochondria. The determination of ATPase activity as an index of mitochondrial damage should be performed at pH 8.5, measuring the basal activity, Mg-ATPase, and DNP-ATPase in the absence of albumin (with 0.05-0.1 m m DNP) and in the presence of albumin (with 0.25-0.4 m m DNP). It is necessary to use several concentrations of DNP because the concentration of the uncoupler required to induce a maximal stimulation of the ATPase activity might vary with the original state of the mitochondrial preparation. Mitochondria isolated from normal human liver presented a low basal activity, a small stimulation by Mg 2+, and a high stimulation by DNP. In the case of a “reversible” damage of mitochondria, the Mg-stimulated activity can be greater than the DNP-stimulated ATPase without albumin, and a reversal of the ratio DNP-ATPase Mg-ATPase is obtained in the presence of albumin. In pathological conditions an “irreversible” damage of mitochondria was noted. This is characterized by high values of the basal activity and of the Mg-ATPase, while the stimulation by DNP is small whether albumin is added or not. These results provide a basis for a critical use of ATPase activity as an index of mitochondrial damage in a variety of diseases.