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PubMed1981

Histochemical findings suggesting that methylazoxymethanol, a liver and kidney carcinogen, is a substrate for hepatic and renal choline dehydrogenase.

Methylazoxymethanol (MAM) is a potent carcinogen and induces tumors predominantly in rat liver, colon and kidney. The findings reported in this paper suggest that MAM is a substrate for the enzyme choline dehydrogenase located in rat hepatocytes and in the terminal portion of the renal proximal convoluted tubule. As with the natural substrate choline, this reaction with MAM did not require NAD+, was not inhibited by pyrazole and was dependent on the electron transfer reagent, phenazine methosulfate. The product of this reaction is probably the same as that obtained from the metabolism of MAM by alcohol dehydrogenase, namely, an unstable aldehydic derivative which decomposes rapidly to carbonium ions. The reaction with alcohol dehydrogenase offered an explanation for the organotropic effects of this carcinogen in liver and colon and the current report provides a mechanism for the induction of kidney tumors as well as another possible means for production of liver tumors.

Alcohol Oxidoreductases↗
PubMed2006

In vitro toxicity evaluation of silver soldering, electrical resistance, and laser welding of orthodontic wires.

The long-term effects of orthodontic appliances in the oral environment and the subsequent leaching of metals are relatively unknown. A method for determining the effects of various types of soldering and welding, both of which in turn could lead to leaching of metal ions, on the growth of osteoblasts, fibroblasts, and oral keratinocytes in vitro, is proposed. The effects of cell behaviour of metal wires on osteoblast differentiation, expressed by alkaline phosphatase (ALP) activity; on fibroblast proliferation, assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenil)-2H-tetrazolium-phenazine ethosulphate method; and on keratinocyte viability and migration on the wires, observed by scanning electron microscopy (SEM), were tested. Two types of commercially available wires normally used for orthodontic appliances, with a similar chemical composition (iron, carbon, silicon, chromium, molybdenum, phosphorus, sulphur, vanadium, and nitrogen) but differing in nickel and manganese content, were examined, as well as the joints obtained by electrical resistance welding, traditional soldering, and laser welding. Nickel and chromium, known as possible toxic metals, were also examined using pure nickel- and chromium-plated titanium wires. Segments of each wire, cut into different lengths, were added to each well in which the cells were grown to confluence. The high nickel and chromium content of orthodontic wires damaged both osteoblasts and fibroblasts, but did not affect keratinocytes. Chromium strongly affected fibroblast growth. The joint produced by electrical resistance welding was well tolerated by both osteoblasts and fibroblasts, whereas traditional soldering caused a significant (P < 0.05) decrease in both osteoblast ALP activity and fibroblast viability, and prevented the growth of keratinocytes in vitro. Laser welding was the only joining process well tolerated by all tested cells.

Cells, Cultured↗
PubMed1989

In-vitro toxoplasmacidal activity of cationic electron carriers.

Exposing murine macrophages infected with the protozoan parasite Toxoplasma gondii to micromolar concentrations of some cationic electron carriers (dyes), resulted in complete killing of the intracellular parasites at concentrations at which these compounds did not seem toxic for the macrophages. The 50% inhibitory concentrations (with 95% confidence limits) were calculated as 0.26 (0.18-0.37), 1.35 (1-2.25), 0.45 (0.13-1.50), and 1.52 (0.91-2.53) microM for crystal violet, phenazine methosulphate, methylene blue and brilliant cresyl blue, respectively. The effects of these electron carriers did not appear to be the result of an enhancement of the natural antitoxoplasmic activity of the macrophages. None of the tested compounds was active against extracellular Tox. gondii as measured by ability to reinfect murine macrophages; thus, these dyes seem to act primarily on actively metabolizing, intracellular, Tox. gondii. Our data also suggest that the killing effect of the electron carriers was not related to the generation of reactive oxygen intermediates as judged by the inability of scavengers of these intermediates to block the activity against intracellular Tox. gondii. Further studies with related redox compounds would have an interesting chemotherapeutic perspective for treating toxoplasma infections.

Animals↗
PubMed2002

Fitness of in vitro selected Pseudomonas aeruginosa nalB and nfxB multidrug resistant mutants.

Overproduction of multidrug resistance (MDR) efflux pumps is involved in the resistance to a wide range of compounds in bacteria. These determinants extrude antibiotics, but also bacterial metabolites like quorum-sensing signals. Non-regulated extrusion of bacterial metabolites might produce a metabolic burden, so that MDR-overproducing mutants could have a reduced fitness when compared with their parental strains. To test such a possibility, we have compared the behaviour of two MDR Pseudomonas aeruginosa in vitro selected mutants (nalB and nfxB) with their isogenic parental strain with respect to some properties with potential relevance for the survival in the environment and the virulence of this bacterial species. Overproduction of the MDR determinants MexABOprM (nalB mutant) and MexCDOprJ (nfxB mutant) decreased the survival in water, the production of phenazines and proteases, and the virulence (using a Caenorhabditis elegans model system) of the P. aeruginosa mutants. In contrast, the capability of forming biofilms was not impaired. The simple models tested in the present work can enable the analysis of the fitness of large numbers of antibiotic-resistant bacteria by using more realistic approaches than the in vitro competition assays currently used.

Drug Resistance, Multiple, Bacterial↗
PubMed1984

Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds.

Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-soluble fragments. A plot of DNA breakdown as a function of concentration was bell-shaped for each of the active compounds, i.e. as the concentration increased, DNA breakdown was enhanced initially, but above a certain concentration, the proportion of DNA degraded declined, often to zero. One of the compounds, acriflavine, when tested also inhibited DNA breakdown following ultraviolet irradiation. A study, by sedimentation methods, of DNA single-strand breakage in phleomycin-treated E. coli cells, using 3 representative compounds, Crystal Violet, 3,6-diaminoacridine and Methylene Blue, revealed a consistent increase in DNA strand breaks as concentration of compound increased. In similar experiments with ethidium bromide the breakage yield/concentration curve exhibited a maximum. In general, however, it seems that the inhibition of DNA-breakdown observed at higher concentrations of these amplifying compounds is not explicable by an effect on the primary breakage event, but is due to suppression of exonucleolytic activity in the cells.

Acriflavine↗
PubMed1991

Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon.

The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative stress regulon in response to superoxide-generating (redox-cycling) agents. We have cloned the soxR locus, which is positioned near the uvrA gene at 92.2 min on the genetic map, by monitoring complementation of a delta soxR mutation. Subclones from the soxR region in the delta soxR strain simultaneously restored cellular resistance to the redox-cycling agent phenazine methosulfate and inducibility of at least two of the regulon proteins, glucose-6-phosphate dehydrogenase and endonuclease IV, by paraquat, another redox-cycling agent. DNA sequence analysis revealed the presence of two genes involved in activating the soxR regulon. These genes, named soxR and soxS, are arranged divergently with their 5' ends separated by only 85 bp. The predicted 12.9-kDa SoxS protein is related to the AraC family of one-component gene regulators, but corresponds only to the putative DNA-binding regions of these proteins. The 17.1-kDa SoxR protein bears significant homology only to the MerR family of proteins including a predicted DNA-binding helix-turn-helix and a cluster of cysteine residues positioned similarly to those that regulate the activity of MerR in response to Hg2+. This suggests that SoxR could be a metal-binding gene regulator that acts as the intracellular sensor for superoxide. SoxS is evidently the proximal activator of the regulon genes: antibiotic resistance and high-level expression of at least three of the regulon proteins was effected in vivo by the individual expression of SoxS, but not of SoxR, whether or not the cells were exposed to paraquat. These data, together with the recently reported paraquat-inducibility of the soxS gene (Wu, I., and Weiss, B. (1990) J. Bacteriol. 173, 2864-2871), indicate that SoxR and SoxS may constitute a novel type of two-component regulatory system in which the two proteins act sequentially to activate transcription of the various regulon genes in response to superoxide stress.

Amino Acid Sequence↗
PubMed1993

Synthetic RNA-cleaving molecules mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts.

RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.

Base Sequence↗
PubMed2001

DNA electrochemical sensor using osmium complex.

The [Os(DA-bpy)2DPPZ]2+ complex (DPPZ; dipyrido [a:2',3'-c] phenazine, DA-bpy; 4,4'-diamino-2,2'-bipyridine) had a lower half-wave potential (E1/2) of 147 mV (vs. Ag|AgCl) and higher binding affinity with DNA (binding constant, K = 3.1 x 10(7) M-1) than those of DPPZ type metal complexes. With a single stranded DNA (ssDNA) immobilized gold electrode, the hybridization signal (delta I) of the [Os(DA-bpy)2DPPZ]2+ complex was linear in the concentration range of 1.0 pg ml-1-0.12 microgram ml-1 for the targeted DNA with a regression coefficient of 0.999. The detection limit was 0.1 pg ml-1. The 400 bp yAL3 gene was also detected with good sensitivity and selectivity using the [Os(DA-bpy)2DPPZ]2+ complex.

Biosensing Techniques↗
PubMed1986

Purification and properties of glucoside 3-dehydrogenase from Flavobacterium saccharophilum.

A membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270,000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66,000. The enzyme reacted with various artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromercuribenzoate. D-Glucose and methyl-alpha- and beta-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.

Carbohydrate Dehydrogenases↗
PubMed1991

3-keto-5 alpha-steroid-delta 4-dehydrogenase from Nocardia corallina: purification and characterization.

The inducible 3-keto-5 alpha-steroid-delta 4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5 alpha-steroid at the 4- and 5-position, e.g. the conversion of 5 alpha-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5 alpha-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4 beta, 5 alpha-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5 alpha-steroids but not 3 beta-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5 alpha-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-delta 1-dehydrogenase.

Chromatography, Affinity↗
PubMed1991

Intermediate and mechanism of hydroxylation of o-iodophenol by salicylate hydroxylase.

Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzes the hydroxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol. The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively. The reaction stoichiometry, as to NADH oxidized, oxygen consumed, and catechol formed, is 2 : 1 : 1, respectively. The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I(+)-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine. The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture. The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol. On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio. o-Phenylenediamine decreased it, being converted to phenazine. This suggests that o-benzoquinone is formed in the oxidation of o-iodophenol as a nascent product. The quinone was detected spectrophotometrically by means of the stopped-flow method. Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH. A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed.

Hydroxylation↗
PubMed1976

Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium.

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.

Adenosine Triphosphate↗
PubMed1978

Methylamine dehydrogenase of Pseudomonas sp. J. Reconstitution from its subunits.

Conditions for the restoration of catalytic activity from heavy and light subunits which had been isolated from methylamine dehydrogenase of Pseudomonas sp. J were investigated in vitro. Maximal restoration of the activity was obtained in 0.8 M potassium phosphate buffer, pH 7.0-9.3, at 30 degrees C with equimolar concentrations of the two subunits. Under the optimal conditions, the recovery of enzyme activity was 86% and the time required for half-maximal recovery was about 3 min. Addition of bovine serum albumin or p-chloromercuribenzoate to the incubation mixture had no effect on the rate or extent of recovery of the enzyme activity. When the heavy subunit was added to the light subunit, the absorption spectrum of the light subunit changed to a form similar to that observed for the native enzyme. The concentration of methylamine required to change the spectrum of the light subunit was greatly decreased in the presence of the heavy subunit. Reconstituted enzyme was prepared from the isolated subunits and purified by gel chromatography. The reconstituted enzyme resembled the native enzyme in specific activity, molecular weight, substrate specificity, reaction mechanism, Michaelis constants for methylamine and phenazine methosulfate, susceptibility to inhibitor, and absorption, fluorescence and ESR spectra. However, it was less stable than the native enzyme to thermal and pH treatments. The CD spectrum was also slightly different from that of the native enzyme.

Circular Dichroism↗
PubMed1978

Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.

Biological Transport↗
PubMed1980

Methylamine dehydrogenases of Methylomonas J and Pseudomonas AM1. Hybridization of light and heavy subunits and some properties of an isolated hybrid enzyme.

Hybridization reactions were carried out in vitro between the light and heavy subunits of two methylamine dehydrogenases obtained from Methylomonas J (formerly Pseudomonas sp. J) and Pseudomonas AM1. Enzymatic activity was restored with a combination of L3(MW: 13,000) and HA(MW: 40,000) but not with LA(MW: 13,000) and HJ(NW: 40,000). This active hybrid enzyme was isolated with a Sephadex G-150 column after incubation of LJ and HA. Its molecular weight was nearly identical to that of the two native enzymes (MW: 105,000). The electrophoretic pattern of proteins cross-linked by dimethyl suberimidate showed the formation of a tetrameric structure. Its absorption spectrum was similar to that of the native enzymes. The specific activity was 4.3 and the substrate specificity resembled that of the Methylomonas J enzyme rather than that of the Pseudomonas AM1 enzyme. Michaelis constants were 290 microM for methylamine and 14 microM for phenazine methosulfate. The circular dichroism spectrum and thermal stability resembled those of the Pseudomonas AM1 enzyme. No appreciable amount of an inactive hybrid enzyme was formed in the reaction between LA and HJ, there being neither restoration of the enzymatic activity nor formation of cross-linked tetrameric proteins with dimethyl suberimidate. Hybridization of LJ and HA was further confirmed by the appearance of three enzymatic active bands on a gel after electrophoresis of an incubation mixture of LJ, HJ, and HA. The results are consistent with a previous observation; the two methylamine dehydrogenases from either obligate of facultative metaylotrophs are the alpha 2 beta 2-type and homologous enzymes. It is also suggested that substrate specificity and catalytic activity are directed by the light subunit, and binding affinity of the substrate to the active site, electrophoretic mobility, and thermal stability of the enzyme are endowed by the heavy subunit.

Kinetics↗
PubMed1980

Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1.

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.

Amino Acids↗
PubMed1981

Methanol dehydrogenase of Methylomonas J: purification, crystallization, and some properties.

A methanol dehydrogenase [EC 1.1.99.8] was purified and crystallized from methanol-grown Methylomonas J (formerly Pseudomonas sp. J), an obligate methylotroph. Its molecular weight was estimated to be 135,000 by gel chromatography. The sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed two bands and their molecular weights were approximately 60,000 and 10,000. The enzyme was relatively stable at pH 6 and 10, and was unstable at pH 8. The enzyme activity was lost at pH 4.0; however, the prosthetic group was not liberated from the enzyme. Its isoelectric point was pH 9.3. The visible-ultraviolet absorption, fluorescence, CD, and ESR spectra were measured. The amino acid composition was analyzed after separation of the two components. Primary alcohols and formaldehyde served as substrates. Phenazine methosulfate (PMS) was an effective electron acceptor of the enzyme in the presence of either NH4Cl or methylamine. The enzyme was inhibited partially by metal chelators and completely by Mn2+ and Co2+.

Alcohol Oxidoreductases↗