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At least 649 records · Page 36Linked to original sources
PubMed1989

A micellar model for investigating the chemical nature of hydrogen transfer in NAD(P)H-dependent enzymatic reactions.

Aqueous micelles of Triton X-100 were shown to catalyse the redox reaction between NADH and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) at the neutral pH. The transfer of reducing equivalents between the reactants in the micellar system appeared to be direct and quantitative. N-tert-butylphenyl-alpha-nitrone, a lipophilic free-radical scavenger which can enter micelles, and superoxide dismutase did not alter the stoichiometry of the reaction. The oxidation product of NADH was found to be 100% enzymatically active. The IR spectrum of INT-formazan (i.e., the product of INT reduction) showed an absorbance at 3,100-3,700 cm- due to NH-stretching. The presence of NH proton, confirmed further by IH-NMR, together with the above observations suggests that INT, as part of the over-all redox process, abstracts a C(4) hydrogen of the dihydropyridine nucleus of NADH with a simultaneous cleavage at N(2-3) position of its 1,2,3,4-tetrazole ring system and that the redox events are confined to a microenvironment as in the case of NAD(P)H-dependent enzymatic reactions.

Colloids↗
PubMed1990

Induction of a 70 kD protein associated with the selective cytotoxicity of beta-carotene in human epidermal carcinoma.

Beta-carotene and canthaxanthin at concentrations of 70 or 300 microM were shown to inhibit the proliferation of cultured human squamous cells (SK-MES lung carcinoma and SCC-25 oral carcinoma) in a 5 hr cell density assay. Responses were similar for both tumor cell lines, ranging from 71-84% inhibition. In contrast, equimolar concentrations of alpha-tocopherol gave only 19-36% inhibition of SCC-25, but 50-75% inhibition of SK-MES cell density. Equimolar reduced glutathione resulted in 4-15% stimulation of SCC-25 and 22-25% inhibition of SK-MES cell proliferation. With cultured normal keratinocytes, treated final cell densities did not differ significantly from those of controls. Two additional assays measuring the metabolic generation of formazan (MTT assay) and [5-3H]thymidine incorporation were in substantial agreement with the growth inhibition pattern. Thus both continuous and cyclic cellular processes are involved in the tumor-specific response. Onset of the response to beta-carotene alone or in combination with alpha-tocopherol is signalled within 1-2 hours of treatment by the appearance of a unique 70 kD heat-shock protein.

Canthaxanthin↗
PubMed1991

Cytotoxicity and activation of CB1954 in a human tumour cell line.

CB1954 (5-aziridin-1-yl-2,4-dinitrobenzamide) is a monofunctional alkylating agent, to which Walker 256 cells are very sensitive. These cells express a nitroreductase which reduces CB1954 to a bifunctional crosslinking agent 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. In vitro testing on the human colon line LS174T showed that the differential cytotoxicities between the monofunctional agent (CB1954), and the active species (generated in situ by the addition of NADH and the Walker rat nitroreductase) were smaller than anticipated due to the unexpected toxicity of CB1954 (IC50 value for CB1954 on LS174T cells of 78 microM). The toxicity of the chemically synthesised active form was less than if it had been generated in situ (on LS174T cells). Further experiments showed that NADH was toxic at the levels used to generate the active species (500 microM). Gel filtration and electrophoresis experiments demonstrated that the human colon carcinoma and choriocarcinoma cell lines MAWI and JAR, as well as LS174T express an enzyme of similar molecular weight to that of the 33 kD Walker cell nitroreductase, which is capable of reducing CB1954 to its toxic metabolite, and reducing MTT to its insoluble formazan salt. The expression of this enzyme presumably accounts for the unexpected toxicity of CB1954.

Animals↗
PubMed1991

Differential effects of oltipraz and its oxy-analogue on the viability of Schistosoma mansoni and the activity of glutathione S-transferase.

Adult worms of Schistosoma mansoni recovered from mice treated with oltipraz (OPZ) showed a significant diminution in their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, a measure of parasite viability. Incubation of glutathione S-transferase (GST) from S. mansoni with OPZ resulted in a time- and concentration-dependent inhibition of enzyme activity. RP 36,642 (an inactive oxy-derivative of OPZ) had a minimal effect on the viability of the worms and no effect on GST activity. The structural integrity of OPZ, particularly the thione sulphur, appears to be necessary for expression of the antischistosomal effects of the drug. OPZ-induced inhibition of GST was non-competitive with either reduced glutathione (GSH) or 1-chloro-2,4-dinitrobenzene (CDNB), indicating that the drug is not a substrate for GST-catalysed conjugation reactions. In addition, the inhibition of GST could not be reversed by dialysis or repurification of the enzyme via a GSH-agarose affinity column. The effects of OPZ on GST activity could render the parasite vulnerable to damage by host-derived reactive oxygen species and aldehydic products of lipid peroxidation. The effects of OPZ on GST activity may play a role in the antischistosomal action of OPZ.

Animals↗
PubMed1992

Drug transport, viability and morphology of isolated rat hepatocytes preserved for 24 hours in University of Wisconsin solution.

Isolated hepatocytes are a valuable tool to study liver functions. Suitable methods to preserve the isolated cells with good maintenance of viability and functions are crucial to extend experiments with hepatocytes from a single isolation over 2 or more consecutive days. We investigated whether University of Wisconsin (UW) solution, which was designed to preserve organs for transplantation, is also suitable for preservation of isolated rat hepatocytes. Viability, as determined by Trypan blue exclusion and reduction of the tetrazolium 3(4,5-dimethyl-thiazoyl-2-yl)2,5 diphenyltetrazolium bromide to a purple formazan, morphological appearance using electron microscopy, ATP levels and uptake and storage of three model drugs ([3H]vecuronium, [3H]taurocholic acid and [3H]ouabain) were determined directly after isolation and after 22 hr of storage in UW solution at 0-4 degrees. The present study shows that cold storage of rat hepatocytes for 22 hr in UW can be performed without significant loss of viability and with maintenance of proper morphology, cellular ATP and transport functions. In contrast, after storage in Krebs-Henseleit buffer the normal morphology, ATP content and transport functions were strongly affected. These results imply that hepatocytes from a single isolation and stored in UW solution can be used for experiments on 2 consecutive days.

Adenosine Triphosphate↗
PubMed1983

Localization of 2',3'-cyclic nucleotide-3'-phosphohydrolase within the vertebrate retina.

Histochemical and immunochemical techniques are used to locate 2',3'-cyclic nucleotide-3'-phosphohydrolase (2',3'-cNMP-3'ase) within cells of the vertebrate retina. A new histochemical method is described which links the hydrolysis of 2',3'-cNADP to the formation of a reduced, insoluble tetrazolium formazan. Photoreceptors from fish, bovine, and rat retinas are stained by this procedure. The reaction is blocked by 2'-AMP, a known inhibitor of 2',3'-cNMP-3'ase. Rabbit antibodies prepared against 2',3'-cNMP-3'ase from bovine brain are found to cross-react with bovine and rat retinal enzymes. Peroxidase-labeled antibody shows by light microscopy the greatest staining along the inner segments of the photoreceptors. Electron microscopy of the same preparations confirms binding to the plasma membrane of the inner segments of both rods and cones. Retinal 2',3'-cNMP-3'ase is thus predominantly associated with the photoreceptors, suggesting some role for 2',3'-cyclic nucleotides as substrates in visual function.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1988

Ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in the cat cerebral cortex, amygdala and caudate nucleus.

The ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in cat cerebral cortex, amygdala and caudate nucleus was investigated by electron microscopy using a modified method applicable to aldehyde-fixed tissues. These NADPH diaphorase-positive neurons were morphologically similar to neurons immunohistochemically positive for somatostatin. They had large amounts of electron-dense formazan reaction products scattered through the whole cytoplasm but not in the mitochondria or nucleus. Similar electron-dense reaction products were visible in the dendrites of these neurons. The results indicate that NADPH diaphorase histochemistry is a useful method for the ultrastructural examination of particular groups of neurons.

Amygdala↗
PubMed1992

Effect of nitric oxide on mitogenesis and proliferation of cerebellar glial cells.

In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesis and cell proliferation of the cerebellar glial cells were assessed. NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO-generating agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernatants. However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitochondrial activity in the glial cells. These results demonstrate that NO inhibits serum-induced mitogenesis and cell proliferation of cultured rat cerebellar glial cells.

Animals↗
PubMed1977

Determination of ethanol in serum by an enzymatic PMS-INT colorimetric method.

A simple, rapid and accurate colorimetric method for the determination of ethanol in serum was studied. Ethanol is converted to acetaldehyde by alcohol dehydrogenase, and the NADH formed transfers its hydrogen through the phenazine methosulphate-p-iodonitrotetrazolium violet (PMS-INT) system to produce the red formazan which is stable and absorbs at 505 nm. The method correlates very well with an enzymatic-UV method, r=0.98 (p less than 0.001). The precision (95% limits) of the method for samples ranging from 11 to 145 mg/dl was +/-6.6%, and the recovery averaged 99%.

Colorimetry↗
PubMed1985

A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry.

A method for greatly enhancing the sensitivity of assays employing enzyme labels is described which offers advantages in assays for a wide range of analytes. The principle of the new approach is that the enzyme label gives rise to a catalytic activator for a specific secondary detection system, the activity of which is measured and related back to the amount of label present and thus of the analyte it is being used to determine (C.H. Self, Eur. Pat. Appl. 80303478.4, 15.4.81 exclusively licenced to IQ (Bio) Ltd.). The general principle of enzyme amplification is illustrated by using alkaline phosphatase as the labelling enzyme and nicotinamide adenine dinucleotide phosphate (NADP) as its substrate. The nicotinamide adenine dinucleotide (NAD) formed catalytically activates a strictly NAD specific redox cycle which produces a coloured formazan as the end product. The measured absorbance is at least two orders of magnitude greater than that achieved by conventional methods. The application of this method to immunoassay is demonstrated by a sensitive, rapid and precise assay for human prostatic acid phosphatase (PAP). Some of the many other applications of this methodology are discussed.

Acid Phosphatase↗
PubMed1992

Colorimetric determination of galactose and galactose-1-phosphate from dried blood.

A colorimetric microassay for the simultaneous quantitative determination of galactose (Gal) and galactose-1-phosphate (Gal-1-P) in dried blood spots is described. An enzymatic reaction involving alkaline phosphatase (EC 3.1.3.1) and galactose dehydrogenase (EC 1.1.1.48) produces NADH, which is coupled with diaphorase (EC 1.8.1.4) and iodonitrotetrazolium violet (INT). The colourless INT is converted to a formazan of red colour the intensity of which is quantitated either photometrically by a microplate reader or determined visually with sufficient sensitivity for screening purposes. We evaluated the assay on 200,000 blood samples in a newborn screening program, and were able to distinguish between classical and milder forms of galactosemia with ease.

Alkaline Phosphatase↗
PubMed1989

Monosialoganglioside GM1 protects against anoxia-induced neuronal death in vitro.

Excitatory dicarboxylic amino acid neurotransmitters, particularly glutamate, have been implicated in mediating neuronal cell injury in brain ischemia-anoxia, epilepsy, and stroke. Glutamate neurotoxicity has been demonstrated in several in vitro models, as well as its prevention by a variety of agents, including several sialic acid-containing glycosphingolipid species, gangliosides. We have now examined ganglioside effects in anoxic exposed cultures of granule cells from Postnatal Day 8 rat cerebellum. Cells between 10 and 12 days in vitro were placed into an anoxic atmosphere or subjected to a chemical model of anoxia by a pulse exposure to rotenone. Widespread neuronal degeneration of neuronal cell bodies and their associated neurite network was seen the following day. These effects on cell vitality at the morphological level were quantitatively confirmed by measuring the photometric reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a blue formazan product. This neuronal injury was abolished by the specific N-methyl-D-aspartate receptor noncompetitive antagonists Mg2+, phencyclidine and MK-801, suggesting that this subtype of glutamate receptor is involved in the pathogenesis of anoxic granule cell injury. Pretreatment for 30 to 60 min or more or concurrent treatment with ganglioside GM1 largely prevented the ensuing neuronal death (ED50 = 25 microM), even 4 days later. Degeneration induced by exogenous glutamate was equally reduced. Asialo GM1 (lacking sialic acid) was ineffective. These results are consistent with the observed beneficial effects of the gangliosides in ischemic brain injury models in vivo.

Animals↗
PubMed1995

The first observation of O2- generation at real time in vivo from non-Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication.

Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2- which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2- in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol-induced O2- generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2- generation was completely abolished by GdCl3. Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non-Kupffer cell population, probably liver sinusoid endothelial cell to produce O2-.

Animals↗
PubMed1995

Effect of tauroursodeoxycholic and ursodeoxycholic acid on ethanol-induced cell injuries in the human Hep G2 cell line.

BACKGROUND & AIMS: Taurodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) exert a protective effect in chronic cholestasis. This study reports the effect of TUDCA and UDCA on an in vitro model for ethanol-induced liver damage. METHODS: Hep G2 cells were incubated for 24 hours with 80 mmol/L ethanol in the presence or absence of 50 mumol/L TUDCA or UDCA. Cells were also pretreated with 80 mmol/L EtOH and then exposed to 50 mumol/L bile acids. Cytotoxicity was assessed by the metabolism of formazan (3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide and sodium 3,3'-(phenylamino) carbonyl-3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrase and by the release into the culture medium of different enzymes (aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transferase, and lactate dehydrogenase). RESULTS: The incubation of Hep G2 with EtOH significantly (P < 0.001) increased cytotoxicity. Both TUDCA or UDCA reduced cytoxicity to a similar extent (P < 0.001). Cells pretreated with EtOH and then added with TUDCA or UDCA responded differently because TUDCA was significantly more effective (P < 0.05) than an equimolar dose of UDCA in reversing the damage. Electron microscopic examination revealed that TUDCA and UDCA were both able to prevent mitochondrial damage and to reduce steatosis induced by EtOH. CONCLUSIONS: Low doses of TUDCA and UDCA protect Hep G2 cells from EtOH-induced cytotoxicity. However, TUDCA seems to be more effective than UDCA in reversing the damage.

Cell Survival↗
PubMed1995

Lipid peroxidation by activated platelets: a possible link between thrombosis and atherogenesis.

Generation of reactive oxygen species (ROS) by platelets was detected by a cyano-tetrazolium dye (CTC) which forms fluorescent formazan on the cell surface upon reduction. Fluorescence was quantitated by a densitometric device. Resting platelets in plasma produced significant fluorescence (P < 0.0001), and addition of thrombin enhanced the fluorescence of the coagulated platelet mass even further (by 2 h, a 6- and 8-fold increase over fluorescence of platelet-free plasma was measured, respectively). Blood containing CTC was perfused through a glass capillary tubing and the action of shear forces resulted in the formation of an occlusive platelet thrombus. Such thrombi (formed either from whole blood or platelet-rich plasma) were intensely fluorescent, indicating formation of ROS in the platelet mass (a 10- and 8-fold increase in fluorescence over coagulated plasma, respectively). Lipid peroxide content of resting platelets in platelet-rich plasma was doubled over 24 h storage, while addition of thrombin caused a 7.4-fold increase (P < 0.0001) of lipid peroxides in the retracted platelet-rich plasma-clot. Transition metal chelator and antioxidant prevented lipid peroxidation by platelets in response to thrombin. Thrombin activation of (washed) platelets in plasma-free medium caused only 1.4-fold increase in oxidation of added low density lipoprotein (LDL). In contrast, thrombin activation of platelets suspended in de-lipidated autologous plasma resulted in a 5.25-fold increase (P < 0.0001) in LDL oxidation. Generation of ROS and lipid peroxides by platelets can be an important mechanism through which thrombotic events contribute to atherogenesis.

Arteriosclerosis↗
PubMed1982

Separation and detection of nitroblue tetrazolium-reducing enzymes from human polymorphonuclear leukocytes.

A technique for detecting the nitroblue tetrazolium-reducing activity of human polymorphonuclear leukocyte enzymes separated on polyacrylamide gels is reported. Slices of gel are incubated in buffer containing nitroblue tetrazolium and either NADPH or NADH. Enzymes capable of reducing nitroblue tetrazolium form a dark band due to the formation of the insoluble formazan. By this technique the soluble fraction of human polymorphonuclear leukocyte sonicates was shown to contain several enzymes capable of reducing nitroblue tetrazolium in the presence of NADPH and NADH.

Chemical Fractionation↗
PubMed1985

Enzyme amplification can enhance both the speed and the sensitivity of immunoassays.

Enzyme immunoassays (EIA) are now used for the quantitation of a wide range of clinically important analytes and have, in many cases, replaced radioimmunoassays, though without improving on the sensitivity of the latter technique. We describe a general enzyme-amplification method which can be used to increase both the speed and the sensitivity of EIA. In this method, the enzyme label is used to catalyse the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP+); the NAD+ so formed then catalytically activates an NAD+-specific redox cycle, yielding an intensely coloured formazan dye. The application of this new enzyme detection method has made possible an assay for human thyroid-stimulating hormone (TSH) with a sensitivity of 1 X 10(-5) IU/1 and a progesterone assay which takes only 15 min to complete.

Animals↗
PubMed1985

Homogeneous colorimetric enzyme inhibition immunoassay for cortisol in human serum with Fab anti-glucose 6-phosphate dehydrogenase as a label modulator.

The Fab fragment of rabbit IgG antibody to bacterial glucose 6-phosphate dehydrogenase covalently linked to the cortisol retained the capacity to inhibit the enzyme completely. In optimal conditions the antibody to cortisol effectively bound the cortisol residues of the cortisol-Fab conjugate, making it incapable of inhibiting the enzyme. The enzyme modulatory properties of the cortisol-Fab conjugate were exploited to set up a direct competitive homogeneous enzyme immunoassay for cortisol in human serum. The procedure involved the use of the auxiliary enzyme diaphorase, specific for NADH, which converts the nitro blue tetrazolium salt to a colored formazan. The procedure detects modulated glucose 6-phosphate dehydrogenase activity by a single-point measurement without serum interference. The assay working range was between 20 and 640 micrograms/1 of cortisol and used 50 microliters of sample.

Antibody Specificity↗