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PubMed1991

Multilocus enzyme analysis of the genus Aeromonas and its use for species identification.

A total of 153 Aeromonas strains were analyzed by multilocus enzyme electrophoresis. All 11 genetic loci were polymorphic with three to 14 alleles per locus (average 6.55). The genetic diversity of each locus varied between 0.095 for indophenol oxidase and 0.881 for the fast variety of malic enzyme. Cluster analysis of the 122 enzyme types revealed a good correlation with taxonomic groupings as determined by DNA-DNA hybridization. In conjunction with biochemical analysis, as few as two enzymes may be sufficient for the identification of all species in the genus Aeromonas.

Aeromonas↗
PubMed2003

Simultaneous measurements of oxygen consumption and ammonia-N excretion in embryos and larvae of marine invertebrates.

The quantification of oxygen consumption and ammonia-N excretion rates is essential in determining energy requirements for development of larval invertebrates. In larval energetics, there is a need for accurate and uncomplicated techniques to quantify metabolic rates. A method for simultaneous measurements of oxygen and ammonia-N concentrations is presented. It employs sealed respirometric chambers (ca. 30 ml) in which embryos and larvae are incubated. Analysis is carried out in end-point samples by Winkler's titration and indophenol-blue for oxygen and ammonia-N, respectively. Water is sampled into volume-calibrated glass syringes and oxygen consumption and ammonia-N excretion rates were determined by the difference between experimental and control (no animals) units. The method was successfully used to measure metabolic rates in embryo and larval stages of the shrimp Farfantepenaeus paulensis and in veliger of the mussel Perna perna. The accuracy denoted by the coefficient of variation is comparable to previous results on larval metabolic rates. A biomass: volume (microg ml(-1)) is proposed to extend its application to further species of marine invertebrates. The method is simple to operate, involves non-expensive material and is portable enough for field work. A substantial number of replicates can be analyzed at the same time and O:N ratio, an indicator of the catabolized substrate, can be calculated.

Ammonia↗
PubMed2001

Expression and activity of NAD(P)H:quinone oxidoreductase (NMO1) in human osteoblastic cells.

NAD(P)H:quinone oxidoreductase (NMO1; EC 1.6.99.2), also called DT-diaphorase, is involved in the reduction of coenzyme Q, an important cellular lipophilic antioxidant that can function as an intermediate electron carrier in plasma membrane-associated electron transport regulating cell growth. We examined the ability of normal human trabecular osteoblastic cells to express NAD(P)H:quinone oxidoreductase (NMO1) and studied its modulation during cell proliferation and growth arrest. We found that confluent primary human trabecular osteoblastic cells derived from healthy individuals constitutively express NMO1 activity, measured using 2-6 dichlorophenol indophenol (DCI) or menadione (vitamin K3) as substrate, and NADPH or NADH as electron donor. We also found that NMO1 activity was related to osteoblastic cell growth. NMO1 activity increased with osteoblastic cell density. Confluent growth-arrested cells expressed an eightfold higher NMO1 specific activity than proliferative human osteoblastic cells. Reverse-transcription polymerase chain reaction analysis showed that NMO1 mRNA levels did not differ in growth-arrested confluent cell and growing cells, suggesting that the increased NMO1 activity with cell density was due to posttranslational events. Harvesting and replating the cells at low density resulted in a 93.4% loss of NMO1 enzymatic activity. Removal of serum from high-density growth-arrested cells resulted in a 48.5% decrease in NMO1 activity. NMO1 activity does not appear to be related to induction of osteoblast differentiation because treatment with the differentiating agent 1,25(OH)(2) vitamin D(3) had no effect on NMO1 activity. The finding that human osteoblastic cells express NMO1 constitutively and that NMO1 activity increases with density-dependent growth inhibition suggest a role for NAD(P)H:quinone oxidoreductase in the control of growth arrest in normal human osteoblastic cells.

Aged↗
PubMedDate not supplied

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Animals↗
PubMed1995

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

Anions↗
PubMed2004

Investigations of the reactions of monochloramine and dichloramine with selected phenols: examination of humic acid models and water contaminants.

Our paper reports on the reactivities and orientations of two common phenols, phenol (2) and m-cresol (3), and some of their chlorinated intermediates with aqueous monochloramine, NH2Cl, and dichloramine, NHCl2. We also examined the further reactivity of 2,4,6-trichlorophenol (4) with the chloramines. The phenols are an important area of investigation because they are substituents in the humic acids and are common contaminants in water. m-Cresol (3) was found to be more reactive than phenol (2)with both chlorinating agents. Both NH2Cl and NHCl2were sufficiently reactive to chlorinate all positions ortho and para to the hydroxyl groups. Mono- and dichloramine showed the same orientation with 2 but different orientations in their reactions with the substituent phenols. Indophenol (as its salt) was formed to a minor extent at high pH but not at pH 9. Both NH2Cl and NHCl2 rapidly replaced the parachlorine in 2,4,6-trichlorophenol (4) to give a mixture of 2,6-dichloro-1,4-benzoquinone-4-(N-chloro) imine (5) and 2,6-dichloro-1,4-benzoquinone (18). Similar reactions occurwith 2,4,6-trichloro-m-cresol (17) and 2,4,6-trichloro-3-methoxyphenol (29). The products for 17 were confirmed by mass spectrometry (El and Cl), 1H NMR, 13C NMR, and IR; the products for 29 were confirmed by mass spectrometry (El and Cl) and IR. An ion radical mechanism is suggested to account for the chlorine replacement by the chloramines. [No side chain oxidation of the methyl group in 17 in H20 or ether occurred, with or without ultraviolet radiation.] Both 5 and 18 underwent further chlorination with NH2Cl or NHCl2. Imine 5 did not function as a chlorinated agent.

Chloramines↗
PubMed2005

Reconstitution of apo-glucose dehydrogenase on pyrroloquinoline quinone-functionalized au nanoparticles yields an electrically contacted biocatalyst.

An electrically contacted glucose dehydrogenase (GDH) enzyme electrode is fabricated by the reconstitution of the apo-GDH on pyrroloquinoline quinone (PQQ)-functionalized Au nanoparticles (Au-NPs), 1.4 nm, associated with a Au electrode. The Au-NPs functionalized with a single amine group were attached to the Au surface by 1,4-benzenedithiol bridges, and PQQ was covalently linked to the Au-NPs. The apo-GDH was then reconstituted on the PQQ cofactor sites. The surface coverage of GDH corresponded to 1.4 x 10(-12) mol cm(-2). The reconstituted enzyme revealed direct electrical contact with the electrode surface, and the bioelectrocatalytic oxidation of glucose occurred with a turnover number of 11,800 s(-1). In contrast, a system that included the covalent attachment of GDH to the PQQ-Au-NPs monolayer in a random, nonaligned, configuration revealed lack of electrical communication between the enzyme and the electrode, albeit the enzyme existed in a bioactive structure. The bioelectrocatalytic function of the later system was, however, activated by the diffusional electron mediator 2,6-dichlorophenol-indophenol. The results imply that the alignment of GDH on a Au-NP through the reconstitution process leads to an electrically contacted enzyme-electrode, where the Au-NP acts as a charge-transfer mediator.

2,6-Dichloroindophenol↗
PubMed2005

Effect of roasting on properties of the zinc-chelating substance in coffee brews.

ApV is a brownish polymer with zinc-chelating activity in brewed coffee. We investigated in this study the effects of roasting on the zinc-chelating, reducing, and antioxidative activities of ApV from light-, medium-, and dark-roasted coffee. We also discuss the effect on the zinc-chelating activity of adding milk to the brewed coffee. The chelating activities of ApVs were evaluated by the tetramethyl murexide method. As the intensity of roasting increased, the yield of ApV increased, and the brown color and molecular weight of ApV respectively became darker and higher. Increasing the degree of roasting also decreased the zinc-chelating activity of ApV. The reducing activities of ApVs estimated by the indophenol method were stronger than those of ascorbic acid. Both the antioxidative activity estimated by the ABTS assay and the reducing activity of ApV increased with roasting. When milk was added to instant coffee and its ApV was prepared, the zinc-chelating activity of ApV was not changed.

Amino Acids↗
PubMed2006

Pachypodol from Croton ciliatoglanduliferus Ort. as water-splitting enzyme inhibitor on thylakoids.

A bioactivity-guided chemical study of aerial parts of Croton ciliatoglanduliferus Ort. led to the isolation for the first time of the flavonoids retusin (5-hydroxy-3,7,3',4'-tetramethoxyflavone) (1) and pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) (2) from the n-hexane extract. Compounds 1 and 2 were separated by preparative thin-layer chromatography. Compound 2 was the most active compound on ATP synthesis inhibition. The I50 value was 51 microM. Pachypodol behaves as a Hill reaction inhibitor. It inhibited the uncoupled electron flow on photosystem II partial reaction from water to dichlorophenol indophenol (DCPIP) and from water to sodium silicomolybdate. However, the uncoupled partial reaction from diphenylcarbazide to DCPIP and the uncoupled photosystem I from DCPIPred to MV were not inhibited by 2. These results were corroborated by fluorescence decay data. Therefore, pachypodol inhibits the water-splitting enzyme activity. Compound 1 with a 4'-methoxy group was a weak inhibitor, indicating that the 4' free -OH group is important for strong inhibition.

2,6-Dichloroindophenol↗
PubMed2002

Subspecies characterization of porcine Campylobacter coli and Campylobacter jejuni by multilocus enzyme electrophoresis typing.

Enteropathogenic Campylobacterjejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated foodstuffs. C jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and L-phenylalanyl-L-leucine peptidase giving 5. 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C jejuni isolates and 19 unique ETs were detected for the 23 C coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C jejuni and C coli. respectively. Alleles were shared by C jejuni and C coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C coli in porcine livers. In certain cases, up to four phenotypically different strains of C coli were isolated from one liver, indicating multiple infections.

Animals↗
PubMed1995

EO9: relationship between DT-diaphorase levels and response in vitro and in vivo.

EO9 [3-hydroxy-5-aziridinyl-1-methyl-2(1H-indole-4,7-dione)-prop-beta-en- alpha-ol] was selected for clinical trial in Europe because of its preclinical profile but also because of its distinct mechanism of bioactivation. Several studies have shown that cells rich in DT-diaphorase may be particularly sensitive to EO9. The present study examined the relationship between DT-diaphorase activity and sensitivity to EO9 in a panel of cell lines largely derived from human and rodent leukaemias/lymphoma and solid tumours. A possible relationship between chemosensitivity and enzyme activity was demonstrated (correlation coefficient 0.796). A number of the human cell lines were established as xenografts in nude mice but, with the exception of HT29, DT-diaphorase specific activity was greatly reduced compared with the corresponding cell lines. These data suggest that in vitro studies of bioactivation of drugs by specific enzymes is unlikely to be relevant for the same tumour in vivo. Except for HCLO, all xenografts failed to respond to EO9 as a single dose. HT29 tumours in vivo had similar DT-diaphorase activity [359 nmol of 2,6-dichlorophenol-indophenol (DCPIP) reduced per min per mg of protein] to the cell line (337) but failed to respond to a single dose or daily dose schedule. A preliminary attempt to investigate an hourly dose schedule demonstrated a modest anti-tumour effect accompanied by enhanced toxicity. Attempts to optimise EO9 exposure parameters to potentiate activity in tumours with high DT-diaphorase activity are under way, but as yet the relevance of this particular enzyme for in vivo EO9 activity requires further investigation.

Animals↗
PubMed2000

Fast urinary screening for paracetamol using on-line microwave assisted hydrolysis and spectrophotometric detection.

A fully automated urinary screening system for paracetamol and its metabolites is proposed. The method comprises on-line acid microwave assisted hydrolysis of the drug to p-aminophenol followed by reaction with o-cresol in alkaline medium. The indophenol blue dye formed can be continuously monitored at 620 nm. The detection limit achieved, 0.1 microgram ml-1, allows a high dilution of the samples, thus reducing potential interferences from the sample matrix (mainly protein degradation during urine hydrolysis). The proposed screening system also possesses an adequate selectivity, as the major interferent, epinephrine, is tolerated at concentrations higher than those that could be found in the positive urine samples. The reproducibility, expressed as relative standard deviation, was 3.0% and the sample frequency 20 h-1. The reliability of the method was established at five concentrations (between 0.5 and 4 times the detection limit). Finally, it was applied to the screening of several human urine samples. The results obtained were compared with those provided by batch acid hydrolysis, and were similar in all instances.

Acetaminophen↗
PubMed1965

Effects of carbon dioxide-bicarbonate mixtures on oxidative phosphorylation by cauliflower mitochondria.

1. Carbon dioxide-bicarbonate mixtures markedly inhibited oxidation and phosphorylation rates of mitochondria prepared from cauliflower. Inhibition occurred with succinate, malate, citrate, isocitrate and NADH as substrates. 2. Indophenol-reductase systems with malate, succinate, isocitrate and NADH as substrates were inhibited by 5% and 15% carbon dioxide. Cytochrome c oxidase was not inhibited by 15% carbon dioxide.

Bicarbonates↗
PubMed1967

Purification and properties of a soluble reduced nicotinamide-adenine dinucleotide (phosphate) dehydrogenase from the hepatopancreas of Octopus vulgaris.

1. The oxidation of NADH and NADPH catalysed by the soluble supernatant from the hepatopancreas of Octopus vulgaris is due to a single enzyme, which has been purified approximately 100-fold. The enzyme reacts rapidly with potassium ferricyanide, and more slowly with 2,6-dichlorophenol-indophenol. No activity is obtained with oxygen, cytochrome c, lipoic acid, vitamin K(1), vitamin K(3), ubiquinone-30, p-benzoquinone, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride or methylene blue. 2. GSH, cysteine and mercaptoethanol stimulate the enzymic activity up to fivefold. GSSG is without any apparent effect. When stimulated by GSH the enzyme becomes sensitive to dicoumarol, which produces an inhibition competitive with respect to the activator. 3. The purified enzyme contains an acid-removable flavine component, which has been identified as FMN by spectrofluorimetry and chromatography in three solvent systems. After acid ammonium sulphate treatment the enzymic activity is lost, but it can be almost fully restored by incubation with FMN. FAD produces only a partial reactivation.

Animals↗
PubMed1969

Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride.

1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.

Animals↗
PubMed1970

Mammalian D-2-hydroxy acid dehydrogenase. Effect of inhibitors and reaction sequence.

1. The reaction of d-2-hydroxy acid dehydrogenase with d-lactate and 2,6-dichlorophenol-indophenol (DCIP) at pH8.6 yields reciprocal plots of 1/rate versus 1/[d-lactate], at different DCIP concentrations, which appear to be parallel. However, at pH7.55, or in the presence of the competitive inhibitor oxalate at pH8.6, the plots are convergent. This is inconsistent with the mechanism previously proposed for this enzyme. 2. The pattern of inhibition by the product, pyruvate, is consistent with either an Ordered mechanism or an Iso Theorell-Chance mechanism. 3. The observation that the enzyme forms a complex with d-lactate favours the Ordered reaction. In this, first d-lactate and then DCIP bind to the enzyme to form a ternary complex, from which pyruvate and reduced DCIP dissociate in that order.

Alcohol Oxidoreductases↗
PubMed1974

Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enxyme by sulphide and by selenide.

1. Turkey liver xanthine dehydrogenase engaged in catalysing the oxidation of xanthine by dichlorophenol-indophenol was progressively inactivated by methanol. This inactivation was reversible by NAD(+). 2. Reaction with arsenite and with cyanide, in each case first-order with respect to enzyme, resulted in characteristic alterations in the visible absorption spectrum of the enzyme. The rate of spectral change on reaction with either agent paralleled the rate of loss of enzyme activity. 3. Cyanide inactivation was accompanied by elimination from the enzyme of sulphur as thiocyanate. Partial restoration of activity was effected by incubation with sulphide or with selenide. The results suggest that turkey liver xanthine dehydrogenase, like milk xanthine oxidase (Massey & Edmonson, 1970), contains at the active centre a cyanolysable persulphide group essential to catalytic activity and that selenium may replace sulphur in this group to give an active enzyme. 4. Incubation of the native enzyme with sulphide or with selenide resulted in the rapid loss of half of the xanthine-oxidizing activity, apparently by disrupting the molybdenum and (Fe/S)II loci. This may indicate non-equivalence of the intramolecular electron-transfer systems.

Animals↗