Ionophore Carbonyl Cyanide M-chlorophenylhydrazone Research Articles

Cyclo(Phe-Pro) produced by Vibrio species passes through biological membranes by simple diffusion.

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.

Exploring the Impact of PARK2 Mutations on the Total and Mitochondrial Proteome of Human Skin Fibroblasts.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson’s disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

Membrane Potential is Vital for Rapid Permeabilization of Plasma Membranes and Lipid Bilayers by the Antimicrobial Peptide Lactoferricin B

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by the antimicrobial peptide lactoferricin B

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Ribosome Hibernation Facilitates Tolerance of Stationary-Phase Bacteria to Aminoglycosides

Upon entry into stationary phase, bacteria dimerize 70S ribosomes into translationally inactive 100S particles by a process called ribosome hibernation. Previously, we reported that the hibernation-promoting factor (HPF) of Listeria monocytogenes is required for 100S particle formation and facilitates adaptation to a number of stresses. Here, we demonstrate that HPF is required for the high tolerance of stationary-phase cultures to aminoglycosides but not to beta-lactam or quinolone antibiotics. The sensitivity of a Δhpf mutant to gentamicin was suppressed by the bacteriostatic antibiotics chloramphenicol and rifampin, which inhibit translation and transcription, respectively. Disruption of the proton motive force by the ionophore carbonyl cyanide m-chlorophenylhydrazone or mutation of genes involved in respiration also suppressed the sensitivity of the Δhpf mutant. Accordingly, Δhpf mutants had aberrantly high levels of ATP and reducing equivalents during prolonged stationary phase. Analysis of bacterial uptake of fluorescently labeled gentamicin demonstrated that the Δhpf mutant harbored increased intracellular levels of the drug. Finally, deletion of the main ribosome hibernation factor of Escherichia coli, ribosome modulation factor (rmf), rendered these bacteria susceptible to gentamicin. Taken together, these data suggest that HPF-mediated ribosome hibernation results in repression of the metabolic activity that underlies aminoglycoside tolerance. HPF is conserved in nearly every bacterial pathogen, and the role of ribosome hibernation in antibiotic tolerance may have clinical implications.

Abstract 1682: Radiation-induced autophagy is dependent on Sirtuin 3.

Abstract Sirtuin family members (Sirtuin 1 to 7) are highly conserved proteins, both structurally and functionally, and have either NAD+ dependent enzymatic activity (Sirtuin 1,2, 3, 5 and 7) for deacetylation of proteins or ADP ribosyl transferase activity (Sirtuin 4 and 6). Their functions in metabolic processes and chromatin remodeling have been conserved throughout evolution. Sirtuin 1 and Sirtuin 2 have been shown to be linked with several neurological disorders. The role of other Sirtuins in neurodegenerative disorders is not known. Radiation therapy (RT), one of the preferred and widely accepted treatments for intracranial primary and secondary metastatic tumors, may lead to long-term irreversible neurotoxicity in susceptible individuals with cognitive dysfunction similar to that observed in other neurodegenerative disorders. There are ample data which demonstrate that aberrant autophagic process and dysfunctional mitochondria are important factors leading to several neurological diseases, such as Huntington's disease and Parkinson's dementia. Among the Sirtuins, Sirtuin 3, 4 and 5 are localized within mitochondria, however, their role in radiation-induced neurotoxicity has not been evaluated. Using Sirtuin 3 knockout (KO) mice generated through genomic deletion allowed us the opportunity to study its role in autophagy and mitochondrial function during exposure to radiation. Preliminary reports show Sirtuin 3 in the adult mouse brain to be localized primarily within hippocampus and subcortical plate. Through flow cytometry and immunofluorescence stainin of mouse embryonic fibroblasts (MEFs) derived from Sirtuin 3 KO mice, we demonstrate that loss of Sirtuin 3 alters rates of basal autophagy. Furthermore, immunoblot analysis of autophagic marker LC3B I &II in the presence and absence of hydrolase inhibitors (E64d & Pepstatin A) exhibits that loss of Sirtuin 3 stimulates autophagy. Radiation treatment of Sirtuin 3 wild-type and knockout MEFs exhibited dose dependent induction of autophagy. Staining with mitotracker red and JC1 illustrates that MEFs lacking Sirtuin 3 retain mitochondria with abnormal morphology and disturbed membrane potential which can further enhanced by radiation treatment and exposure to the ionophore Carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Enrichment of lower form of LC3B in Sirtuin 3 KO cells upon treatment with CCCP and radiation suggests that loss of Sirtuin 3 sensitizes cells towards enhanced autophagy via mitophagy. Taken together, this study indicates that Sirtuin 3 is an important player in deciding cell fate via mitophagy during radiation treatment and has important implications in further studies of radiation-induced neurotoxicity. Citation Format: Sadhanshu Shukla, DeeDee Smart. Radiation-induced autophagy is dependent on Sirtuin 3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2013-1682

Kinetics of NH 4 + uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100μM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.

Ethidium bromide efflux by Salmonella: modulation by metabolic energy, pH, ions and phenothiazines

The main efflux pump of Salmonella enterica serotype Enteritidis, which obtains its energy for the extrusion of noxious agents from the proton-motive force, was studied with the aid of an ethidium bromide (EtBr) semi-automated method under conditions that define the role of metabolic energy, ions and pH in the extrusion of the universal substrate EtBr. The results obtained in this study indicate that in minimal medium containing sodium at pH 5 efflux of EtBr is independent of glucose, whereas at pH 8 metabolic energy is an absolute requirement for the maintenance of efflux. In deionised water at pH 5.5, metabolic energy is required for the maintenance of efflux. The inhibitory effect of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on efflux is shown to be minimised by low pH, and at high pH by metabolic energy. Similarly, thioridazine, an inhibitor of metabolic enzymes, inhibits efflux of EtBr only at pH 8 and the degree of inhibition is lessened by the presence of metabolic energy.

Histamine-induced Ca2+ release in bovine adrenal chromaffin cells.

The histamine-induced biphasic increase of the intracellular free [Ca2+] ([Ca2+]i) was studied in bovine adrenal chromaffin cells using fura-2 microfluorimetry and the whole-cell patch-clamp technique. Both the rapid, transient Ca2+ rise and the sustained plateau component of elevated [Ca2+]i were independent of extracellular Ca2+. Incubation with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blocker thapsigargin diminished histamine-induced changes in [Ca2+]i. When Ca2+ release was either stimulated by IP3 or blocked with the competitive inhibitor heparin, histamine was unable to elicit the typical Ca2+ rise. Ryanodine, tetracaine and ruthenium red, all blockers of Ca2+ release from caffeine-sensitive stores, had only minor effects on the agonist-induced Ca2+ changes. The contribution of mitochondria in shaping the histamine-induced Ca2+ increase was studied using ruthenium red and the two proton ionophores carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Both mitochondrial uncouplers reversibly increased [Ca2+]i and induced an inward current leading to cell membrane depolarisation. In summary, these results indicate that Ca2+ from IP3-sensitive stores is essential for the generation of both the transient increase and secondary elevation in [Ca2+]i.

The Effect of Metabolic Inhibitors, Sugars and Fusicoccin on the Electrical Potential Difference Arising Across an Intact Chenopodium Rubrum L. Plant

An analysis of the effect of metabolic inhibitors, sugars, and fusicoccin on the trans-plant electrical potential difference arising across one-week-old green or herbicide-treated Chenopodium rubrum L. plants was performed. The substances were applied either to the solution bathing the root or in the form of drops to the stem. The respiratory inhibitors (KCN and salicylhydroxamic acid), sulfhydryl agents (N-ethylmaleimide and p-chloromercuribenzene sulfonic acid) and proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) affected the electrical potential, the kinetics of the induced changes varying with different inhibitors and site of application. None of the applied sugars (sucrose, glucose or sorbitol), ATPase stimulator fusicoccin or inhibitor vanadate exerted any appreciable effect on the electrical potential. An effect of sucrose could be observed in the case of its application immediately following de-rooting, especially in the case of herbicide-treated plants. These results we explain by non-participation of the sucrose transporter or the proton ATPase in the generation of the electrical potential difference across intact plants (apoplast-apoplast configuration).

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress.

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

Electrogenicity of the Na +-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H + countertransport

Sodium accumulation by the Na +-ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by Δ Ψ generation across the vesicle membrane (positive inside) and H + efflux from the vesicle lumen. Na + accumulation was assayed with 22Na +; Δ Ψ generation was detected by recording absorption changes of oxonol VI; H + efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na + uptake and H + ejection were increased by the H + ionophore carbonyl cyanide m-chlorophenylhydrazone (ClCCP) while Δ Ψ was collapsed. The lipophilic anion tetraphenylboron ion (TPB −) inhibited H + ejection from the vesicles and abolished Δ Ψ. Based on the effects of ClCCP and TPB − on H + ejection and Δ Ψ generation, the conclusion was drawn that H + countertransport observed during Na +-ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na + translocation across the vesicle membrane. Increasing Na + concentrations stimulated H + efflux and caused the decrease in the Δ Ψ observed, thus indicating that Na + is likely a factor controlling H + permeability of the vesicle membrane.

NH4+ transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1.

NH4(+) transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [14C]methylammonium ion (14CH3NH3+) into the intact cells. 14CH3NH3+ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH4Cl instead of glutamate. Vibrio ABE-1 did not utilize CH3NH3+ as a carbon or nitrogen source. NH4Cl and nonradiolabeled CH3NH3+ completely inhibited 14CH3NH3+ uptake. These results indicate that 14CH3NH3+ uptake in this bacterium is mediated via an NH4+ transport system and not by a specific carrier for CH3NH3+. The respiratory substrate succinate was required to drive 14CH3NH3+ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H+ and/or Na+ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated 14CH3NH3+ uptake. The 14CH3NH3+ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0 degrees and 15 degrees C, and the apparent Km value for CH3NH3+ of the uptake did not change significantly over the temperature range from 0 degrees to 25 degrees C. Thus, the NH4+ transport system of this bacterium was highly active at low temperatures.

Medium- and long-chain fatty acid uptake and utilization by Streptomyces coelicolor A3(2): first characterization of a gram-positive bacterial system.

The first characterization of fatty acid uptake in a Gram-positive bacterium is reported. Streptomyces coelicolor A3(2) utilizes fatty acids of different chain length (C4-C18) as sole carbon and energy sources. In vivo beta-oxidation studies and the assay of two enzymes of the beta-oxidation cycle proved that fatty acid degradation is constitutive in this micro-organism. Uptake of the medium-chain fatty acid octanoate showed the characteristics of simple diffusion, whereas the uptake of palmitate, a long-chain fatty acid, occurred by both simple diffusion and active transport. After correcting for non-mediated transport, palmitate uptake measured over a wide range of concentrations followed Michaelis-Menten kinetics. The apparent Km for palmitate was 97.8 microM and the Vmax was 19.3 nmol min-1 (mg protein)-1. Competition experiments showed specificity of the mediated transport component for long-chain fatty acids (> C10). Metabolic inhibitors such as oligomycin, NaF and vanadate, and the ionophores gramicidin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited palmitate uptake to different degrees, consistent with the existence of an active transport mechanism. Uptake rates measured at different pH values indicated that both the ionized and the unionized forms of octanoate crossed the cytoplasmic membrane by simple diffusion. Palmitate in its ionized form appears to be transported by an active mechanism, whereas the unionized molecule diffuses through the membrane. When present in the medium, glucose stimulated the degradation of long-chain fatty acids by increasing the rate of uptake and the level of acyl-CoA synthetase.

Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 expresses inducible bile acid 7alpha-dehydroxylation activity via a multistep pathway. The genes encoding several of the inducible proteins involved in the pathway have been previously mapped to a bile acid-inducible (bai) operon in Eubacterium sp. strain VPI 12708. We now report the cloning, sequencing, and characterization of the baiG gene, which is part of the bai operon. The predicted amino acid sequence of the BaiG polypeptide shows significant homology to several membrane transport proteins, including sugar and antibiotic resistance transporters, which are members of the major facilitator superfamily. Hydrophilicity plots of BaiG show a high degree of similarity to class K and L TetA proteins from gram-positive bacteria, and, like these classes of TetA proteins, BaiG has 14 proposed transmembrane domains. The baiG gene was cloned into Escherichia coli and shown to confer an energy-dependent bile acid uptake activity. Primary bile acids were preferentially transported into E. coli cells expressing this gene, with at least sevenfold and fourfold increases in the uptake of cholic acid and chenodeoxycholic acid, respectively, over control reactions. Less transport activity was observed with cholylglycine, 7-oxocholic acid, and deoxycholic acid. The transport activity was inhibited by the proton ionophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and nigericin but not by the potassium ionophore valinomycin, suggesting that the transport is driven by the proton motive force across the cell membrane. In summary, we have cloned, sequenced, and expressed a bile acid-inducible bile acid transporter from Eubacterium sp. strain VPI 12708. To our knowledge, this is the first report of the cloning and expression of a gene encoding a procaryotic bile acid transporter.

Intracellular pH does not affect drug extrusion by P-glycoprotein

The intracellular pH (pH i) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-VI/KB-3-I system in this paper. The possible role of an elevated pH i in Pgp-related MDR has been investigated by shifting back the pH i of the MDR + cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked ΔpH i on relative daunorubicin (DNR) accumulation was similar in the case of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pH i on Pgp pumping efficiency. However, an indirect connection between pH i regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR + cells.

Application of in vivo bioluminescence to the study of ionophoretic action.

Ionophores (carbonyl cyanide m-chlorophenylhydrazone; valinomycin; and the hop-derived compounds colupulone, trans-isohumulone and trans-humulinic acid) reduced the rate of dodecanal-dependent light emission from IuxA/B-transformed cells of Lactococcus lactis subsp. diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose. This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non-destructive technique in real time.

Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology

A method has been developed to continuously measure the intracellular pH (pH(i)) of cells cultivated in a bioreactor in an on-line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pH(i) indicator 9-aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pH(i) from measured 9AA-fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pH(i) (7 microM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA-fluorescence. The pH(i) change in yeast cell following the addition of a proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well-established pH(i), indicator (which is however unsuitable for on-line applications in a bioreactor) bis-carboxyethyl carboxy fluorescein (BCECF). The pH(i) of yeast under substrate starved conditions was 6.4 units. The responses of pH(i) of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pH(i) increased by 0.12 unit following a 100-ppm glucose pulse addition and by 0.25 unit following a 300-ppm ethanol pulse addition. Under anaerobic condition, pH(i) increased by 0.1 unit following a 500-ppm glucose pulse addition. Comparison of pH(i) with other indicators of cellular metabolic state suggests that pH(i) is a cellular metabolic state indicator.

Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo.

We have utilized a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae, in addition to performing a series of in vivo experiments in yeast, to investigate the coupling between cytosolic protein synthesis and protein transport into mitochondria. We found that the import of bulk mitochondrial proteins was inhibited in both the homologous in vitro reaction and in vivo upon arrest of cytosolic protein synthesis with the addition of cycloheximide. Tight coupling of synthesis and import was also demonstrated in vivo for the beta subunit of the mitochondrial F1-ATPase. We also investigated the effect of the antifolate methotrexate on the import of a fusion protein consisting of the mitochondrial targeting signal of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (the COXIV-DHFR fusion protein). Methotrexate has previously been shown to inhibit posttranslational import of COXIV-DHFR by preventing the DHFR moiety from unfolding. However, we found that antifolate addition had no inhibitory effect on the import of COXIV-DHFR in vivo, suggesting that its import into mitochondria in yeast cells occurs cotranslationally. Further, when we treated yeast with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone to collapse the mitochondrial membrane potential and induce the accumulation of extramitochondrial precursor pools, we found that the ability to be imported by a strictly posttranslational mechanism upon reestablishing the membrane potential varied from one precursor to another, suggesting that cotranslational import may be mandatory for the import of some proteins in vivo. In summary, our findings are entirely consistent with the notion that import of proteins into yeast mitochondria occurs cotranslationally under normal conditions in vivo.

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.