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Kinetics of the reversible tight-binding inhibition of pig liver catechol-O-methyltransferase by [2-(3,4-dihydroxy-2- nitrophenyl)vinyl]phenyl ketone.

The inactivation of partially purified pig liver catechol-O-methyltransferase (COMT) by [2-(3,4-dihydroxy-2-nitrophenyl)vinyl]phenylketone has been studied. The results demonstrated that COMT is inhibited in a reversible tight-binding fashion with an apparent Ki of 0.2 microM. IC50 values were determined at different concentrations of both substrates of the enzymatic reaction, pyrocatechol and S-adenosyl-L-methionine (AdoMet). The plot of IC50 versus pyrocatechol concentration gave a straight line, suggesting competitive inhibition. However the nitrocatechol derivative showed an uncompetitive inhibition pattern when measured as a function of AdoMet concentration.

Animals↗

Inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures.

A large number of compounds including actinobolin, adrenochrome, amicetin, anisomycin, aurintricarboxylic acid, blasticidin S, chartreusin, chlortetracycline, cycloheximide, doxycycline, edeine A1, edeine complex, emetine, fusidic acid, gougerotin, GppCH2p, oxytetracycline, pactamycin, polydextran sulphate, puromycin, pyrocatechol violet, sparsomycin and tubulosine have been tested for inhibitory effects on protein synthesis in cultured cells from both mouse fibroblasts (3T6 cells) and chick embryo fibroblasts (CEF). Essentially, similar results were obtained with both cell types with the most effective inhibitors being pactamycin, emetine, tubulosine, anisomycin and cycloheximide and with no significant inhibitory activity being detected with edeine complex, edeine A1, GppCH2p, polydextran sulphate, aurintricarboxylic acid, pyrocatechol violet and adrenochrome. The concentration of pactamycin required to produce 50% inhibition of protein synthesis approximated 5 X 10(-9) M, but for most of the inhibitors it ranged from 5 X 10(-6) M to 5 X 10(4) M. The molecular basis underlying these differences may be related, in addition to their intrinsic inhibitory power, to differences in permeability of the cells towards the various drugs tested. Alternatively, active accumulation of the drugs by the cells may be the variable parameter.

Animals↗

Isolation and characterization of catechol 1,2-dioxygenases from Rhodococcus rhodnii strain 135 and Rhodococcus rhodochrous strain 89: comparison with analogous enzymes of the ordinary and modified ortho-cleavage pathways.

Catechol 1,2-dioxygenases of the ordinary ortho-cleavage pathway have been isolated from strains Rhodococcus rhodnii 135 and Rhodococcus rhodochrous 89 grown on phenol as the sole source of carbon and energy. The activities of the catechol 1,2-dioxygenases with 3- and 4-methylpyrocatechols were 1.3-1.5 times higher than those with pyrocatechol. The rate of oxidation of 3-chloropyrocatechol catalyzed by both enzymes was 20% of the rate of oxidation of unsubstituted pyrocatechol. The enzymes are homodimers composed of 37-kD subunits.

Catechol 1,2-Dioxygenase↗

[Microbial degradation of components of waste water from phenol-producing industry].

Processes of aerobic biodegradation of components of phenol production sewage (phenol, acetophenone, dimethylphenylcarbinol, cumene hydroperoxide, alpha-methylstyrene, benzoate, and p-hydroxybenzoate) by bacterial strains obtained from the collection of Saratov Institute of Biocatalysis were studied. The metabolic reactions were shown to be oxidative and have a common catabolic sequence (cumene hydroperoxide-dimethylphenyl-carbinol alpha-methylstyrene-acetophenone-phenyl acetate-phenol-pyrocatechol-aromatic ring breakage). Benzoate and p-hydroxybenzoate were degraded through the formation of pyrocatechol and protocatechuate, respectively. Metabolic pathways were similar in model mixtures of components and sewage samples.

Biodegradation, Environmental↗

[Determination of activator aluminum by catalytic kinetic spectrophotometry].

In this paper, a new catalytic kinetic spectrophotometric method for the determination of activator aluminum was studied. The method was based on the activation effect of aluminum (III) on the copper (II) which catalyzes the oxidation of pyrocatechol by hydrogen peroxide in acetic acid-sodium acetate buffer solution at pH 5.0. The increase in absorbance of pyrocatechol oxidation product was monitored at 360 nm. The optimization of the operating conditions regarding measuring wavelength, concentrations of the reagents, reaction temperature, reaction time and interfering ions were also investigated and chosen. Under the optimum experimental conditions, the linear range of the determination was 0.0040-0.20 mg.L-1, the detection limit of the method was 8.2 x 10(-7) g.L-1. Combined with separation of extraction of dithizone-carbon tetrachloride solution, the method was applied to the determination of aluminum in water and tea samples with satisfactory results.

Aluminum↗

[Measurement of aluminum (III) in crops by synergistic chromatic reaction].

In the presence of OP, a sensitive color reaction of aluminum (III) on pyrocatechol violet and rhodamine 6G has been studied. In a NH4Ac-HAc (pH = 6.5) medium, aluminum (III) ions form an ions-associate complex with pyrocatechol violet and rhodamine 6G. The composition of the complex is Al (III) : PV: Rh6G = 1 : 2 : 4. The maximum absorption of the ions-associate complex is at 580 nm with an apparent molar absorptivity of 6.8 x 10(4) L x mol(-1) x cm(-1). Beer's law is obeyed in the range of 0-7 microg/25 mL of aluminum. The method is highly selective and has been applied to the determination of trace aluminum in crops with satisfactory results.

Aluminum↗

[Degradation of aniline by Delftia tsuruhatensis 14S in batch and continuous processes].

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of purification works of OAO Volzhskii Orgsintez. The strain grew on pyrocatechol and p-hydroxybenzoic acid, but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the pyrocatechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.

Aniline Compounds↗

Radiochemical detection of dihydrodiol dehydrogenase: distribution of the enzyme in male Sprague-Dawley rat tissues and its sensitivity to inhibition by indomethacin and 6-medroxyprogesterone acetate.

Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP+-dependent oxidation of (-)-7R,8R-dihydroxy-dihydro-benzo(a)pyrene and (+)-7S,8S-dihydroxy-dihydro-benzo(a)pyrene, which are potent proximate carcinogens (Smithgall, Harvey, and Penning, J. Biol. Chem., 261: 6184-6191, 1986). Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a model substrate for these reactions, dihydrodiol dehydrogenase can be assayed in rat liver cytosol by measuring the change in absorbance of the pyridine nucleotide. This method lacks the sensitivity to detect the enzyme in extrahepatic tissues. Here we describe a sensitive radiochemical assay for dihydrodiol dehydrogenase in which the oxidation of benzenedihydrodiol to pyrocatechol is coupled to O-methylation catalyzed by catechol-O-methyltransferase (EC 2.1.1.6). In this manner the pyrocatechol formed in the oxidation step can be radiolabeled using S-adenosyl[methyl-3H]methionine as methyl donor. The resulting tritiated product, guaiacol, is readily extracted into toluene and quantified by scintillation counting. Using S-adenosyl[methyl-3H]methionine at a specific activity of 0.1 microCi/nmol, the assay provides a 5000-fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the Km and Vmax values obtained for the 40-75% (NH4)2SO4 fraction of rat liver cytosol with those measured spectrophotometrically. There was close agreement between values determined radiochemically (Km = 0.77 +/- 0.11 mM, Vmax = 2.14 +/- 0.13 nmol/min/mg protein) and determined spectrophotometrically (Km = 0.96 +/- 0.10 mM, Vmax = 6.31 +/- 0.50 nmol/min/mg protein). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in extrahepatic sites of polycyclic aromatic hydro-carbon metabolism: lung greater than small intestine greater than testis greater than bladder greater than prostate. Specific activities varied over 50-fold (0.866-0.017 nmol/min/mg protein) and did not show a strict inverse correlation with organ susceptibility to PAH-induced chemical carcinogenesis. Four tissues predominantly concerned with trans-dihydrodiol oxidation (liver, lung, small intestine, and testis) contain dihydrodiol dehydrogenase which is potently inhibited by indomethacin, two of these tissues (liver and small intestine) contain dehydrogenase sensitive to inhibition by 6-medroxyprogesterone acetate. These observations suggest that indomethacin and 6-medroxyprogesterone acetate may prevent the oxidation of trans-dihydrodiol proximate carcinogens in major tissues involved in their further metabolism and activation.

Alcohol Oxidoreductases↗

Assessment of catechol-O-methyltransferase activity and its inhibition in erythrocytes of animals and humans.

A non-radiometric method to measure the catechol-O-methyltransferase (COMT) activity in erythrocytes was modified to increase its sensitivity four-fold as well as its reproducibility and applicability. The method is based on the COMT-mediated O-methylation of 4-(naphtho [1,2-d] thiazol-2-yl) pyrocatechol, the product of which was determined fluorometrically. COMT activities down to less than 1% of those present at baseline could be measured precisely and accurately. The intra- and inter-assay coefficients were below 3 and 5.3%, respectively. Basal COMT activity and the distribution between soluble and membrane-bound COMT was shown to be variable among different species (ten species tested). The applicability of the method was demonstrated by the characterization of COMT activity-time courses in human erythrocytes after oral administration of the COMT inhibitor tolcapone. The assay developed will be useful in the rapid screening and clinical development of new COMT inhibitors.

Animals↗

Enhancement effect of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and cationic surfactant on the chemiluminescence of poly(3, 4, 5-trihydroxybenzoate ester)dendrimers.

In the presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the chemiluminescence (CL) intensities of poly(3, 4, 5-trihydroxybenzoateester)dendrimers, 1 and 2, having 1,2-pyrocatechol and 1,3,5-trihydroxybenzene as core molecules and also six and nine gallic acid units in the periphery, were respectively 7.4- and 2.4-fold stronger than those of 1 and 2 in the absence of EDC. Similarly, the CL intensities of 1 and 2 in the presence of cetyltrimethylammonium bromide (CTAB) were respectively 4- and 1.7-fold stronger than those of 1 and 2 in the absence of CTAB.

Ethyldimethylaminopropyl Carbodiimide↗

Waste-free and facile solid-state protection of diamines, anthranilic acid, diols, and polyols with phenylboronic acid.

Phenylboronic acid (2) reacts quantitatively by ball-milling in the solid state with o-phenylendiamine, 1,8-diaminonaphthalene, anthranilic acid, pyrocatechol, pyrogallol, pinacol, bicyclic cis-diols, mannitol, and inositol to form the five- or six-membered cyclic phenylboronic amides or esters. Catalysts or other auxiliaries are strictly excluded as they are not required and would have to be removed after the reactions. These varied model reactions provide pure protected products without the necessity of further purifying workup and the potential for protection chemistry is demonstrated. Some of the reactions can also be quantitatively performed if stoichiometric mixtures of the reactants are co-ground or co-milled and heated to appropriate temperatures either below the eutectics or above the melting points. The temperatures are much higher in the latter case. Similar reactions in solution suffer from less than 100 % yield of the mostly sensitive compounds that are difficult to purify and thus create much waste. The hydrolysis (deprotection) conditions of the products are rather mild in most cases. Therefore, this particularly easy access to heteroboroles, heteroborolanes, heteroborinones, heteroborines, and heteroborinines is highly valuable for their more frequent use in protective syntheses.

Journal Article↗

Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem.

The efferent connections of two types neurons in the dorsal motor nucleus of the vagus nerve (DMV) were studied in the cat by light and electron microscopy following horseradish peroxidase (HRP) injections into the cervical vagus nerve or brainstem. After injections of HRP into the vagus nerve, up to 80% of medium-sized neurons averaging 26 x 20 micrometers in 1-micrometer-thick sections were retrogradely labeled while no small neurons were labeled in the DMV. Incubation with either diaminobenzidene (DAB) or p-phenylenediamine-pyrocatechol (PPD-PC) chromogens yielded electron-dense reaction products localized mainly in lysosomes. Identification of label at the ultrastructural level was facilitated by omitting lead citrate staining and by counting numbers of lysosomes, which were higher in labeled neurons. Quantitative comparisons of the dimensions of labeled and unlabeled somata demonstrated that retrograde transport and incorporation of HRP had no effect on cell size within the 2-3-day survival times used in this study. In order to determine whether neurons in the DMV project to higher levels of the brain stem, large injections of HRP (1-3 microliters) were made into the pons, mesencephalon, hypothalamus, and amygdala. After injections of HRP into the brainstem, only small neurons, measuring 17 x 10 micrometers, were retrogradely labeled. Approximately 90% of the small neurons remained unlabeled following the HRP injections. The ultrastructural features of the labeled small neurons included an invaginated nucleus, low cytoplasmic/nuclear ratio, and relatively fewer organelles than the medium-sized neurons. A quantitative analysis of labeled and unlabeled small neurons demonstrated that the labeled neurons were significantly larger than the unlabeled small neurons. Thus, two populations of small neurons may exist in the DMV. One population appears to have ascending projections to higher levels of the brainstem while the other more numerous population may be interneurons or project for only short distances.

Amygdala↗

Reduction of blue tetrazolium by corticosteroids.

Pseudo-first-order rate constants were observed for the reaction of corticosteroid or corticosteroid esters with blue tetrazolium. The data indicate that the reactivity of corticosteroids is, in part, a function of their geometry in that corticosteroid reactivity toward blue tetrazolium increases with increasing planarity of the steroid molecule and that corticosteroid esters must be hydrolyzed as a necessary prerequisite to reaction with blue tetrazolium. Evidence is presented indicating that free radicals are not involved in the blue tetrazoliun reaction with corticosteroids. Certain pharmaceutically important compounds such as pyrocatechol derivatives and hydroquinone appear to reduce blue tetrazolium by the anion free radical mechanism proposed previously. A spectrophotometric method for determining the number of reduction units transferred to blue tetrazolium per molecule is described.

Adrenal Cortex Hormones↗

Differential identification of mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors in plasma clots.

Because benzidine and its derivatives have possible carcinogenic activity, a safe method is needed to demonstrate endogenous peroxidase activity. Colonies derived from mouse bone marrow cells in plasma clot culture were classified as granulocyte (CFU-g) or macrophage (CFU-m) precursors by peroxidase and naphthol AS acetate (NASA) esterase staining, respectively. Endogenous peroxidase activity was measured using benzidine or p-phenylenediazine-pyrocatechol (PPD-PC). The effectiveness of peroxidase staining with both reagents was evaluated under several conditions, and the enzyme property was confirmed by inactivation with a variety of inhibitors. The level of peroxidase activity did not differ significantly between PPD-PC and benzidine. Colony number and number of cultured cells were strongly correlated (P greater than 0.983). We conclude that PPD-PC safely demonstrates peroxidase activity in cultured cells and is as accurate, reliable, and efficient as benzidine.

Animals↗

Effect of phenobarbital pretreatment on benzene biotransformation in the rat. II. 9,000 g supernatant and isolated perfused liver versus living rat.

Factors responsible for different quantitative effect of phenobarbital (PB) pretreatment (sodium phenobarbital, 50 mg kg-1 day-1 for 3 days) on benzene metabolism to phenol in vivo and in vitro were studied in male Wistar rats. A more than 4-fold increase of benzene metabolism was observed wih 9,000 g supernatant of liver homogenate, 2.8- to 4-fold increase with isolated perfused liver; phenol formation in vivo after oral benzene was increased by PB 2-fold, but only shortly following benzene administration and the enhancement rapidly diminished to 1.15-fold increase in the total excreted phenol. Benzene concentrations i 9,000 g supernatant incubations were 2 mM, those with isolated perfused livers were up to 4 mM, but those in blood in vivo were below 0.3 mM; the effect of PB induction in vivo disappeared along with decreasing benzene and increasing phenol blood concentrations which surpassed benzene 2-3 h after oral benzene administration. The effect of benzene concentration on the manifestation of PB induction is also supported by almost a 2-fold increased phenol formation in PB rats over controls in vivo after repeated administration of benzene. The elimination of radioactive metabolites of orally administered benzene-14C, 3 mmoles kg-1, in urine was markedly inhibited by intraperitoneal administration of phenol (1.2 mmol kg-1), but not by pyrocatechol, resorcinol or hydroquinol (0.6 mmole kg-1, respectively) suggesting that phenol might inhibit benzene metabolism in vivo especially when its concentration exceeds that of benzene.

Animals↗

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry.

Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3'-diaminobenzidine (DAB), p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co2+ and Ni2+ ions. The results indicate that DAB-imidazole and DAB-Co2+ and Ni2+ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.

Alkaline Phosphatase↗

Medusa cells: cytostructure and cytochemistry of amoeboid eosinophils with pseudopod-like processes.

Eosinophils having one or more pseudopod-like processes of various lengths are observed in bone marrow, peripheral blood, sputum, nasal smears, and in other exfoliative cytology and tissue specimens after fixation and staining for histochemical study; they are called medusa cells. Although the conformations and lengths of the processes vary, they resemble protozoal pseudopodia. The form which first called our attention to these cells is a conical determinate projection that tapers to a fine tip, much like a protozoal axopod. A basal specialization in the form of a vesicle or thickening may be frequently observed where the process protrudes from, or appears attached to, the cell body. The processes of these eosinophil variants appear morphologically specialized to interact with other cellular elements of the blood and are occasionally seen in contact with, or engulfing, erythrocytes, platelets or other leukocytes. Two hydroperoxidases have been elucidated in eosinophils and medusa cells by virtue of different substrate specificities, subcellular localizations and inhibitor sensitivities. One of these hydroperoxidases is shown by 3,3'-diaminobenzidine, is cyanide resistant, and is never observed in granules or rods in the medusa cell processes; it is frequently polarized to the sites of contact of medusa cells with other cellular elements of the blood. The other hydroperoxidase is revealed by p-phenylenediamine-pyrocatechol, is sensitive to cyanide and is frequently observed in granules and rods in medusa cell processes as well as in a population of larger granules in the cell bodies; the granules in the processes appear to be precursors to the rods, which may be related to Charcot-Leyden crystals. The extrusion of medusa cell processes is facilitated by the divalent cations calcium and magnesium and is inhibited by anions which sequester them such as phosphate, EDTA, citrate and oxalate. Medusa cells have been observed in samples from both rodents and humans and can be very prominent when eosinophilia accompanies allergy, parasitosis and malignancy.

Asthma↗