Leakage Of Metabolites Research Articles

Inhibitory metabolites production by the cyanobacterium Fischerella muscicola

Broad-spectrum inhibitory metabolites were produced by a benthic cyanobacterium Fischerella muscicola (UTEX 1829) in batch culture. These metabolites inhibited the growth of eukaryotic algae, cyanobacteria and eubacteria. The effect of culture age on the production and leakage of these inhibitory metabolites from the cyanobacterium was studied. Confirmation of the presence of an allelochemical, possibly fischerellin was achieved using high performance liquid chromatography (HPLC). The cyanobacterium produced inhibitory metabolites intracellularly at all stages of its growth cycle.

Electropermeabilization of cell membranes

A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. When cells are submitted to short lived electric field pulses with an overcritical intensity, a local membrane alteration is induced, which may reseal. Its molecular definition remains unknown. A free exchange of hydrophilic molecules takes place across the membrane. A leakage of cytosolic metabolites is present. However, a loading of polar drugs into the cytoplasm is obtained. A short description of the processes affecting the cell membrane organization is given. Lipids appear as the primary target of the field effect as in the case of liposomes. Nevertheless membrane proteins appear to be affected by a direct or by a back effect. The permeabilized state is long lived. The cell metabolism plays indeed a critical role in the recovery. The cell viability can be nevertheless preserved.

Morphological and chemical alterations inBotrytis cinereaexposed to the dicarboximide fungicide vinclozolin

Treatment of actively growing Botrytis cinerea hyphae with micromolar concentrations of the dicarboximide fungicide vinclozolin resulted in significant alterations in the growth rate, morphology, and chemical composition of the cells. The addition of vinclozolin resulted in an immediate and severe reduction in the hyphal growth rate and a retardation in the emergence of the second germ tube. Cells treated with vinclozolin had a lower content of pool metabolites than control cells, and this difference increased with time of exposure to the fungicide. In contrast, vinclozolin-treated cells had a higher chitin concentration than control cells. These biochemical alterations were followed by the disorganization and clearing of cells, and by the appearance of dense and dark masses outside the hyphae, presumably composed of cell debris. Hyphae exposed to vinclozolin were more curved and branched and had shorter cells than the controls. The results indicate that vinclozolin causes a slow but generalized leakage of pool metabolites; this release precedes cell lysis and is not the result of a rapid and gross damage to the cytoplasmic membrane.Key words: vinclozolin, Botrytis cinerea, pool metabolites, membrane damage.

Mechanism of inhibition of glycolysis in Streptococcus mutans NCIB 11723 by chlorhexidine

Inhibition of the rate of acid production from glucose by the cells of Streptococcus mutans NCIB 11723 was directly related to the concentrations of 0.075 to 0.20 mM chlorhexidine. Lactate production was inhibited to a greater extent than acetate and formate. Quantification of glycolytic intermediates revealed that the steps in glycolysis inhibited by chlorhexidine were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. However, the activities of these enzymes were not decreased in cells treated with the inhibitor. It was demonstrated that chlorhexidine caused leakage of metabolites from the cells. Our results indicate that the decreased rate of glycolysis caused by chlorhexidine is due to the leakage of metabolic intermediates and not to direct effects on enzymes involved in glycolysis by S. mutans NCIB 11723.

Polyamines in various rice (Oryza sativa) genotypes with respect to sodium chloride salinity

Physiological responses of various rice genotypes were studied in relation to salt (NaCl) stress. Cultivars CSR‐1 and Dular germinated well in different NaCl regimes compared to cvs Rupsail, Assam Getu and M‐1–48. At 100 mM NaCl, the lowest germination was observed in cv. M‐1–48. Cvs CSR‐1 and Dular were relatively effective in maintaining high concentrations of polyamines as well as arginine decarboxylase (EC 4.1.1.19) activity in coleoptiles and roots in a non‐stressed condition. The activities of two biodegradative enzymes, diamine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.4.3.4), were lowest in cv. CSR‐1. The polyamine content was not significantly altered when seedlings of cv. CSR‐1 were exposed to 100 mM NaCl. However, in cv. M‐1–48 enhancement of arginine decarboxylase activity with concomitant accumulation of polyamines was observed. Leakage of metabolites and changes in the levels of Na+ and Cl− were prominent in cv. M‐1–48 under saline conditions. The results suggest a correlation between polyamine and salt stress‐induced responses in rice genotypes.

Regulation of nutrient transfer between host and fungus in vesicular-arbuscular mycorrhizas.

Although the overwhelming majority of non-aquatic vascular plants form vesicular-arbuscular (VA) mycorrhizal associations, the extent of colonization of the host root by any given fungal symbiont varies considerably depending on host and environmental factors. Because VA mycorrhizal fungi are obligate biotrophs, transfer of photosynthate from host to fungus may be an important factor in regulating the extent of VA mycorrhizal formation. Host metabolites must cross the plasma membrane before becoming available to the fungus. Several studies on rates of root exudation under various environmental conditions show a strong correlation between rates of root exudation and percent of root length colonized by VA mycorrhizal fungi. However, passive leakage of simple metabolites from roots as the sole means of regulating fungal colonization seems improbable for an obligate biotroph which has not yet been successfully cultured on any artificial medium. So far there has been insufficient investigation of hormone interactions between symbionts, and of the interference by the fungus in host cell wall synthesis, to evaluate the possible role of these factors in controlling growth of VA mycorrhizal fungi. Cytochemical studies of the host-fungus interface suggest modification of host plasma membrane ATPase activity as arbuscules develop, but the function of this altered activity remains unresolved. The presence of a linked P1 -photosynthate exchange mechanism on the host plasma membrane analogous to the P1 -photosynthate translocator known to exist in the outer membrane of chloroplasts remains an uninvestigated possible mechanism for balancing photosynthate demand by the fungus with enhanced P uptake.

Electric-field-induced permeabilization and fusion of embryonic amphibian cells.

The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different animal, vegetal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of great interest for embryological studies. Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae, either freshly dissociated or cultured for 5 days, can be permeabilized when submitted to an external electric field of sufficient intensity: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells. Permeabilization was indicated by both the leakage of metabolites (ATP) from the cells and the uptake of exogenous compounds (pyranin) into the cells. With the use of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained. Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was observed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally. For the cultured plated cells, cell viability fell with increasing electric-field strength, and for a given electric field value, cell viability decreased with the age of the culture after pulsing. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.

A simple, leaky cell growth model for plant cell aggregates

A simple growth model is proposed for plant cell aggregates which accounts for leakage of a single intermediate metabolite from the aggregates to the medium. This model predicts a lag phase in the growth curve whose extent is determined by the intermediate metabolite leakage coefficient and its equilibrium distribution coefficient between the medium and the cell aggregates, the size of the inoculum relative to the system total water content, and the initial intermediate metabolite content in the medium. The model thus provides for an interaction between growing plant cells and their environment in a way that has heretofore been unquantified. Preliminary validation of the model has been made against literature data of Dioscorea deltoidea grown in batch suspension cell culture on sucrose, yielding a correlation coefficient of 0.997. The predicted glucose + fructose concentration in the medium agrees reasonably well with experimental measurements after ca, 3.5 days of culture, although a discrepancy exists between model prediction and experiment immediately after startup. Further validation of the model is suggested on this and other plant species.

MPP +-induced increases in extracellular potassium ion activity in rat striatal slices suggest that consequences of MPP + neurotoxicity are spread beyond dopaminergic terminals

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces symptoms similar to idiopathic Parkinson's disease in primates. A metabolite of MPTP, MPP + (1-methyl-4-phenylpyridinium), is actively accumulated by dopaminergic (DA) terminals and selectively destroys nigrostriatal DA neurons. The mechanism of this effect remains unknown but reports that MPP + inhibits electron transport in isolated mitochondria and increases oxidation of cytochrome b in striatal slices suggest that depression of ATP production is involved. To relate metabolic effects of MPP + with tissue electrophysiology, extracellular potassium ion activity [K +] 0 was measured by microelectrodes simultaneous to optical monitoring of reduction/oxidation (redox) activity of cytochrome b during superfusion of MPP + onto rat striatal and hippocampal slices. MPP + increased oxidation of cytochrome b and increased [K +] 0 in slices of striatum. These increases were greater than expected from a selective effect of MPP + on DA terminals which likely comprise no more than 3% of the total striatal mass. These effects of MPP + were slowed by a dopamine uptake inhibitor (mazindol) and did not occur in hippocampal slices. These findings indicate that MPP + influences ion transport as well as metabolic activity and that these actions require the presence of functioning DA terminals. However, the large amplitudes of the MPP +-induced changes suggest that consequences of MPP +-neurotoxity are not ultimately confined to DA terminals. Two hypothesis are proposed: that energy failure in DA terminals results in leakage of neurotoxic substances or metabolites altering membrane conductance properties of adjacent cells and thereby placing additional demand upon ion transport pumps and mitochondrial oxidative phosphorylation; or that there is secondary uptake of MPP + leading to mitochondrial inhibition in cells neighboring DA terminals.

CMP-N-acetylneuraminic acid: Is it synthesized in the nucleus

RadioactiveN-acetylmannosamine was injected intravenously into rats to labelN-acetylneuraminic acid (NeuAc) and CMP-NeuAc. Nuclei were isolated from the livers using a non-aqueous technique to prevent leakage of polar metabolites. A preparation was obtained, which was eight times enriched in nuclei based on the ratio DNA/RNA. Free NeuAc and CMP-NeuAc were isolated from this nuclear fraction and from whole liver, and the specific radioactivities were determined. It appeared that at all time points studied, i.e. 1.5, 9.5, and 18 min after injection, the specific radioactivities of free NeuAc as well as of CMP-NeuAc in the nuclear preparation were lower than those in whole liver. Also no significant differences were found between free NeuAc and CMP-NeuAc in the ratio of specific radioactivities in the nuclear fraction/whole liver. Furthermore, no enzyme involved in the synthesis of NeuAc was enriched in the nuclear preparation as compared to various other cytosolic and non-cytosolic enzymes.

Metabolism and excretion of carnitine and acylcarnitines in the perfused rat kidney

Rat kidneys were perfused for 30 min with a Krebs-Henseleit bicarbonate buffer with 5 mM glucose. Albumin proved superior to pluronic polyols as oncotic agent with regard to carnitine reabsorption in the perfused kidney. The reabsorption of 30 μM (−)-[ methyl- 3H]carnitine was approx. 96% during the first 10 min. At 750 μM the reabsorption decreased to 40%. The tubular reabsorptive maximum ( T max) was approx. 170 nmol/min per kidney. The fractional reabsorption and clearance of (+)-carnitine, γ-butyrobetaine, and carnitine esters did not deviate significantly from that of (−)-carnitine. (+)-Carnitine was not metabolized by the perfused kidney. In perfusions with (−)-carnitine or (−)-carnitine plus 10 mM α-ketoisocaproate or α-ketoisovalerate increased amounts of acetylcarnitine, isovalerylcarnitine and isobutyrylcarnitine were found. Propionate (5 mM) inhibited acetylcarnitine formation. Isovalerylcarnitine, isobutyrylcarnitine and propionylcarnitine were actively degraded to free (−)-carnitine. In urine, we found a disproportionally high excretion of carnitine or carnitine esters formed in the kidney, compared to the same derivatives when ultrafiltrated. Leakage of metabolites formed in the kidney into preurine may explain this phenomenon.

The short-term partitioning of carbon-14 assimilate between zooxanthellae and polyp tissue in Acropora formosa

The usefulness of Fluorinert for the extraction of Acropora formosa polyp tissue and zooxanthellae was demonstrated. The latter remain intact, with no leakage of metabolites, and the polyp tissue can be extracted in a minimal volume. Intact A. formosa and its isolated zooxanthellae were incubated in the light with sodium [14C]bicarbonate for 5 s to 15 min and the kinetics of carbon-14 fixation was determined. The isolated zooxanthellae showed a linear response for carbon fixation, whilst the zooxanthellae in the intact association showed a lag period of 1 to 2 min, containing only 12% of the total fixed carbon in the first 1 min. After 10 min, the distribution of fixed carbon between the symbiotic partners was approximately even and the total carbon fixed was in a range similar to that fixed by the isolated zooxanthellae. A pulse-chase experiment showed rapid movement of fixed carbon from the polyp tissue to the zooxanthellae after the 30 s pulse. The paper discusses two possible explanations for the observed results.

Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis CP4.

Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugar conversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can be attributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanol decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essential cofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage of intermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanol on fermentation in Zymomonas mobilis.

Sequential onset of permeability changes in mouse ascites cells induced by sendai virus

(1) The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. (2) The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. (3) Depending on virus: cell ratio, Ca 2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. (4) A model for the induction of permeability changes is presented.

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C4 species was demonstrated from 14C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing 14CO2 under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total 14C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO2 fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since 14C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α‐hydroxypyridine‐methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [14C]‐glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells.Operation of ribulose‐1,5‐bisphosphate oxygenase activity in competition with ribulose‐1,5‐bisphosphate carboxylase is demonstrated by the lowering of total 14CO2 fixation when O2 is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose‐1,5‐bisphosphate labelling when the O2 level was decreased. This was accompanied by an increase in CO2 fixation. This could indicate an increased rate in synthesis of ribulose‐1,5‐bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.

The Effect of Cycloheximide on Nitrogen and Carbohydrate Metabolism of Cladosporium cladosporioides and Cunninghamella echinulata grown on different nitrogen sources

Summary Cycloheximide induced a reduction in the uptake of nitrates, sugars and amino acids by the growing fungal mycelia of Cladosporium cladosporioides and Cunninghamella echinulata . As indicated in the experimental results, reducing sugars and total soluble-N including amino-N were partly recovered in the metabolism solutions of the differently treated samples in presence of cycloheximide. This was considered as evidence of leakage of metabolites from the growing mycelia and of inversion of sucrose in the respective media, prior to its uptake.

The ATP4- receptor of rat mast cells.

The concentration-dependence on exogenous ATP of activation and inhibition of mast-cell histamine secretion, phosphatidylinositol labelling and leakage of metabolites shows that all these functions are regulated by the free acid ATP4-. Maximal histamine secretion and phosphatidylinositol labelling occur with ATP4- at approx. 2 microM, but higher concentrations, which cause inhibition of secretion and phosphatidylinositol labelling, are required to maximize leakage of 32P-labelled metabolites. Both enhancement and inhibition of phosphatidylinositol labelling (due to low and high concentrations of ATP4- respectively) are rapid in onset; histamine secretion is characterized by a delay, especially at low concentrations of ATP4- (approx. 1 microM). Phosphatidylinositol labelling and histamine secretion are dependent on extracellular Ca2+. Metabolite leakage due to the presence of exogenous ATP4- is slow and does not require Ca2+. Of 18 analogues of ATP that were tested, only four were agonists for secretion, and only these four permitted leakage of 32P-labelled metabolites. It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor. For mast cells stimulated with ATP4- enhancement of phosphatidylinositol metabolism is not sufficient by itself to cause Ca2+-dependent secretion.

Intramitochondrial and Extramitochondrial Concentrations of Adenine Nucleotides and Inorganic Phosphate in Isolated Hepatocytes from Fasted Rats

1 The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393–399] was used to determine the concentrations of adenine nucleotides and of inorganic phosphate in the mitochondrial and cytosolic compartments of hepatocytes isolated from the livers of starved rats. 2 Using glycerol 3-phosphate as a cytosolic marker, it was concluded that leakage of cytosolic metabolites was complete after 15 s exposure of the hepatocytes to 2 mM digitonin. The recovery of the adenine nucleotides after digitonin fractionation was measured. The recovery of ATP was about 100% and that of ADP and AMP 88–109%. Radioactively labelled ATP or ADP were added to the fractionation medium in order to test whether redistribution or metabolism of adenine nucleotides occurs during the fraction procedure. No significant redistribution of adenine nucleotides occurs during the fractionation. Although slight metabolic activity appears to occur in the cytosolic fraction this leads to a change of only 20% in ADP and no change in ATP. It is concluded that the digitonin fractionation procedure can be used to obtain an accurate estimation of the content of adenine nucleotides in the cytosolic and mitochondrial fractions of isolated rat-liver cells. 3 In isolated hepatocytes from fasted rats, 60% of total adenine nucleotides is found in the cytosolic fraction. 4 When isolated hepatocytes from fasted rats are incubated with alanine, the ATP/ADP ratio is 8.76 in the cytosolic fraction and 1.77 in the mitochondrial fraction. On addition of oleate there is no change in cytosolic ATP/ADP. 5 In hepatocytes incubated with alanine or alanine + oleate, the concentration of inorganic phosphate is 5-times higher in the mitochondrial fraction than in the cytosol. 6 It can be calculated that in isolated hepatocytes incubated with alanine the phosphorylation state ([ATP]/[ADP]x[Pi]) in the mitochondrial fraction is 105 M−1 (ΔGc= 12.0 kJ/mol) (2.87 kcal/mol) and that in the cytosolic fraction 2630 M−1 (ΔGc= 20.3 kJ/mol) (4.85 kcal/mol). The addition of oleate did not alter the value of the cytosolic phosphorylation state whereas that in the mitochondria was increased by about 20%. 7 The concentrations of the complexed and uncomplexed forms of the adenine nucleotides and inorganic phosphate in the cytosol were calculated assuming [K+]free= 100 mM, [Mg2+]free= 0.4 mM, pH = 7.0 and I= 0.25 M. The cytosolic concentration of uncomplexed ATP was calculated to be 170 μM, and that of uncomplexed ADP 115 μM. The latter value is about 10-times greater than the Km for ADP that has been reported for the adenine nucleotide translocator at low temperatures.

On describing microbial growth kinetics from continuous culture data: Some general considerations, observations, and concepts

Analysis of continuous culture methodology suggests that this potentially powerful tool for kinetic analysis can be improved by minimizing several inherent shortcomings. Medium background substrates - organic carbon, phosphate, and manganese - were shown to dominate kinetic observations at concentrations below chemical detection methods. Reactor wall growth, culture size distribution changes, sample removal-induced steady state perturbations, and limiting substrate leakage from organisms are treated in terms of kinetic measurement errors. Large variations in maximal growth rates and substrate uptake rates found are attributed to experimental protocol-induced transient states. Relationships are presented for correcting limiting substrate concentrations for lability during sampling, contamination with unreacted medium, and background substrate effects. Analytical procedures are discussed for improved measurement of limiting substrate kinetics involving enzymes, isotopes, and material balance manipulation. Relaxation methods as applied to continuous culture are introduced as a means for isolating separate rate constants describing net substrate transport and for evaluating cellular metabolite leakage. Low velocity growth, multiple substrate metabolism, and endogenous metabolism are discussed along with measurements showing that 1-month generation times for aquatic microorganisms can be quite normal and that the kinetics are compatible withμg/liter limiting substrate concentrations. The concept of regarding growth kinetics as the sum of several net accumulation processes is suggested.

Some physiological effects of near-maximum growth temperatures on an obligately psychrophilic marine bacterium.

The heat inactivation of the obligately psychrophilic marine bacterium Ant-300 was investigated in terms of glucose uptake, the oxidation of glucose to CO2, and permeability control. At 13C, the maximum temperature for growth, and at slightly higher temperatures, CO2 evolution decreased with time during the oxidation of exogenously supplied glucose. The decrease in CO2 evolution appeared to be a result of heat-induced restrictions on glucose uptake. Leakage of intracellular metabolites apparently contributed to the cells decreased ability to take up glucose at elevated temperatures. A consequence of these heat-induced changes seemed to be the acceleration of cell starvation.