Biological effect of alkoxymethylene trimethylammonium chlorides on yeast Saccharomyces cerevisiae

Six compounds of the group of quaternary ammonium salts have been tested for their biological activity using yeasts as a biological system. They have an inhibitory effect on respiration, cell growth and amino acid transport. A destroying action on protoplast regeneration and respiration has been also observed. The studied chemicals appear to have very pleiotropic action, focused on a damage of mitochondrial and cell plasma membranes.     

Transport of the highly charged myo-inositol hexakisphosphate molecule across the red blood cell membrane: a phase transfer and biological study

To address the problem of delivering highly charged small molecules, such as phytic acid (InsP(6) or IHP), across biological membranes, we investigated an approach based on a non-covalent interaction between transport molecule(s) and IHP. Thus, we synthesized a collection of compounds containing IHP ionically bound to lipophilic (but non-lipidic) ammonium or poly-ammonium cations. First, we assessed the ability of these water-soluble salts to cross a biological membrane by measuring the partition coefficients between human serum and 1-octanol. In view of the ability of IHP to act as potent effector for oxygen release, the O(2)-hemoglobin dissociation curves were then measured for the most efficient salts on whole blood. From both the biological and the physical properties of IHP-ammonium salts we determined that cycloalkylamines (or poly-amines) were the best transport molecules, especially cycloheptyl- and cyclooctylamine. Indeed, the octanol/serum partition coefficient of IHP undecacyclooctylammonium salt, is superior to 1, which is very favorable for potential uptake into the red blood cell membrane. A qualitative correlation was found between the partitioning experiments and the biological evaluations performed on whole blood.     

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

Early metabolic effects and mechanism of ammonium transport in yeast

Studies were performed to define the effects and mechanism of NH+4 transport in yeast. The following results were obtained. Glucose was a better facilitator than ethanol-H2O2 for ammonium transport; low concentrations of uncouplers or respiratory inhibitors could inhibit the transport with ethanol as the substrate. With glucose, respiratory inhibitors showed only small inhibitory effects, and only high concentrations of azide or trifluoromethoxy carbonylcyanide phenylhydrazone could inhibit ammonium transport. Ammonium in the free state could be concentrated approximately 200-fold by the cells. Also, the addition of ammonium produced stimulation of both respiration and fermentation; an increased rate of H+ extrusion and an alkalinization of the interior of the cell; a decrease of the membrane potential, as monitored by fluorescent cyanine; an immediate decrease of the levels of ATP and an increase of ADP, which may account for the stimulation of both fermentation and respiration; and an increase of the levels of inorganic phosphate. Ammonium was found to inhibit 86Rb+ transport much less than K+. Also, while K+ produced a competitive type of inhibition, that produced by NH4+ was of the noncompetitive type. From the distribution ratio of ammonium and the pH gradient, an electrochemical potential gradient of around -180 mV was calculated. The results indicate that ammonium is transported in yeast by a mechanism similar to that of monovalent alkaline cations, driven by a membrane potential. The immediate metabolic effects of this cation seem to be due to an increased [H+]ATPase, to which its transport is coupled. However, the carriers seem to be different. The transport system studied in this work was that of low affinity.     

Mediated and Residual Methionine Transport by Brewer's Yeast

The characteristics of methionine uptake by brewer's yeast (S. carlsbergentis “Schwehat No. 2”) and the regulation of the process have been studied. The methionine uptake by brewer's yeast grown on wort is a complex process Composed of a mediated transport process saturating at low concentration and permeation that is in linear dependence on the concentration. The mediated transport process is under regulated control and can be repressed by (a) certain amino acids added to wort media or applied to phosphate buffer in shorter pre-treatment, (b) ammonium ions added to a synthetic nutrient solution or, in shorter pre-treatment, by ammonium ions applied to a synthetic nutrient solution containing glutamic acid.     

Effects of some cyclic elements containing amphiphilic compounds on stability and transport properties of model lecithin membranes

The paper presents results of studies on changes in model membrane properties induced by some single-chain amphiphilic quaternary ammonium salts with fungicidal activity, N-dodecyl-N-methylmorpholinium chloride (IVC), N-dodecyloxymethylene-N-methylmorpholinium chloride (IVD), N-dodecyl-N-methylpiperidinium chloride (VIC), N-dodecyloxymethylene-N-methylpiperidinium chloride (VID) and N-dodecyl-N,N-dimethyl-N-benzy-lammonium chloride (IB). Two different lipid systems were used, namely, unilamellar liposomes and black planar membranes (BLM). All the compounds studied interfered with sulphate ion transport across liposomal membranes as well as with calcium ion desorption from the membranes. The compounds were found also to change the stability of black lecithin membranes. Generally the sequence of effectiveness of the salts studied on both models used was: IB greater than VID greater than IVD greater than VIC greater than IVC, although there were some differences for particular processes. The results obtained are discussed in view of a possible mechanism of the interaction between the salts studied and the lipid model including the role the salt head group structure and charge distribution can play in this interaction.     

Toxic effects of fatty acids on yeast cells: possible mechanisms of action.

As shown in a previous paper, threshold concentrations of lower and intermediate fatty acids inhibit the uptake of inorganic phosphate, growth, and cell division in yeast cells. This demonstrates that, apart from these effects, the acids cause an increase in the respiration quotient (RQ), inhibition of CO2 fixation, production of ethanol at the expense of anabolic processes, and inhibition of active amino acid transport in the yeast Candida utilis. On the other hand, the threshold concentrations have no effect on intracellular pH. The inhibition of the inorganic phosphate uptake cannot be the sole primary mode of action of fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought about by fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought by fatty acids at concentrations still premitting some phosphate uptake. Although 2,4-dinitrophenol and caproic acid at low concentrations cause an analogous decrease in biomass yield, their combination does not bring about any marked increase in the effect. Considering the physicochemical properties of fatty acids and their preferential action on energy-requiring processes, one of the key sites of action can be assumed to be the mitochondrial membrane. Fatty acids might inhibit the transport of anions, especially phosphate, across the membrane, and disturb the membrane potential by affecting the transport protons. The physiocochemical properties of fatty acids may also give rise to their binding to other intracellular membranes and to a subsequent interference with the function of the corresponding organelles.     

Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.     

Degradation of Benzothiophene and Related Compounds by a Soil Pseudomonas in an Oil-Aqueous Environment

Pseudomonas aeruginosa PRG-1, an isolate from oil-contaminated soil, degrades benzothiophene (BT) and other related compounds in a 5% oil-basal medium system. The organism cannot grow on BT alone; 0.05% yeast extract is a suitable substrate for its growth and for its attack on BT. Although BT is partially toxic to the bacteria, toxicity is reduced when BT is added in this oil system. The oil phase is emulsified by bacterial action during the process. Oxygen uptake studies with washed cell suspensions show increased respiration in the presence of BT. Endogenous respiration is markedly decreased by p-hydroxy-mercuribenzoate, whereas respiration due to BT is scarcely affected, suggesting that oxygen is added directly to BT. Results obtained both in direct degradation and in respiration studies indicate that 3-methyl-thiophene is more rapidly and extensively degraded than BT and other related compounds.     

Interaction between model membranes and a new class of surfactants with antioxidant function.

The effect of two series of amphiphilic quaternary ammonium salts on some properties of phospholipid membranes was studied. The compounds of one series, N-benzyl-N,N-dimethyl-N-alkyl ammonium bromides, exert a destructive effect on membranes and are treated as reference compounds. The compounds of the other series, N-(3,5-di-t-butyl-4-hydroxy)benzyl-N,N-dimethyl-N-alkyl ammonium bromides, are derivatives of the former ones, exhibit antioxidant properties, and do only relatively slight damage to the membranes. The aim of the work was to explain the difference in molecular interaction with membranes between the two kinds of hydrophobic compounds. Thermodynamic methods, a new mixing technique, and monolayer and quantum calculation methods were used. It has been shown that the antioxidant molecules are less hydrophobic than those of the reference compounds and disturb the membrane organization to a lesser extent. On the basis of monolayer data, we suggest that the studied antioxidant behaves like a substitutional impurity, whereas the reference behaves like an interstitial one.     

Preparation and Toxicological Assessment of Functionalized Carbon Nanotube-Polymer Hybrids

Novel Carbon Nanotube-Polymer Hybrids were synthesized as potential materials for the development of membranes for water treatment applications in the field of Membrane Bioreactors (MBRs). Due to the toxicological concerns regarding the use of nanomaterials in water treatment as well as the rising demand for safe drinking water to protect public health, we studied the functionalization of MWCNTs and Thin-MWCNTs as to control their properties and increase their ability of embedment into porous anisotropic polymeric membranes. Following the growth of the hydrophilic monomer on the surface of the properly functionalized CNTs, that act as initiator for the controlled radical polymerization (ATRP) of sodium styrene sulfonate (SSNa), the antimicrobial quaternized phosphonium and ammonium salts were attached on CNTs-g-PSSNa through non-covalent bonding. In another approach the covalent attachment of quaternized ammonium polymeric moieties of acrylic acid-vinyl benzyl chloride copolymers with N,N-dimethylhexadecylamine (P(AA12-co-VBCHAM)) on functionalized CNTs has also been attempted. Finally, the toxicological assessment in terms of cell viability and cell morphological changes revealed that surface characteristics play a major role in the biological response of functionalized CNTs.     

Effect of chromium on the rate of carbon dioxide evolution fromSaccharomyces cerevisiae

The rate of carbon dioxide evolution by whole yeast cells was measured under conditions of both chromium starvation and availability. Preincubation experiments show that there is no evidence for the view thatSaccharomyces cerevisiae can synthesize a biologically important form of chromium from simple chromium(III) salts. Chromium enters the yeast cell, possibly by way of the glucose transport system, and exerts a slight inhibitory effect on the fermentation rate but does not affect the rate of cell growth. Glucose tolerance factor fractions show a stimulatory effect in the yeast fermentation assay irrespective of whether the yeast is chromium depleted. The evidence shows that chromium is not an essential trace element for normal yeast metabolism and hence the activity of glucose tolerance factor preparations is unlikely to be due to a chromium complex as is commonly assumed. Some other as yet unidentified compound or compounds must be responsible for the observed increase in the rate of carbon dioxide evolution.     

Effect of different nitrogen sources on the biosynthesis of lipase by Oospora lactis

The effect of inorganic and organic nitrogen compounds on the synthesis of biomass and extracellular lipase by Oospora lactis was studied. Among the inorganic nitrogen sources ammonium sulphate and ammonium secondary phosphate and among the organic nitrogen sources yeast autolysate proved to be most beneficial for the lipase synthesis. Lipase activity and biomass accumulation in the medium containing yeast autolysate were greater than in the media containing the above ammonium salts. Lipase synthesis reached maximum in the nutrient medium containing yeast autolysate (0.7%) and ammonium sulphate (0.3%).     

Carborane derivatives of 1,2,3-triazole depolarize mitochondria by transferring protons through the lipid part of membranes

Boron containing polyhedra (carboranes) are three-dimensional delocalized aromatic systems. These structures have been shown to transport protons through lipid membranes and mitochondria. Conjugation of carboranes to various organic moieties is aimed at obtaining biologically active compounds with novel properties. Taking advantage of 1,2,3-triazoles as the scaffolds valuable in medicinal chemistry, we synthesized 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazole (c-triazole) and 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazolium iodide (c-triazolium). Both compounds interacted with model lipid membranes and exhibited a proton carrying activity in planar bilayers and liposomes in a concentration- and pH-dependent manner. Importantly, mechanisms of the protonophoric activity differed; namely, protonation-deprotonation reactions of the triazole and the o-carborane moieties were involved in the transport cycles of c-triazole and c-triazolium, respectively. At micromolar concentrations, c-triazole and c-triazolium stimulated respiration of isolated rat liver mitochondria and depolarized their membrane potential, with c-triazole being more potent. In living K562 (human chronic myelogenous leukemia) cells, both c-triazolium and c-triazole altered the mitochondrial membrane potential as determined by a decreased intracellular accumulation of the potential-dependent dye tetramethylrhodamine ethyl ester. Finally, cell viability testing demonstrated a cytotoxic potency of c-triazolium and, to a lesser extent, of c-triazole against K562 cells, whereas non-malignant fibroblasts were much less sensitive. In all tests, the reference boron-free benzyl-4-pentyl-1,2,3-triazole showed little-to-no effects. These results demonstrated that carboranyltriazoles carry protons across biological membranes, a property potentially important in anticancer drug design.     

New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Photodynamic damage of yeast subcellular fraction induced by elevated levels of endogenous protoporphyrin IX

The 2,2'-dipyridyl-induced accumulation of protoporphyrin IX in Saccharomyces cerevisiae cells was shown to be accompanied by the photoinhibition of cell respiration and the enhancement of the photoinduced permeability of plasma membranes to the fluorescent dye primuline. The visible-light illumination (at 400-600 nm) of the mitochondria and plasma membranes isolated from yeast cells with a high level of endogenous protoporphyrin IX intensified lipid peroxidation in these subcellular organelles. Comparative studies showed that the rad 52 mutant cells, which are deficient in the postreplicative recombinational DNA repair system, are considerably more sensitive to the inactivating action of visible light than are the wild-type cells and the rad 3 mutant cells, which are deficient in the excision DNA repair system. The contribution of photodynamic damage to the yeast subcellular organelles to the lethal photodynamic effect is discussed.     

Effect of cultivation conditions on the activity of respiration of Saccharomyces bayanus]

The effect of sugar and ammonium nitrogen content in the initial nutrient medium as well as the cultivation temperature on the respiration activity and respiratory quotient of yeast used in the champagne manufacture was studied. An addition of ammonium nitrogen stimulated respiratory processes in the yeast cell. The respiration activity increased with an increase of pH to 4.0. With an elevation of the sugar content of the substrate the respiratory quotient increased resulting in an uneconomic utilization of sugar. The maximum respiration activity and minimum respiratory quotient occurred at a temperature of 20 degrees.