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At least 289 records · Page 16Linked to original sources
PubMed1994

Transport of C4-dicarboxylates by anaerobically grown Escherichia coli. Energetics and mechanism of exchange, uptake and efflux.

Transport activities for uptake, efflux and exchange of C4-dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4-dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles. Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter-exchange. The external substrate was determined as divalent fumarate2- (or succinate2-) at pH 6-9, whereas monovalent H-fumarate dominated as the substrate at pH 3-4. The exchange was not inhibited by dissipation of delta p or constituents of it (delta psi or delta pH). We conclude that this transport mode functions as an electroneutral exchange of C4-dicarboxylates. The uptake of C4-dicarboxylates did not depend on internal counter-substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5-6. The uptake was inhibited by dissipation of delta p or delta psi. Artificially imposed delta pH, delta psi or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport. Independent of the presence of an external counter-substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of delta p or delta psi. An imposed K(+)-diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de-energized membrane vesicles generated a delta psi of -70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.

Anaerobiosis↗
PubMed1985

Solubilization and detergent effects on interactions of some drugs and insecticides with the t-butylbicyclophosphorothionate binding site within the gamma-aminobutyric acid receptor-ionophore complex.

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.

Animals↗
PubMed1990

Aluminum uptake by neuroblastoma cells.

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.

Aluminum↗
PubMed1983

The contractile response to and the release of noradrenaline by transmural nerve stimulation in the guinea-pig vas deferens and a comparison with the response to noradrenaline.

The release of noradrenaline from the guinea-pig vas deferens by transmural stimulation was measured electrochemically after separation by h.p.l.c. Variations in release with different durations of stimulation were then compared with the mechanical response during stimulation. During long periods of 270 s of stimulation at 10 Hz the average release of noradrenaline in the final 250 s remained at about 50% of that released during the first 20 s yet the mechanical response throughout the later time declined to near zero. Superfusion with constant concentrations of noradrenaline produced mechanical responses similar to those from transmural nerve stimulation, an initial rapid contraction declining to a low level of maintained activity. During the later period, when the response to infused noradrenaline was low, the twitch response to transmural stimulation was potentiated. Phentolamine 10(-6)M reduced the potentiation of the twitch response to transmural stimulation by noradrenaline and propranolol 10(-6)M enhanced the potentiation. These results suggest that the decline in the adrenergic component of the response to prolonged transmural nerve stimulation is not the result of a decline in noradrenaline release but mainly due to desensitization to the contractile effect of noradrenaline. Since the similar desensitization produced by infused noradrenaline does not reduce the twitch response to transmural nerve stimulation it is unlikely that the transmitter responsible for that component is noradrenaline.

Animals↗
PubMed1985

Influence of five different anticoagulants on human blood monocyte isolation and functional activities.

The recovery of blood monocytes after centrifugation with metrizoate/polysucrose was high with EDTA (97.0%) and heparin (91.6%) as anticoagulants, moderate with EGTA and citrate, but low with oxalate (52.3%; P less than 0.05 compared with heparin). There were no differences in viability, spontaneous migration, chemotaxis, or phagocytosis and killing of Candida albicans by the monocytes isolated from blood anticoagulated with heparin, citrate, oxalate, EDTA, or EGTA. However, heparin in concentrations above 20 IU/ml decreased the spontaneous migration and chemotaxis. It is concluded, that EDTA and heparin below 20 IU/ml are the most satisfactory anticoagulants for the functional study of blood monocytes isolated by metrizoate/polysucrose density centrifugation.

Adult↗
PubMed1986

Characteristics of monoclonal antibody measurements in human peripheral blood.

Three areas of monoclonal antibody measurements using flow cytometry have been presented. These include a description of a dual immunofluorescent method for measuring two antibodies simultaneously, the effects of blood storage on enumeration of helper (H) and suppressor (S) cells, and the relationship between absolute lymphocyte count and H/S ratio in both control and AIDS patients. These studies reveal that a dual immunofluorescent labeling method is useful for enumerating lymphocytes from peripheral blood which bear the helper, suppressor and/or thymus-derived (T) cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate helper T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for enumerating suppressor T-lymphocytes. Dual immunofluorescently stained lymphocytes, prepared from whole blood, were analyzed by flow cytometry. Two light scatter parameters, (forward and 90 degree scatter) were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper and suppressor distributions from 167 control patients were as follows: The average percentage +/- SD of the helper and suppressor cells were 42.8 +/- 7.5 and 21.6 +/- 6.4, respectively. The H/S ratio was 2.17 +/- .75. These studies show that the H/S ratio can be determined in a single preparative sample and analyzed by dual immunofluorescence in a single flow cytometric analysis even though the H/S ratio may vary from normal during a disease condition. The dual immunofluorescent assay enables one to correlate the activities of two antibodies against cell surface receptors and allows the measurement of a large number of samples in a minimal time. This study also compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor T-lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. Marked changes were observed in the percentages of helper cells at 4 degrees C, whereas the percentages of suppressor cells did not change appreciably regardless of the anticoagulant storage time or temperature. Finally, the relationship between ALC and the H/S ratio in control and AIDS patients was determined. The ALC varied considerably in both control and patient populations as a function of time. Conversely, the H/S ratio remained constant.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1992

A neutral glycoprotease of Pasteurella haemolytica A1 specifically cleaves O-sialoglycoproteins.

A neutral metalloprotease with marked specificity for an O-sialoglycoprotein has been isolated from culture supernatants of Pasteurella haemolytica A1. The 35-kDa enzyme cleaves human erythrocyte glycophorin A, which is O glycosylated, but does not cleave N-glycosylated proteins or nonglycosylated proteins. Glycophorin A was cleaved when it was present in situ in erythrocyte ghost plasma membranes or when it was free in solution. The glycoprotease did not hydrolyze glycophorin A from which sialate residues had been removed by neuraminidase treatment. An immobilized preparation of the enzyme cleaved glycophorin A at several positions, with a major site of cleavage at Arg-31-Asp-32. The glycoprotease is inhibited by EDTA, citrate, and ascorbate, but inhibition appears to be due to the masking of metal ion activators rather than to their removal. The enzyme is not inhibited by phosphoramidon, an inhibitor of other bacterial neutral metalloproteases.

Ascorbic Acid↗
PubMed1992

Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins.

Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents.

Bacterial Adhesion↗
PubMed1972

Feedback regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase from a marine bacterium, Vibrio MB22.

A marine bacterium, Vibrio MB22, has been studied to determine the pattern of feedback regulation of the first enzyme unique to the biosynthesis of the aromatic amino acids, 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. The crude extract was used to study response of the enzyme to various salts as well as possible feedback inhibitors. Ethylenediaminetetraacetic acid was found to be inhibitory to enzyme activity, and only CoCl(2), of the salts tested, allowed full recovery as well as apparent stimulation of the DAHP synthetase activity. The DAHP synthetase activity was inhibited solely by the aromatic amino acids, tyrosine, tryptophan, and phenylalanine, of the possible effectors tested. Further work demonstrated the existence of three isozymes of DAHP synthetase, each primarily inhibited by one of the aromatic amino acids.

Aldehyde-Lyases↗
PubMed1972

Nature of the elimination of the penicillinase plasmid from Staphylococcus aureus by surface-active agents.

Growth of Stapylococcus aureus in various ionic surface-active agents resulted in loss of the ability to produce penicillinase, whereas growth in nonionic surface-active agents had no effect on penicillinase production. The curing effect of various alkyl sulfates was found to be dependent upon the chain length. Curing by surface-active agents could be inhibited by magnesium. Reciprocal transduction experiments showed that curing by a surface-active agent was a property of the plasmid, not of the bacterial strain in which the plasmic resides.

Culture Media↗
PubMed1973

D-alanine oxidase from Escherichia coli: participation in the oxidation of L-alanine.

Cell wall-membrane preparations of Escherichia coli, prepared by the ethylenediaminetetraacetic acid-lysozyme method, contain enzymes which catalyze the oxidation of d-alanine and, to a lesser extent, l-alanine into pyruvate and ammonia without the formation of hydrogen peroxide. The kinetic parameters were (i) pH optima of 8.3 to 8.4 for l- and d-alanine and (ii) a K(m) value of 6.6 +/- 0.2 mM for d-alanine. Several coenzymes were without effect when added to the reaction mixture. The participation of d-alanine oxidase in the oxidation of l-alanine was demonstrated. The evidence is based on (i) results of cellular fractionation; (ii) labeling experiments; (iii) inhibition studies with aminooxyacetate and cycloserine; (iv) denaturation experiments; and (v) demonstration of the presence of an active racemase.

Acetates↗
PubMed1975

Uptake of C4 dicarboxylates and pyruvate by Rhodopseudomonas spheroides.

The uptake of C4 dicarboxylates by cells from exponential cultures of Rhodopseudomonas spheroides followed saturation kinetics at concentrations below 100 muM with Km values for succinate, malate, and fumarate of 2.7, 2.3, and 0.8, respectively. Corresponding Vmax values of 50, 52, and 67.5 nmol/min per mg of protein at 20 C were obtained. Each of these compounds interfered competitively with uptake of the others, and a common transport system appears to be involved. Fructose-grown cells took up C4 dicarboxylates only at very low rates, and pyruvate-grown cells took up C4 dicarboxylates at one-third the rates found with succinate-grown cultures. Malonate and maleate inhibited uptake less severely, and aspartate and alpha-ketoglutarate had no effect at 100-fold excess. Divalent metals stimulated uptake. Light or respiration was required for uptake, and entered materials were rapidly converted to other metabolities, notably amino acids. Pyruvate entry appeared to be mediated by several systems, of which only one could be resolved kinetically. This system had a Km of 13 muM and Vmax of 5.6 nmol/min per mg of protein at 20 C. A number of related mono- and dicarboxylates interfered with pyruvate uptake. The pyruvate uptake system was distinguishable from the C4 dicarboxylate system by the absence of divalent cation stimulation and by substrate and inhibitor specificity.

Acetates↗
PubMed1976

Isolation and characterization of T-even ghost-tolerant mutants of Escherichia coli.

Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.

Acridines↗
PubMed1979

Alterations in peptidoglycan chemical composition associated with rod-to-sphere transition in a conditional mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae Mir M7 is a spontaneous parentless morphology mutant which grows as cocci at pH 7 and as rods at pH 5.8. This strain has been characterized as defective in lateral wall formation (at pH7). Data suggest that the cell wall is mainly made up of poles of the rods (G. Satta, R. Fontana, P. Canepari, and G. Botta, J. Bacteriol. 137:727--734, 1979). In this work the isolation and the biochemical properties of the peptidoglycan of both Mir M7 rods and cocci and a nonconditional rod-shaped Mir M7 revertant (strain Mir A12) are described. The peptidoglycan of Mir M7 (both rods and cocci) and Mir A12 strains carried covalently bound proteins which could be easily removed by pronase treatment in Mir M7 rods and Mir A12 cells, but not in Mir M7 round cells. However, when the sodium dodecyl sulfate-insoluble residues of Mir M7 cocci were pretreated with ethylenediaminetetraacetic acid (EDTA), pronase digestion removed the covalently bound proteins, and pure peptidoglycan was obtained. EDTA treatment of the rigid layer of Mir M7 cocci removed amounts of Mg2+ and Ca2+, which were 10- and 50-fold higher, respectively, than the amount liberated from the rigid layer of Mir M7 rods and Mir A12 cells. Amino acid composition was qualitatively similar in both strains, but Mir M7 cocci contained a higher amount of alanine and glucosamine. Mir M7 cocci contained approximately 50% less peptidoglycan than rods. Under electron microscopy, the rigid layer of the Mir M7 rods and Mir A12 cells appeared to be rod-shaped and their shape remained unchanged after EDTA and pronase treatment. On the contrary, the Mir M7 cocci rigid layer appeared to be round, and after EDTA treatment it collapsed and lost any definite morphology. In spite of these alterations, the peptidoglycan of Mir M7 cocci still appeared able to determine the shape of the cell and protect it from osmotic shock and mechanical damages. The accumluation of divalent cations appeared necessary for the peptidoglycan to acquire sufficient rigidity for shape determination and cell protection. We concluded that the coccal shape in Mir M7 cells is not due to loss of cell wall rigidity but is a consequence of the formation of a round peptidoglycan molecule. The possibility that the alterations found in the Mir M7 cocci rigid layer may reflect natural differences in the biochemical composition of the septa and lateral wall of normally shaped bacteria is discussed.

Amino Acids↗
PubMed1988

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.

Citrates↗