Binding of Germanium to Pseudomonas putida Cells

The binding of germanium to Pseudomonas putida ATCC 33015 was investigated by using whole intact cells grown in a medium supplemented with GeO₂ and catechol or acetate. Electron-microscopic examination of the control and metal-loaded samples revealed that germanium was bound within the cell envelope. A certain number of small electron-dense deposits of the bound element were found in the cytoplasm when the cells were grown in the presence of GeO₂ and catechol. The study of germanium distribution in cellular fractions revealed that catechol facilitated the intracellular accumulation of this element.     

Germanium accumulation byPseudomonas stutzeri AG259

Summary The influence of pH, temperature and catechol concentration on germanium (Ge) accumulation byPseudomonas stutzeri AG259 was investigated. Increasing the incubation temperature or pH of the culture medium markedly enhanced Ge accumulation. High amounts of Ge were accumulated at pH 11 and at 50°C, conditions under whichP. stutzeri cells were non-viable. Ge accumulation was unaffected by treatment with toluene or 2,4-dinitrophenol. These results indicate that Ge was accumulated by an energy-independent process. Ge accumulation increased as the catechol concentration increased. The use of autoclaved catechol solutions consistently increased the amount of Ge accumulated at all concentrations of catechol tested. It is possible that Ge enters the bacterial cells as a Ge-catechol complex and this uptake is enhanced by autoclaved catechol.     

The highly toxic oxyanion tellurite (TeO<Stack><Subscript>3</Subscript><Superscript>2−</Superscript></Stack>) enters the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> via an as yet uncharacterized monocarboxylate transport system

The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 μM acetate. Conversely, tellurite at 100 μM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.     

Iron Transport in Escherichia coli: Uptake and Modification of Ferrichrome

During the transport of iron as ferrichrome complex into cells of Escherichia coli K-12, the ligand was modified and excreted into the medium. The rate of the formation of the modified product corresponded with the rate of iron transport. The modified product showed a decreased affinity for ferric iron and did not serve as an effective iron ionophore. After all of the ferrichrome had been converted, the modified product was taken up into the cell in an iron-free form. The uptake of ferrichrome and of the modified product depended on the transport system specified by the tonA and tonB genes. The modified product could be converted back into ferrichrome by mild acid or alkaline hydrolysis. One mole of acetate was released per mole of ferrichrome. It is proposed that one N-hydroxyl group of ferrichrome is acetylated to explain the low affinity for iron as the N-hydroxyl groups form the ligands for iron (III). A weak ester linkage by which the acetyl group is covalently bonded would account for the easy hydrolysis. The iron-free form of ferrichrome, deferri-ferrichrome, was also rapidly converted when incubated with cells with a functional transport system. It is therefore likely that iron is released from ferrichrome by reduction before modification takes place. The conversion of the ligand could be a mechanism by which cells rid themselves of a potentially deleterious ligand for iron in the cytoplasm. A possible role in ferrichrome transport is discussed.     

Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed on a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action.

Feeding lactating rats on high-fat cheese crackers in addition to laboratory chow increased the dietary intake of fat from 2 to 20% of the total weight of food eaten and decreased mammary-gland lipogenesis in vivo by approx. 50%. This lipogenic inhibition was also observed in isolated mammary acini, where it was accompanied by decreased glucose uptake. These inhibitions were completely reversed by incubation with insulin. Insulin had no effect on the rate of glucose transport into acini, nor on pyruvate dehydrogenase activity as estimated by the accumulation of pyruvate and lactate, suggesting that these are not the sites of lipogenic inhibition. Insulin stimulated the incorporation of [1-14C]acetate into lipid in acini from high-fat-fed rats. In the presence of alpha-cyanohydroxycinnamate, a potent inhibitor of mitochondrial pyruvate transport, and with glucose as the sole substrate, neither [1-14C]glucose incorporation into lipid nor glucose uptake were stimulated by insulin. Insulin did stimulate the incorporation of [1-14C]acetate into lipid in the presence of alpha-cyanohydroxycinnamate, and this was accompanied by an increase in glucose uptake by the acini. This indicated that increased glucose uptake was secondary to the stimulation of lipogenesis by insulin, which therefore must occur via activation of a step in the pathway distal to mitochondrial pyruvate transport. Insulin stimulated acetyl-CoA carboxylase activity measured in crude extracts of acini from high-fat-fed rats, restoring it to values close to those of chow-fed controls. The effects of insulin on acetyl-CoA carboxylase activity and lipogenesis were not antagonized by adrenaline or dibutyryl cyclic AMP.     

Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent K_m of 18 μM) and carnitine and a low affinity for proline (apparent K_m of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in L. plantarum and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by trans substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.     

Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies

When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Evaluation of acetate uptake and its relative conversion to acetylcholine in Torpedo electric organ synaptosomes under different ionic and metabolic conditions

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10^?4 M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis.Acetate uptake displayed a marked requirement for external Na⁺ and was decreased following depolarization of the synaptosomes by an elevated K⁺ concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca²⁺, while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself.The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.     

Effect of acetate on transport of organic acid (fluorescein) in renal proximal tubules of frog

The effect of acetate on active fluorescein transport in intact proximal tubules of surviving frog kidney was studied. When the kidneys were incubated in a 120 mM Na⁺ medium, 10 mM acetate stimulated fluorescein uptake in the tubules. The stimulation was more pronounced if the kidneys had been previously preincubated for 3 h in the substrate-free solution. Lowering of the Na⁺ concentration in the bathing medium to 10 mM resulted in the disappearance of the acetate effect. Preincubation of the kidneys with acetate at 2–4°C gave rise to stimulation of the fluorescein transport only in the 120 mM Na⁺ acetate-free medium. The acetate effect on the fluorescein uptake was partially prevented by ouabain. The stimulation of the uptake by acetate in the 120 mM Na⁺ medium correlated with an increase in the extent of reduction of pyridine nucleotides in the tubules. The pyridine nucleotides were reduced more markedly after incubation of the kidneys in the 10 mM Na⁺ medium, when acetate had no effect on the fluorescein transport. In both the 120 mM and the 10 mM Na⁺ media, the cold preincubation of the kidneys with 2.5 mM ADP or 2.5 mM ATP resulted in only slight stimulation of the fluorescein uptake. But in both media the uptake was significantly enhanced after cold preincubation of the kidneys with 2 mM NADH. After the cold precincubation with ADP, stimulation of the fluorescein transport by acetate was observed in the case of the 10 mM Na⁺ medium also. The absence of any stimulatory effect of acetate on the organic acid transport in the 10 mM Na⁺ medium is explained as the result of the transformation of mitochondria in the tubular cells into the inactive state 4 due to a decrease in the intracellular ADP level. Reducing equivalents are supposed to take part in energization and/or regulation of transport processes in plasma membranes of the renal proximal tubules.     

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations

The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K⁺. Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO₃⁻, fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg²⁺ decreases both of these values whereas p-chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.     

Active transport of chloride by Anacystis nidulans

Chloride uptake by the cyanobacterium Anacystis nidulans at 38°C is energy dependent showing maximum rate (around 5.10^-7 mol Cl^-xml cell water^-1xmin^-1) and accumulation (up to 160 fold) in light and air. The respective values in air and darkness were 40–70% lower. In the dark under N₂ no uptake was found. Chloride transport had an optimum at pH 6.7 and a K _M of 2.10^-5 M which was pH-independent. It was inhibited by carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide in the light and in the dark, and also to a lesser extent by valinomycin. 3-(3,4-dichlorophenyl)-1,1-dimethylurea in the light caused a moderate stimulation. To obtain information about the energy source of active chloride transport the action of the four inhibitors on membrane potential (determined through the distribution of triphenylmethylphosphonium) and ATP level (determined by the firefly method) was examined. It was found that a high negative membrane potential was unfavorable for chloride accumulation probably by stimulating passive efflux. On the other hand a good correlation between ATP level and chloride transport activity was obtained. Attempts to induce chloride uptake by sudden acidification of the external medium in presence of N,N′-dicyclohexyl-carbodiimide or during anaerobiosis were not successful. Two mechanisms of chloride uptake are discussed:

1. primary active transport by an ATP-dependent pump, and
2. “chemiosmotic” secondary active transport linked to a proton gradient, the present data favoring mechanism a.

     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

Role of glycogen in acetate uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic activated sludge with a minimized polyphosphate content

The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.     

Membrane Potential Changes Related to Active Transport of Glycine in Lemna gibba G1

Accumulation of ¹⁴C-labeled glycine and microelectrode techniques were employed to study glycine transport and the effect of glycine on the membrane potential (Δψ) in Lemna gibba G1. Evidence is presented that two processes, a passive uptake by diffusion and a carrier-mediated uptake, are involved in glycine transport into Lemna cells. At the onset of active glycine uptake the component of Δψ which depended on metabolism was decreased. The depolarized membrane repolarized in the presence of glycine. This glycine-induced depolarization followed a saturation curve with increasing glycine concentration which corresponded to carrier-mediated glycine influx kinetics. The transport of glycine was correlated with the metabolically dependent component of Δψ. It is suggested (a) that the transient change in Δψ reflects the operation of an H⁺-glycine cotransport system driven by an electrochemical H⁺ gradient; and (b) that this system is energized by an active H⁺ extrusion. Therefore the maximum depolarization of the membrane consequently depended on both the rate of glycine uptake and the activity of the proton extrusion pump.     

Sucrose uptake by developing soybean cotyledons

Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K_m of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN₃ or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro-mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH₀. Since sucrose-induced depolarization of the membrane potential and carrier-mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K_m for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed.     

Accumulation of sulfate labelled with S35 by rat tissue in vitro

Rat kidney slices incubated in vitro may show, in parallel with other shifts in electrolyte content, a striking capacity for accumulating sulfate ion (sulfate labelled with S(35)). The uptake is reversed or reduced by CN(-), cooling to room temperature, and by interference with adequate oxygenation. Under the conditions of the experiment, the presence in the medium of sodium acetate and glucose as substrates was found to be without measurable effect on the accumulation. The extent of sulfate uptake is related to the ionic composition of the medium in which the tissue is incubated, for the uptake occurs optimally only in the presence of a K(+) level of about 0.04 M, and is decreased as the concentration of Na(+) rises. Likewise, when Ca(++), Mg(++), or choline is present in the medium, sulfate accumulation may be depressed. In addition to renal cortex, kidney medulla and liver showed capacity for sulfate accumulation, whereas no convincing evidence for significant uptake was obtained with strips of aorta, colon, or diaphragm.     

Comparative Study of Uptake and Cellular Distribution of Hg203-Labeled Phenyl-Mercuric Acetate and Mercuric Acetate by Pea Roots

Uptake and cellular distribution of mercury²⁰³ from dilute mercuric acetate or phenylmercuric acetate solutions by excised pea roots (Pisum sativum) have been investigated. The time course of uptake showed that the amount of mercury uptake was increased with the time of incubation, and was similar for inorganic mercury or phenylmercuric acetate. The trend of mercury²⁰³ incorporation into cellular components from mercuric acetate and phenylmercuric acetate differed greatly as the time of incubation increased. The concentrations of mercuric acetate and phenylmercuric acetate solutions or the temperature of incubation also affected the mercury²⁰³ uptake as well as its cellular distribution. Longer time of exposure or higher concentration resulted in a greater mercury incorporation into mitochondrial fraction from phenylmercuric acetate than from inorganic mercury. This difference in intracellular distribution may be responsible for the degree of toxicity between inorganic mercury and phenylmercuric acetate in biological systems.     

Role of the Glycine Betaine and Carnitine Transporters in Adaptation of Listeria monocytogenes to Chill Stress in Defined Medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.