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At least 199 records · Page 11Linked to original sources
PubMed2002

Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT.

Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Animals↗
PubMed1985

Peroxidase-mediated oxidation of isoniazid.

Oxidation of isonicotinic acid hydrazide (isoniazid) by horseradish peroxidase at the expense of H2O2 yielded reactive species which were able to reduce nitroblue tetrazolium and bleach p-nitrosodimethylaniline. Nicotinic acid hydrazide oxidation did not cause these effects. At slightly alkaline pH, oxidation of isonicotinic acid hydrazide by horseradish peroxidase proceeded at the expense of molecular O2, and the reaction was oxygen consuming. The addition of H2O2 abolished O2 consumption. Bovine liver catalase enhanced the rate of nitroblue tetrazolium reduction and decreased the maximal velocity of the reaction proportionately to catalase concentration. During oxidation of isonicotinic acid hydrazide by horseradish peroxidase-H2O2, splitting of the heme group of horseradish peroxidase took place as shown by the disappearance of the Soret and minor bands in the visible region of the spectrum.

Catalase↗
PubMed1985

Enzymatic and nonenzymatic superoxide-generating reactions of isoniazid.

During the course of horseradish peroxidase-mediated oxidation of either o-dianisidine or 2-2'-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS), no O2 consumption took place. When isonicotinic acid hydrazide (isoniazid) (INH) was included in the reaction mixture, O2 was consumed in amounts linearly related to the INH concentration. Nicotinic acid hydrazide at equimolar concentrations induced lower rates of O2 consumption. Superoxide dismutase activated O2 consumption. At equimolar concentrations, INH, nicotinic acid hydrazide, and phenylhydrazine induced bleaching of p-nitrosodimethylaniline in the horseradish peroxidase mediation of ABTS oxidation. Bleaching was not inhibited by hydroxyl radical (. OH) scavengers. After a short lag period, INH reacted with NADH at alkaline pH to produce superoxide radical (O2-), as detected by superoxide dismutase-inhibitable Nitro Blue Tetrazolium reduction. Nicotinic acid hydrazide with NADH caused a lower rate of O2- production after a longer lag period than INH.

Catalysis↗
PubMed1997

Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining.

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to a fluorescently labeled universal 16S rRNA probe; (sup3)H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4(prm1),6-diamidino-2-phenylindole) staining; staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a compound supposed to indicate oxidative cell metabolism; and total bacterial counts (classical DAPI staining), taken as a reference. For the universal-probe counts, we used an image intensifying and processing system coupled to the epifluorescence microscope. All of the above-mentioned methods yielded lower cell counts than DAPI total counts. Universal-probe counts averaged about half of the corresponding DAPI count and were highly correlated to autoradiography counts (r(sup2) = 0.943; n = 7). Nucleoid-containing cell counts could be lower than DAPI counts by as much as 1 order of magnitude but sometimes matched autoradiography or probe counts. CTC counts were 2 orders of magnitude below DAPI counts. Universal 16S rRNA probe counts correlated well with autoradiography results, indicating a population with at least minimal metabolic activity. The greater variability of the nucleoid-containing cell counts calls for further investigation of the processes involved, and CTC counts were well below the range of the other methods tested.

Journal Article↗
PubMed1996

Myocardial uptake of 99mTc-tetrofosmin, sestamibi, and 201Tl in a model of acute coronary reperfusion.

BACKGROUND: To investigate whether tetrofosmin uptake is affected by myocardial viability as has been noted for 201Tl and sestamibi, we analyzed the initial and delayed distribution patterns of tetrofosmin in a rat coronary artery occlusion-reperfusion model. METHODS AND RESULTS: Animals were intubated and ventilated, and their arterial pressures were monitored. A left thoracotomy was performed. After 1-hour occlusion and 1-hour reperfusion of a major branch of the circumflex artery, 201Tl and either tetrofosmin or sestamibi were injected intravenously. Radiolabeled microspheres were used to document the area at risk and reperfusion. Five minutes or 1 hour after administration of the diffusible tracers, the animals were killed. Tracer distribution was determined by use of segmental tissue analysis, and tissue viability was determined by use of histochemical staining. Both the initial and delayed retention of tetrofosmin were sensitive to myocardial viability, as shown by significantly lower uptake (30 +/- 14%) and retention (24 +/- 12%) of tetrofosmin in the nonviable segments compared with the viable segments. In addition, the initial myocardial distribution of tetrofosmin was similar to that noted for 201Tl, but after 1 hour of tracer circulation, the tetrofosmin tissue distribution appeared unchanged compared with the initial regional blood flow distribution. This is in direct contrast to our present observations of significant 201Tl redistribution and some changes in sestamibi distribution as well. CONCLUSIONS: The clinical implication of these observations suggests that initial and delayed imaging after tetrofosmin administration would reflect both the initial regional blood flow pattern and myocardial viability.

Animals↗
PubMed1991

Do the copper complexes of histamine, histidine and of two H2-antagonists react with O2-?

The effects of cimetidine, ranitidine, histamine and histidine, as well as of their copper complexes, have been examined in an enzymic and chemical O2- generated systems. Copper complexes like CuZnSOD inhibited both the reduction of cytochrome c and NBT2+ in xanthine-xanthine oxidase systems, but their inhibitory action was due to a certain extent to the copper-induced inhibition of xanthine oxidase. EDTA abolished the inhibitory effect of all copper complexes studied. Luminol chemiluminescence in NADH2-PMS system was inhibited by CuZnSOD while it was enhanced by copper complexes. The copper-accelerating effect gradually increased up to about 1 microM Cu and decreased, reaching the control values up to 10 microM Cu. In the presence of low copper concentrations chemiluminescence was inhibited by CuZnSOD only, while in the presence of high copper concentrations it was inhibited by catalase and mannitol, but not by CuZnSOD. The ligands however, have been ineffective in the two O2- generated systems.

Catalase↗
PubMed1993

Effects of coumarin derivatives on superoxide anion generation.

A number of simple coumarins with different patterns of substitution were tested on activated polymorphonuclear leukocyte superoxide generation and superoxide scavenging using the reduction of nitroblue tetrazolium. The most effective compound was fraxetin, which showed a better potency as an inhibitor of superoxide radical generation than as a scavenger. From the structure-activity study it can be deduced the importance of an ortho-dihydroxyl function, especially at the position 7, 8. Blockade of active hydroxyl groups by methylation or glycosylation decreases the activity. None of the coumarins were cytotoxic on leukocytes as assessed by lactic dehydrogenase activity release. Inhibition of superoxide generation in activated neutrophils may play a role in the vasoprotective effects of simple coumarins.

Animals↗
PubMed1998

Correlations between uptake of technetium-99m Q12 and thallium-201: myocardial perfusion and viability in a model of acute coronary reperfusion.

UNLABELLED: To investigate whether Q12 uptake is affected by myocardial viability, as has been noted for 201Tl and sestamibi, we analyzed the initial and delayed distribution patterns of Q12 in a rat coronary artery occlusion-reperfusion model. METHODS: Animals were intubated and ventilated, and their arterial pressures were monitored. A left thoracotomy was performed. After a 1-hr occlusion and a 1-hr reperfusion of a major branch of the circumflex artery, 201Tl and Q12 were injected intravenously. Radiolabeled microspheres were used to document the areas of risk and reperfusion. The animals were killed at 5 min or 1 hr after administration of the diffusible tracers. Tracer distribution was determined by segmental tissue analysis, and tissue viability was determined by histochemical staining. RESULTS: Both the initial uptake and delayed retention of Q12 are sensitive to myocardial viability as shown by significantly lower uptake (28% +/- 8%) and retention (41% +/- 13%) of Q12 in the nonviable as compared to the viable segments (p < 0.001). In addition, the myocardial retention of Q12 was significantly less in the nonviable tissue when compared to the initial uptake (p < 0.01). CONCLUSION: The clinical implication of these observations suggests that initial and delayed imaging after Q12 administration would reflect both the initial regional blood flow pattern and myocardial viability. Also, delayed imaging of Q12 may reflect viability better than the initial imaging.

Animals↗
PubMed2000

Antioxidant compounds interfere with the 3.

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner. Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product. These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments.

Acetylcysteine↗
PubMed1988

Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds.

A rapid, sensitive and automated assay procedure was developed for the in vitro evaluation of anti-HIV agents. An HTLV-I transformed T4-cell line, MT-4, which was previously shown by Koyanagi et al. (1985) to be highly susceptible to, and permissive for, HIV infection, served as the target cell line. Inhibition of the HIV-induced cytopathic effect was used as the end point. The viability of both HIV- and mock-infected cells was assessed spectrophotometrically via the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The procedure was optimized as to make optimal use of multichannel pipettes, microprocessor-controlled dispensing and optical density reading. The absorbance ratio of the mock-infected control to the HIV-infected samples was about 20. This allowed an accurate determination of the 50% effective doses, as demonstrated for 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddCyd), dextran sulfate and heparin. The technique significantly reduced labor time as compared to the trypan blue exclusion method, and permits the evaluation of large numbers of compounds for their anti-HIV activity.

Acquired Immunodeficiency Syndrome↗
PubMed1991

An application of tetrazolium (MTT) colorimetric assay for the screening of anti-herpes simplex virus compounds.

A rapid and sensitive procedure was developed for in vitro evaluation of anti-herpes simplex virus (HSV) agents. The procedure is based on spectrophotometrical assessment for viability of virus- and mock-infected cells via in situ reduction of a tetrazolium dye MTT, which has already been used for the detection of anti-human immunodeficiency virus (HIV) agents (Pauwels et al., 1988). Monolayer cells such as human embryonic fibroblast, VERO, or HeLa cells were not suitable for this purpose. Among the non-adherent cell lines examined for susceptibility to HSV type 1 (HSV-1), a B-lymphoblastoid cell line NC-37 was found to be the most sensitive. The cell line was found to have a good correlation between the viable cell number and the reduction of MTT. In addition, centrifugation of the virus-infected cells resulted in further increase of the sensitivity of NC-37 cells to HSV-1. After optimization, the method proved to be as sensitive as plaque reduction. The system simplifies significantly the assay procedures and thus permits the evaluation of larger numbers of compounds for anti-HSV-1 activity.

Animals↗
PubMed2005

In vitro activity of cycloSal-nucleoside monophosphates and polyhydroxycarboxylates against orthopoxviruses.

Because variola virus might be used as a pathogen in biological attacks, there is an urgent need to provide effective antiviral drugs for the treatment of orthopoxvirus infections. Thus, the aim of the present study was to test the antiviral activity of 3 pro-nucleotides of the acyclic nucleoside analogues aciclovir (ACV), 3 of penciclovir (PCV) and 38 of the cyclic nucleoside analogue brivudin (BVDU), on the basis of cycloSaligenyl-nucleoside monophosphate approach against vaccinia virus and cowpox virus in vitro. In further experiments, 13 synthetic humic acid-like polymers, so-called polyhydroxycarboxylates, were examined. Antiviral screening was performed by means of the plaque reduction assay and for quantification of the cytotoxicity of the test compounds the XTT-based tetrazolium reduction assay EZ4U was used. As result, three cycloSal-monophosphate derivatives of ACV proved to be potent inhibitors of both vaccinia virus and cowpox virus replication in vitro. Among the tested monophosphate derivatives of cycloSal-PCV and cycloSal-BVDU, selected substances showed a promising antiviral activity against vaccinia virus and cowpox virus. For the polyanionic compounds, no relevant antiviral activity was detected. In conclusion, by the delivery of nucleoside monophosphates from neutral, membrane-permeable prodrugs on the basis of the cycloSaligenyl-nucleotide concept, different ACV, PCV and BVDU derivatives can act as potent and selective inhibitors of orthopoxvirus replication. However, most of the cycloSal-monophosphate derivatives of BVDU had a higher cytotoxicity than their parent nucleosides.

Acyclovir↗
PubMed2000

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC.

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Biological Transport↗