Energy-dependent transport of manganese into yeast cells and distribution of accumulated ions.

Manganese transport into yeast cells is energy-dependent. It is dependent on endogenous sources of energy and is inhibited by olygomycin (12.5-25 microgramg/ml), 2,4-dinitrophenol (1 mM), 2-deoxyglucose (1-50 mM) and sodium azide (1-10 mM), but is stimulated by cyanide and glucose. The stimulating effect of glucose is eliminated by N-ethylmaleimide and iodoacetate, which apparently inhibit the transport of glucose itself. About 75% of the manganese accumulated in the presence of glucose is found in yeast protoplasts and nearly 25% in the cell walls. A major portion of the accumulated manganese is found in vacuoles. The concentration of osmotically free manganese in the cytosol did not exceed 2 mM, but the concentration in vacuoles was up to 14 mM. The tonoplast is assumed to have a transport system for divalent cations, thereby regulating their concentration in the cytosol.     

Shedding of tumor cell surface membranes

Mastocytoma P815 cells are induced to form and shed membrane vesicles (MV) from their surfaces by incubation at low temperature (4 °C) for 1 hr. and subsequently allowing them to warm up to room temperature (22 °C). Within 1–2 hrs. at room temperature, up to 90% of the P815 cells form and shed MV from their surfaces. Both cells and vesicles remain trypan blue-excluding during the MV shedding process. This process is energy dependent in that it can be inhibited by 2-deoxyglucose, sodium azide and 2,4-dinitrophenol. The shed MV can be harvested by centrifugation on a 6% Ficoll cushion and quantitated in terms of protein content. The shedding of membrane vesicles from the tumor cell surfaces results in a significant reduction in the cell size.     

Protection of Nitrogenase in Azotobacter vinelandii

The site or sites that protect nitrogenase from O(2) inactivation in vivo are sensitive to sodium azide or 2,4-dinitrophenol. Both components of nitrogenase can be synthesized when oxidative phosphorylation is disrupted.     

Effect of some inhibitors on kanamycin formation byStreptomyces kanamyceticus

The formation of kanamyein is markedly inhibited by mercuric chloride, sodium iodoacetate, 2,4-dinitrophenol, sodium arsenite and sodium azide particularly when these are added at the start of fermentation. Less inhibition of kanamyein synthesis is observed in case of sodium 5,5-diethylbarbiturate, malonic acid, sodium arsenate and sodium fluoride. Inhibition of kanamycin synthesis is associated with growth inhibition in case of 2,4-dinitrophenol, sodium arsenite and sodium azide. Bacitracin and D-cycloserine have a stimulatory effect on kanamycin synthesis with slight inhibition of cellular growth. This stimulation might be due to accumulation of cell wall intermediates — aminosugar and sugar — which are shunted to the pathway of kanamycin synthesis. Penicillin lowers kanamycin synthesis by 65 percent as compared with 19 percent reduction of cellular growth. Chloramphenicol has a stimulatory effect at lower concentration (20 μg/ml), when it is added at 24 h of fermentation. At higher concentration (60 (μg/ml) chloramphenicol shows marked inhibition of both cellular growth and antibiotic biosynthesis.     

Extranuclear mutant ofSchizophyllum commune

A mutant of the hymenomyceteSchizophyllum commune was isolated which, owing to an extranuclear mutation, did not utilize acetate as the sole carbon source for growth. The growth of the mutant on glucose minimal medium was completely inhibited by sodium azide but was resistant to the effect of 2,4-dinitrophenol or oligomycin. Its endogenous respiration was cyanide-sensitive and was stimulated by 2,4-dinitrophenol to a considerably smaller degree than that of the wild-type strain. The experimental results obtained with this mutant suggest a defect in aerobic phosphorylation.     

A simple method to study colony development in Streptomyces

Abstract When sodium azide was added to cultures of Myxococcus coralloides D a rapid loss in turbidity was observed. The lysis occurred irrespective of the culture age. If the azide was added to cultures which had been division-inhibited with puromycin, lysis was also induced. Other uncoupling agents (2,4-dinitrophenol, methyltriphenylphosphonium bromide and N , N '-dicyclohexylcarbodiimide) were effective to induce lysis, but not the ionophores gramicidin D or valinomycin. Energizing the membrane by the addition of glycerol, glucose or ascorbate to prelytic cultures was a means of preventing the lytic events.     

Morphogenesis of Schizophyllum commune III.Activity of inhibitors of energy metabolism

The effect of azide, 2,4-dinitrophenol, malonate and 2-deoxyglucoseon the physiology and morphology of normal and mutant strainsof Schizophyllum commune was studied. Normal, thin and puffstrains showed a decrease in dry weight and an increase in glucoseutilization in response to azide, dinitrophenol and 2-deoxyglucose.Streak was less sensitive to azide and dinitrophenol and almostcompletely resistant to 2-deoxyglucose. Numerous morphogeneticeffects were caused by the inhibitors. These included the inductionof certain aspects of the morphology of the common-A sexualreaction in normal and thin homokaryons by 2-deoxyglucose, areversion to normal morphology by thin strains in response toazide and dinitrophenol, and a morphogenetic insensitivity tothe inhibitors by the streak mutants. A discussion of the relationshipof these effects to the control of morphology in S. communeis given. ¹This investigation was supported in part by Research GrantAI-06570 from the National Institute of Allergy and InfectiousDiseases of the U. S. Public Health Service and by Grant No.GB-3613 from the National Science Foundation (U.S.A.). ²Present Address: Department of Biology, State College at Bridgewater,Bridgewater, Massachusetts 02324, U. S. A.     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

Pyrophosphate-stimulated uptake of calcium into the germlings of Phytophthora infestans.

The uptake of 1 micrometer calcium into 6-h-old germination tubes of the fungus Phytophthora infestans follows Michaelis-Menten kinetics with a Km of 33 +/- 4 micrometer and a V of 0.3 nmol.min-1.(5 x 10(4) cells)-1.Uptake is inhibited by ruthenium red and lanthanum (both at 1 micrometer) and by the proton conductors 2,4-dinitrophenol (1 mM) and carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone (1--10 micrometer) and also by sodium azide. These data suggest that calcium uptake is dependent on energy and on a carrier. Calcium uptake is stimulated by pyrophosphate but not by ATP, orthophosphate, or polyphosphate. This stimulation is prevented by proton conductors or by incubation at 0 degrees C.     

tight coupling of monosaccharide transport and metabolism inRhodotorula gracilis

A variety of agents have been applied to separate the uphill, metabolically linked transport of monosaccharides from facilitated diffusion inRhodotorula gracilis. The substances used were iodoacetamide, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, sodium azide, sodium fluoride and heavy-metal cations, including Cd²⁺, Hg²⁺, Cu²⁺, UO²⁺ ₂, La³⁺ and Th⁴⁺. No agent examined led to such separation. There was always either a complete inhibition of both cell metabolism (or uncoupling of oxidative phosphorylation) and the transport mechanism or the transport of sugars uphill persisted (when the metabolism was only partly inhibited). It is concluded that the monosaccharide transporting system inRhodotorula gracilis is tightly coupled to cell metabolism and cannot operate without sufficient energy supply even in cases when no uphill movement is involved.     

Differential response of three types of actin filament bundles to depletion of cellular ATP levels

The effect of low levels of ATP on actin filament bundles in PtK2 cells was investigated by using 2-deoxyglucose, together with either sodium cyanide, sodium azide, or 2,4-dinitrophenol. Three actin filament systems were examined: stress fibers, cleavage rings, and dimethyl-sulfoxide (DMSO)-induced actin bundles in the nucleus. Of the three, only stress fibers disassembled when the ATP production was inhibited. The disassembly progressed slowly with the cells losing all stress fibers after about 90 min, but remaining in flat interconnected sheets. Mitotic cells that had progressed as far as metaphase when inhibitors were added, assembled cleavage rings. The process of cytokinesis took place in these cells but at a rate 5 to 10 times slower than normal, and disassembly of the cleavage ring was inhibited after the completion of cytokinesis. DMSO-induced nuclear actin bundles did not disassemble in cells depleted of ATP even when DMSO was eliminated from the medium. The peripheral aggregates of contractile proteins present in these cells became redistributed, however, and the cells flattened in the low ATP environment when DMSO was removed. Nuclear actin bundles did not form in DMSO-treated cells if the ATP inhibitors were present for as little as 5 min prior to DMSO exposure. Thus, the three types of actin filament bundles are affected in different ways by low intracellular levels of ATP. Stress fibers are most sensitive and cleavage rings, the least.     

Growth phase-dependent active transport of pyridoxine in a fission yeast, Schizosaccharomyces pombe

Schizosaccharomyces pombe showed maximum pyridoxine uptake activity around 10 h after starting cultivation. High concentrations of thiamine and pyridoxine in the medium did not affect the activity or the time but changed intracellular levels of vitamin B₆ compounds. Pyridoxine was taken up by a saturable mechanism with two kinds of affinity (K_m 22.4 μM and 118 μM). The uptake depended on the energy produced anaerobically with an optimum pH of 4.5. The uptake was completely inhibited by amiloride, sodium azide or 2,4-dinitrophenol. The uptake system of the fission yeast was different in various respects from that of a budding yeast.     

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 m?g/ml) and colchicine (2 × 10^?5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re-tightening” following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process. © 1995 Wiley-Liss, Inc.     

Naphthalene uptake by a Pseudomonas fluorescens isolate

The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.     

Effects of chemical ischemia in cerebral cortex slices. Focus on nitric oxide

Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1-10 mM) in the presence of 2 mM 2-deoxyglucose ("chemical ischemia"). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5-30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45+/-2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70+/-3.1% of the controls; the N-methyl-D-aspartate receptor antagonist MK-801 (10 microM) as well as the nitric oxide scavengers, haemoglobin (20 microM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 microM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (10 microM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 microM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 microM) or the N-type calcium channel blocker omega-conotoxin (0.5 microM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.     

Suitability of 2-deoxyglucose for measuring initial rates of glucose uptake in isolated adipocytes

The suitability of [3H]-2-deoxyglucose from measuring initial rates of glucose uptake in isolated rat adipocytes was assessed using three approaches. Basal and insulin-stimulated rates of glucose uptake were directly compared in 2 sec and 5 min assays using [14C]-3-O-methylglucose, [3H]-2-deoxyglucose, and [3H]-D-glucose. Equilibrium kinetics of 2-deoxyglucose uptake were compared with those of 3-O-methylglucose through impairment of hexokinase activity by depleting cellular energy with 2,4-dinitrophenol. The equivalence of these glucose analogues in a dynamic system was assessed by measuring the lag time preceding insulin stimulation of glucose uptake, insulin activation rates, and the T 1/2 of insulin activation. Our results demonstrate that no fundamental difference exists in the initial transport of 3-O-methylglucose, 2-deoxyglucose, and D-glucose.     

Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.     

Transport-associated phosphorylation of 2-deoxyglucose in rat adipocytes

The relationship between ATP levels and 2-deoxyglucose uptake was investigated. When the concentration in the medium lies between 1 and 10 mM 2-deoxyglucose uptake causes a marked decrease in ATP level. This could partly be explained by an inhibiting effect of 2-deoxyglucose and 2-deoxyglucose 6-phosphate on ATP synthesis in the mitochondria. A good correlation between the various ATP levels induced by 2,4-dinitrophenol and the rate of uptake of 5 microM and 0.5 mM (but not 5 mM) 2-deoxyglucose was observed. The addition of glucose and 2-deoxyglucose to cells incubated in the presence of trace amounts of 2-deoxy-[1-14C]glucose induced marked changes in the uptake of the tracer that were associated with a rapid decline in ATP level. It appeared that the phosphorylation of 2-deoxyglucose is an important step in the uptake of the sugar. It is hypothesized that the processes of transport and phosphorylation of 2-deoxyglucose are coupled in rat adipocytes.     

Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes

The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.