Presence Of Metabolic Inhibitors Research Articles

Alginate biosynthesis by Pseudomonas aeruginosa: effect of arsenite and other metabolic inhibitors.

Biosynthesis of alginic acid in presence of metabolic inhibitors by resting cells of mucoid Pseudomonas aeruginosa was studied. Among the inhibitors tested, arsenite exhibited very interesting results, while the others showed no remarkable effect. Firstly, arsenite stopped alginate production from all the substrates during initial hours of incubation; secondly, degradation of newly synthesized alginates to smaller molecular weight fragments took place if it was added after a few hours of incubation with the substrate; and thirdly, uncontrolled synthesis of alginate started after several hours of inhibition. Presence of arsenite was needed for the initial inhibitory phase of alginate synthesis; but once the cells were capable of synthesizing alginate after initial hours of inhibition, arsenite may be omitted from the medium.

Protein turnover in Halobacterium cutirubrum and other microorganisms that live in extreme environments

In mesophilic bacteria, turnover of proteins during exponential growth is very low, ca 1% h −1 . Turnover may be much higher in microorganisms that live in more extreme environments. This was previously shown in thermophiles, a psychrotrophic bacterium, and in a moderate halophile. It has now been studied in the extreme halophile, Halobacterium cutirubrum by labelling cellular proteins with ( 14 C) amino acids and then chasing with excess ( 12 C) amino acids. Cells labelled with ( 14 C) proline, a non-essential amino acid for these bacteria, showed low turnover of protein. When cells were labelled with ( 14 C) leucine, an essential amino acid, in the presence of sufficient ( 12 C) leucine to support growth, again very low turnover was observed in the subsequent chase period. However, when cells were labelled with ( 14 C) leucine in the absence of ( 12 C) leucine high turnover rates were observed. These were higher for short-labelled than for long-labelled proteins. Turnover rates were influenced by NaCl concentrations of the labelling and turnover media, and the presence of metabolic inhibitors. Turnover studies with these and other amino acids suggest that when concentrations of essential amino acids are very low, they are incorporated mainly into unstable protein fractions. However, when proteins are labelled with non-essential amino acids or with adequate concentrations of essential ones, incorporation is much higher and the labels enter more stable protein fractions. In contrast to the bulk protein, the bacteriorhodopsin of ( 14 C) leucine-labelled cells did not appear to turn over at all; instead, its specific radioactivity increased (10–20% during a 3 h chase), suggesting its formation from a precursor.

Ascorbic acid transport in mammalian kidney.

Ascorbic acid is known to circulate free in the plasma of several species and is therefore filtered in the kidney; reabsorption subsequently takes place and prevents urinary loss. However, no specific mechanism of renal ascorbic acid transport has previously been presented. In the present study, rat and guinea pig kidney were incubated as slices or as isolated tubules in vitro in the presence of low concentrations of [14C]ascorbic acid. The kidneys of both species handle ascorbic acid similarly. Ascorbic acid accumulates in the renal tissue to a concentration three to four times that present in the bathing media. Recently absorbed ascorbic acid diffuses freely from the kidney and is predominantly nonmetabolized during absorption. Uptake is reduced following replacement of bathing solution sodium by lithium or cesium, or when incubation is performed in the presence of metabolic inhibitors or at low temperatures. The results indicate that ascorbic acid is reabsorbed in the kidney by a sodium-dependent active transport mechanism that operates by concentrating ascorbic acid in the cellular fluid. Renal slices and tubules both appear to transport ascorbic acid and galactose across the brush-border membrane; this indicates that the tubular lumens in these preparations are not collapsed or sealed off.

Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein.

A flow cytometric investigation has been made on the membrane permeability properties that mediate intracellular turnover of fluorogenic substrates. The accumulation and efflux of fluorescein, consequent to the enzymatic turnover of fluorescein diacetate, were assessed in the presence of metabolic inhibitors and after treatment with membrane-active compounds. The metabolic poisons KCN and rotenone greatly inhibited only the fluorescein efflux, reducing the rate constant to as little as one-tenth in relation to control cells; in the presence of glucose such inhibition was partially removed. Glucose availability also affected fluorescein efflux: an increase of the rate constant was observed in cells treated with 20 mM glucose, and a decrease was measured in cells incubated for 1 hr in glucose-free buffer. Membrane-active compounds Triton X-100 and hydrocortisone reduced fluorescein accumulation. Hydrocortisone strongly blocked also the efflux; the addition of glucose did not restore the rate significantly. The major evidence of these results is that fluorescein efflux is dependent on membrane integrity and on availability of metabolic energy. Fluorescein accumulation is only partially related to permeability properties regulating FDA uptake, due to the influence that treatments exhibit at the same time on FDA hydrolysis and/or fluorescein release.

Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin.

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.

Renal transport of gossypol in the rabbit.

The present work was carried out to investigate the transport characteristics of gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, into the rabbit renal cortical slice. The uptake of gossypol increased linearly during a 2-hr incubation after which it leveled off with the average slice-to-medium concentration ratio (S/M) slightly above 20. In the presence of metabolic inhibitors, the S/M gossypol leveled off at about 9, suggesting an extensive binding of gossypol to tissue proteins. The uptake of gossypol was significantly inhibited by p-aminohippurate (PAH), probenecid, ouabain, and DIDS, all of which are known inhibitors of renal organic anion transport. However, the gossypol uptake was not affected by tetraethylammonium (TEA), a prototypical organic cation. Kinetic studies indicated that the apparent Km for gossypol transport is 0.28 mM, and also that probenecid inhibits gossypol transport in a competitive manner. It is concluded that gossypol is transported by the renal tubule through the classic organic anion system.

Immunoradiometric assay to measure the in vitro penetration of sporozoites of malaria parasites into hepatoma cells.

We describe here an immunoradiometric assay to quantitate the in vitro invasion of hepatoma cells by sporozoites. The assay measures levels of circumsporozoite (CS) antigen that remain associated with the hepatoma cells after their incubation with the parasites. Several observations show that these measurements reflect internalized rather than extracellular antigen. For example, when incubations were performed with nonviable parasites (sonicated or heated), or in the presence of metabolic inhibitors, such as sodium azide and deoxyglucose, the amounts of CS antigen found in hepatoma cell extracts were greatly diminished. Moreover, Western blotting experiments revealed a striking difference in the pattern of CS proteins of infected cell extracts as compared with those of free parasites. The assay was used to measure the amounts of intracellular CS antigen for several days after infection of the hepatoma cells. The results confirmed previous microscopic observations, made by using immunofluorescence techniques, showing that the CS antigen in the host's liver cells diminishes progressively while the parasite develops into the exoerythrocytic stage. The immunoradiometric assay should facilitate the evaluation of the effects of drugs on sporozoites and also on studies aimed at the identification of a sporozoite receptor on the hepatocyte.

Antigen-specific suppression of anti-influenza antibody production in man. Possible role of a membrane-antigen complex.

E rosette-forming (E+) cells from human secondary lymphoid tissue were incubated with high dose influenza A virus (Mem-Bel) in an attempt to generate suppressor T cells. Suppression was assayed by transferring the antigen-pulsed E+ cells into effector cultures consisting of E+ and E- cells stimulated with immunogenic amounts of either the inducing virus Mem-Bel) or the non-cross-reacting influenza B virus (B/HK). The transfer resulted in marked inhibition of IgG, IgA and IgM antibody production to Mem-Bel but not to the control antigen, B/HK virus. The suppressive effect was specific at the level of induction as well as expression since E+ cells exposed to high dose Mem-Bel could provide help to an effector culture containing E- cells and optimal dose of B/HK virus. However, metabolically active cells did not appear to be required for suppression. Thus, it could be elicited (a) after only 15 min incubation of E+ cells with high-dose virus and (b) by E+ cells exposed to irradiation, incubated in the presence of metabolic inhibitors, or disrupted by repeated freeze thawing. In contrast, treatment of E+ cells with pronase reversed the suppressive effect. Interestingly, virus heated to 70 degree C failed to induced suppression, while retailing the ability to elicit a normal helper response. Suppression induced by exposure to standard amounts of high-dose antigen was mediated by T cells of both helper/inducer (Leu-3a+) and suppressor/cytotoxic subsets (Leu-2a+), but not by B cells. Two groups of observations pointed to the B cell as the target of suppression. First, suppression could still be transferred to effector cultures in which helper T cells had been replaced by T cell-replacing factor or suppressor T cells removed by irradiation. Second, significant inhibition of antibody production was obtained when the transfer of antigen-pulsed E+ cells was delayed for up to 120 h after initiation of the effector culture. Taken together the results suggest that suppression in this system is due to the formation of an antigen bridge between specific receptor sites on the T cell membrane and the target. Although not dependent on triggering of metabolically active suppressor T cells the phenomenon highlights the need for care in interpreting the mechanism of suppression by high-dose antigen and could, in addition, represent a biologically important control mechanism capable of rapid inhibition of effector T cells and B cells in sites of high antigen concentration.

Differential mechanisms for liposome uptake by vegetative and aggregation competent cells of Dictyostelium discoideum.

Liposomes labelled with 125I-labelled albumin were used as carriers for the introduction of albumin into vegetative and aggregation competent cells of Dictyostelium discoideum. The subcellular distribution of albumin and its degradation products was followed. The results show that 44 and 67% of the radioactivity incorporated by vegetative and aggregation competent cells, respectively, was found in soluble form in the cell. These figures fall to 26 and 34% in the presence of metabolic inhibitors with a concomitant increase of membrane-bound radioactivity. Differences in the mode of liposome--cell interaction in the two stages of development of the organism and the possibility that metabolic inhibitors may operate at the site of the vacuole--liposome fusion step are discussed.

The mouse fibroblast growth hormone receptor: ligand processing and receptor modulation and turnover.

The recent observation that adipose conversion of mouse 3T3 fibroblasts is stimulated by physiological concentrations of human GH (hGH) and rat GH in vitro suggested that this cell line may be suitable for the study of GH-receptor interactions. The aim of this study was to examine the binding and subsequent processing of [125I]iodo-hGH by BALB/c 3T3 mouse fibroblasts. Binding of [125I]iodo-hGH to 3T3 fibroblasts was time and temperature dependent. Apparent steady state binding was achieved after 1 and 2 h at 37 and 30 C, respectively. At 37, 30, and 20 C specifically bound [125I]iodo-hGH became increasingly resistant to removal by acid treatment (0.15 M NaCl/0.05 M glycine, pH 2.5). In contrast at 4 C or at higher temperatures in the presence of metabolic inhibitors, a greater proportion of specifically bound hGH was removed by acid treatment. Inclusion of 0.2 mM chloroquine in the incubation medium resulted in significantly more accumulation of trichloroacetic acid (TCA)-precipitable radioactivity compared to control cells without affecting the shift of radioactivity from the acid-elutable to the acid-inaccessible compartment. After removal of [125I]iodo-hGH from the medium there was a rapid loss of radioactivity (t 1/2 = 36.5 +/- 7.2 min, SE, n = 3) from the cell monolayer with a concomitant appearance in the medium of TCA-soluble radioactive species. Chloroquine reduced the rate of efflux of radioactivity from the monolayer (t 1/2 = 4.5 +/- 0.6 h, n = 3) and the appearance of TCA-soluble material in the medium. The half-time of GH receptor loss after inhibition of protein synthesis with cycloheximide (0.1 mM) was 1.25 +/- 0.14 h, n = 3). In contrast half-time of net receptor synthesis calculated from the recovery of specific [125I]iodo-hGH binding capacity after ligand-induced down-regulation was 10.2 +/- 1.5 h, n = 3). These data reveal that after binding of [125I]iodo-hGH to specific cell surface receptors there is rapid irreversible binding of GH to its receptor with a resultant reduction in receptor concentration. Degradation of [125I]iodo-hGH occurs intracellularly and involves processes which are inhibited by lysosomotropic agents. On the basis of these studies we conclude that the binding and subsequent processing of GH by 3T3 fibroblasts is qualitatively similar to that described for other polypeptide hormones and growth factors in this and other cell lines.

Cell swelling increases a barium-inhibitable potassium conductance in the basolateral membrane of Necturus small intestine.

Previous studies have shown that, immediately after the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, there is a rapid depolarization of the electrical potential difference across the mucosal membrane (psi mc). This is followed by a repolarization of psi mc that is paralleled by an increase in the ratio of the effective resistance of the mucosal membrane to that of the basolateral membrane (rm/rs); the latter was shown to be, at least in part, due to a marked increase in the conductance of the basolateral membrane. We now report the following. (i) Exposure of this epithelium to a 12% hypotonic solution results in a hyperpolarization of psi mc and an increase in rm/rs. These effects are blocked by metabolic inhibitors and by the presence of 5 mM Ba2+ in the bathing solution; indeed, in the presence of Ba2+, psi mc depolarizes and rm/rs decreases to low values. (ii) Addition of 15 mM galactose to the mucosal solution when the serosal solution alone contains 5 mM Ba2+ results in a depolarization of psi mc but no subsequent repolarization of psi mc or increase in rm/rs; however, psi mc repolarizes and rm/rs increases when Ba2+ is subsequently removed from the serosal bathing solution. We conclude that (i) the basolateral membrane normally possesses a Ba2+-inhibitable K conductance, which appears to be reduced in the presence of metabolic inhibitors; (ii) after exposure of the tissue to a hypotonic solution or the addition of galactose to the mucosal solution, this conductance increases; and (iii) these responses can be blocked by metabolic inhibitors. These findings suggest that the delayed response of this tissue to the addition of sugars or amino acids to the mucosal solution may be the result of cell swelling resulting from the intracellular accumulation of these solutes in osmotically active forms.

The control of the strength of the caffeine contracture in frog atrial trabeculae: an activity of the sodium-calcium exchange.

In frog atrial trabeculae low-Na contractures and caffeine contractures evoked in Na-poor solutions are increased by raising the external Ca concentration [( Ca]o), lowering the external Na concentration [( Na]o) or by tissue depolarization in K-rich fluids. The source of activator Ca for the low-Na and the caffeine contractures is probably different because Na-withdrawal contractures are inhibited by Mn2+ but unaffected by local anaesthetics, while the caffeine contractures are potentiated by Mn2+ and inhibited by local anaesthetics. The spontaneous relaxation of the low-Na contracture has a rate constant of 0.055 +/- 0.009 s-1, at room temperature for any [Na]o, [Ca]o or external K concentration [( K]o). The spontaneous relaxation of the caffeine contracture in Na-free fluid has a similar rate constant. However, the rate of spontaneous relaxation is increased if Na+ is present in the bathing medium and analysis of the results of experiments done in solutions with different [Na]o, [Ca]o or [K]o, suggests that two processes are involved in the reduction of the internal Ca concentration [( Ca]i) and the fall of tension. One, also responsible for the spontaneous relaxation of the low Na contracture, is dependent on metabolic energy and may be intracellular in origin. The other depends upon the activity of the sarcolemmal Na/Ca exchange, persists in the presence of metabolic inhibitors and is also activated during a low Na contracture when [Na]o is raised, or [Ca]o is lowered. The contractile response, of frog cardiac muscle to caffeine, would seem to be largely due to a release of Ca2+ from an intracellular store, but the strength of the resulting contracture depends upon a competition between the contractile proteins and the Na/Ca exchange for the released Ca2+.

Microbial Transformation of the Labdanes,cis-Abienol and Sclareol

The microbial transformation of cis-abienol and sclareol by a recently isolated soil bacterium, JTS–131, was studied. In the presence of metabolic inhibitors, three new compounds were isolated from the culture broth as degradation products of cis-abienol. They were determined to be (12Z)-labda-12,14-dien-18-ol, (12Z)-labda-12,14-dien-18-oic acid and its methylester. Two new compounds were also formed from sclareol in the presence of metabolic inhibitors. They were determined to be 13β-hydroxylabd-14-en-18-oic acid and 13β-hydroxylabd-14-en-18-oic acid methylester. As a result of sequence determination experiments, the biotransformation pathways of cis-abienol and sclareol by the bacterium were proposed.

Microbial transformation of the labdanes, cis-abienol and sclareol.

The microbial transformation of cis-abienol and sclareol by a recently isolated soil bacterium, JTS–131, was studied. In the presence of metabolic inhibitors, three new compounds were isolated from the culture broth as degradation products of cis-abienol. They were determined to be (12Z)-labda-12,14-dien-18-ol, (12Z)-labda-12,14-dien-18-oic acid and its methylester. Two new compounds were also formed from sclareol in the presence of metabolic inhibitors. They were determined to be 13β-hydroxylabd-14-en-18-oic acid and 13β-hydroxylabd-14-en-18-oic acid methylester. As a result of sequence determination experiments, the biotransformation pathways of cis-abienol and sclareol by the bacterium were proposed.

Microbial transformation of manool and (12Z)-labda-8(17),12,14-triene by Rhodococcus erythropolis JTS-131.

Rhodococcus erythropolis JTS-131, a cis-abienol and sclareol transformable bacterium, converted manool and accumulated manoyl oxide in the culture broth. In the presence of metabolic inhibitors, 13β-hydroxylabda-8(17),14-dien-18-oic acid and its methylester (a new compound) were accumulated as the transformation products. This strain also transformed (12Z)-labda-8(17),12,14- triene and acqumulated (12Z)-labda-8(17),12,14-trien-18-al in the culture broth. In the presence of metabolic inhibitors, two compounds, (12Z)-labda-8(17),12,14-trien-18-ol and 4,18,19-trinor-3,4- seco-5-oxo-(12Z)-labda-8(17),12,14-trien-3-oic acid were accumulated. Based on the experiments on the degradation sequence, transformation pathways for these two labdanes by the bacterium are proposed.

A New Pathway in the Microbial Transformation ofcis-Abienol byNocardia restrietaJTS-162

cis-Abienol was transformed into a new compound, (12Z)-labda-12,14-dien-17a-oic acid, in the presence of metabolic inhibitors, by Nocardia restricta JTS-162. Based on the experiments with a degradation sequence, a new transformation pathway was proposed. This pathway was not determined by the plasmid, and was suppressed by the other pathway determined by the plasmid.

Calcium metabolism of the rabbit lens

The Ca concentration in the rabbit lens is less than that in its bathing aqueous and vitreous humors. This Ca is not distributed evenly in the tissue; it has a higher concentration in the outer peripheral than the inner parts of the organ. About 60% of the total lenticular Ca is present in a layer extending about 0·5 mm down from the outer surface (25% of the total tissue in the lens). When incubated in a solution containing 45Ca about 40% of the lenticular Ca readily exchanges with the isotope in 3 hr; no further change occurred over the next 21 hr. This exchange appeared to occur across either the anterior or posterior side of the lens. The accumulated 45Ca was found to be present in a surface layer about 1 mm deep. The remaining 60% of the lenticular Ca was difficult to displace. Incubation for 5 hr in a Ca-free solution containing EDTA or following freezing and thawing resulted in a loss of only an additional 20% of tissue Ca. The remaining ‘immobilizable’ Ca (40%) was present in both the nuclear and peripheral zones of the lens. When the Ca concentration of the external medium is raised, lens Ca rises also, and when the external concentration is reduced the tissue level declines. In the presence of metabolic inhibitors (iodoacetate plus cyanide) lenticular Ca rises. This effect is slow; little change was seen in 5 hr. Ca accumulation in the lens was enhanced by the Ca ionophore A23187 but an effect was not detectable in 5 hr. Incubation in Na-free media or ouabain did not influence Ca accumulation suggesting that a CaNa exchange is not involved in regulation. Quercetin and orthovanadate, which can inhibit Ca-activated ATPase in some tissues were without effect on lens calcium. The phenothiazine trifluoperazine (TFP), however, enhanced accumulation of Ca. Efflux of accumulated 45Ca from the lens was rapid; 60% in 10 min and 90% in 3 hr. Lanthanum reduced the latter to 60% loss. Efflux of Ca from the lens was unchanged, under experimental conditions, by metabolic inhibitors, by EGTA, verapamil, TFP and vanadate.

A new pathway in the microbial transformation of cis-abienol by Nocardia restricta JTS-162.

cis-Abienol was transformed into a new compound, (12Z)-labda-12, 14-dien-17α-oic acid, in the presence of metabolic inhibitors, by Nocardia restricta JTS-162. Based on the experiments with a degradation sequence, a new transformation pathway was proposed. This pathway was not determined by the plasmid, and was suppressed by the other pathway determined by the plasmid.

Electrical properties of the plasma membrane of microplasmodia of Physarum polycephalum.

Microplasmodia of Physarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30 mM K+, 4 mM Ca++, 3 mM Mg++, 18 mM citrate buffer, pH 4.7, 22 degrees C), the transmembrane potential difference Vm is around -100 mV and the membrane resistance about 0.25 omega m2. Vm is insensitive to light and changes of the Na+/K+ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN-, N3-), Vm drops to about 0 mV. Under normal conditions, Vm is very sensitive to external pH (pH0), displaying an almost Nernstian slope at pH0 = 3. However, when measured during metabolic inhibition, Vm shows no sensitivity to pH0 over the range 3 to 6, only rising (about 50 mV/pH) at pH0 = 6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1 mM of the sugar. Sugar-induced depolarization was insensitive to pH0. The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981, J. Membrane Biol. 63:165-190). Three transport systems are characterized: 1) An electrogenic H+ extrusion pump with a stoichiometry of 2 H+ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K+ pathways, there is a passive H+ transport system; here the protonated form seems to be positively charged. 3) A tentative H+-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.

Effect of hydroxyurea and 5-fluorodeoxy-uridine on deoxyribonucleoside triphosphate pools early in phytohemagglutinin-stimulated human lymphocytes

The induction of deoxyribonucleoside triphosphate pools was studied in phytohemagglutinin-stimulated human lymphocytes in the presence of metabolic inhibitors. The dATP pool was completely inhibited in cells treated with hydroxyurea, in contrast to the dTTP pool. However, 1-formyl-isoquinoline thiosemicarbazone inhibited the formation of these pools equally. During approximately 3 hr of treatment of stimulated cells with hydroxyurea, the dATP, dGTP and dCTP pools were depleted to the base levels observed in the cells before the pools were induced. The base level of the dTTP pool was achieved only in the presence of 5-fluorodeoxyuridine, but the inhibition was completely prevented by addition of thymidine. It is suggested that, when resting lymphocytes were stimulated to enter the growth cycle, the formation of deoxyribonucleoside triphosphates in the early transformation was due to the de novo pathway.