SEARCH · PubMed Health

Results for “Formazans”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources

PubMed1989

Respiratory burst activity of intestinal macrophages in normal and inflammatory bowel disease.

Macrophages isolated from normal mucosa (greater than 5 cm from tumour) and inflamed mucosa (from patients with inflammatory bowel disease) of colon and ileum were studied for their ability to undergo a respiratory burst as assessed by reduction of nitroblue tetrazolium to formazan. Using phorbol myristate acetate (PMA) and opsonised zymosan as triggers, only a minority (median: 8% for zymosan and 9% for PMA) of macrophages isolated from normal colonic mucosa demonstrated release of oxygen radicals. In contrast, a significantly greater (median: 17% for zymosan and 45% for PMA) proportion of macrophages isolated from inflamed colonic mucosa were able to undergo respiratory burst. Studies with normal and inflamed ileum showed similar results. Stimulation of macrophages isolated from normal colon with interferon-gamma produced only a small increase in the proportion of cells showing release of oxygen radicals. We conclude that the respiratory burst capacity of majority of macrophages isolated from normal colon and ileum is downregulated and a greater proportion of macrophages isolated from inflamed colon and ileum are able to undergo a respiratory burst.

Adolescent↗

PubMed1993

Location of superoxide anion generation in human colonic mucosa obtained by biopsy.

To identify which cells generate superoxide, inflamed human mucosa was tested with nitro-blue tetrazolium as a probe, because it is reduced by strong reducing agents to form insoluble blue formazan, which then precipitates in tissues. Biopsy specimens from control subjects and patients with ulcerative colitis were studied. The specimens were organ cultured with bubbling air or nitrogen, and inhibition of the reduction by catalase (a hydrogen peroxide scavenger), para-benzoquinone (a tissue permeable superoxide scavenger), or superoxide dismutase (a superoxide scavenger) was assayed. The dye was reduced by epithelial cells, vascular endothelium, and infiltrating mononuclear cells of the mucosa. Its reduction by vascular endothelium and infiltrating mononuclear cells was greater in inflamed mucosa. The reduction by vascular endothelium and infiltrating mononuclear cells was inhibited in cultures with nitrogen saturation or with 1 mM para-benzoquinone. The vascular endothelium seems to produce superoxide in the inflamed mucosa, which would exacerbate tissue injury in ulcerative colitis.

Adolescent↗

PubMed1966

Macroscopic identification of early myocardial infarction by dehydrogenase alterations.

A method utilizing a general dehydrogenase reaction has been used to demonstrate early gross myocardial infarctions. The procedure takes advantage of substrate and enzyme loss from the damaged myocardium. In the viable muscle, where endogenous substrates, coenzymes, and dehydrogenases are present, reduction of Nitro-BT yields a dark blue formazan. Necrotic muscle fibres remain unstained by this technique.A survey of 31 human hearts obtained at necropsy disclosed that there is no alteration in the Nitro-BT reaction following acute coronary insufficiency with sudden death or severe congestive heart failure. The earliest myocardial infarct to show loss of dehydrogenase activity was of eight hours' duration. Post-infarction scars and patchy interstitial fibrosis provided very precise information concerning topographic relationships when this method was applied to heart slices.

Humans↗

PubMed2001

Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells.

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Animals↗

PubMed2003

Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis.

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.

Animals↗

PubMed2000

Surfactant components modulate fibroblast apoptosis and type I collagen and collagenase-1 expression.

During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 microg/ml of Survanta; 10, 50, and 100 microg/ml of SP-A; and 500 microg/ml of Survanta plus 50 microg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability approximately 10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression approximately 2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.

Apoptosis↗

PubMedDate not supplied

Ethanol-modulated cytokine production and expression in skin cells exposed to methotrexate.

Evidence suggests a link between alcohol consumption, psoriasis and the response of psoriatic patients to methotrexate (MTX) therapy. Ethanol (EtOH) may play a role in the pathogenesis of psoriasis by upregulating the expression and inducing the local secretion of proinflammatory cytokines, e.g. interleukins IL-1alpha, IL-6, chemokine IL-8 and tumor necrosis factor alpha (TNF-alpha). We investigated whether EtOH or MTX or their combination influence the secretion of these cytokines using normal human primary skin cells (NHPSC) and epidermoid cell line A431. The objectives of this study were: (1) to quantify the differences in cellular changes induced by MTX, (2) to measure the effect of EtOH on MTX toxicity and (3) to determine the relationship between MTX and EtOH exposure and production of proinflammatory cytokines. NHPSC and A431 were incubated with 0-10 mM MTX or alpha-MEM (control) in the presence or absence of 40 mM EtOH. A formazan 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay was used as a marker for cell viability (control was 100%). Significance was calculated by ANOVA. Cytokine release into media was quantitated by ELISA. After 24 h of MTX exposure, the release of IL-1alpha was unchanged. IL-6 increased 1.7 times in both cultures, and IL-8 increased 1.7 times in NHPSC and 2.1 times in A431. TNF-alpha release increased twice in A431 but not in NHPSC. Human recombinant IL-1alpha and IL-6 for 24 h had no effect, while TNF-alpha reduced cytoviability by 30% in NHPSC and 22% in A431. Anti-TNF-alpha reversed the effect produced by TNF-alpha in NHPSC and reduced it in A431 (11.8%, p < 0.05). We concluded that in vitro in normal human primary keratinocytes, toxicity and inflammatory responses are enhanced by EtOH.

Antibodies, Monoclonal↗

PubMed2001

Differential expression of proteoglycan epitopes and growth characteristics of intervertebral disc cells grown in alginate bead culture.

This study assessed the growth characteristics and proteoglycan (PG) production in vitro of cells isolated from various zones; annulus fibrosus (AF), the transitional zone (TZ, an opalescent region situated between the AF and nucleus pulposus, NP) and the NP of the ovine intervertebral disc. The isolated cells were subsequently cultured in calcium alginate microspheres. Cellular metabolic activity was assessed over the 10 days of culture by the bioreduction of a tetrazolium dye substrate to a coloured formazan chromophore which could be measured colorimetrically at 490 nm. These data were correlated with the DNA content of beads measured using the fluorescent dye Hoechst 33258. Viable and non-viable cells in alginate beads were also assessed histochemically using the fluorescent dyes 5-chloromethylfluorescein diacetate and ethidium homodimer-1. PGs synthesised in culture were examined immunohistologically using monoclonal antibodies to defined glycosaminoglycan side chain PG epitopes. The results obtained clearly showed that AF and TZ cells differed from NP cells in the measured indices of cellular metabolism and in the extent of matrix deposition. In contrast to cells from the NP the cells from the AF and TZ were more metabolically active and programmed more to cellular proliferation than to matrix production.

Alginates↗

PubMed2004

Uniform potent activity of the antiproliferative metal chelate trans-bis(resorcylaldoximato)copper(II) against a large panel of human tumor cell lines in vitro.

BACKGROUND: The copper chelate trans-bis(salicylaldoximato)copper(II), CuSAO2, is a powerful antitumor agent in vivo, being capable of drastically increasing the life span of mice bearing Ehrlich ascites carcinoma, and has in many cases even a curative effect. This compound as well as some congeners, most notably the 4-hydroxylated analogue trans-bis(resorcylaldoximato)copper(II), CuRES2, also have potent antiproliferative activity against tumor cells in vitro. METHODS: CuRES2 was tested in vitro against a panel of 37 different types of human tumor cells (leukemias, small cell and non-small cell lung tumors, melanomas, as well as colon, breast, central nervous system, ovarian and renal tumors). The cells were incubated on microculture plates for 6 days, and the amount of viable cells was determined with the agent XTT, whose metabolic reduction gives a colored formazan compound. RESULTS: CuRES2 inhibited the proliferation of all the 37 cell lines studied. The IC50 values ranged between 0.575 and 8.57 microg/ml and the IC90 values between 1.74 and 20.6 microg/ml. Thus, it had distinct and very potent activity against all the cell lines studied, and the differences between the susceptibilities of different tumor types and cell lines were not very large. DISCUSSION: Clearly, CuRES2 is active against all the tumor cell lines studied. None of the cell lines (tumor types) demonstrates a significantly higher or lower susceptibility, but some trends appear to exist. Thus, most (but not all) of the leukemia and melanoma cell lines, the two ovarian cancer lines and one small cell lung cancer and one CNS cancer cell line were somewhat more susceptible than the cell lines on average. The results obtained for the melanomas are of special interest, since CuRES2 (and even more so CuSAO2) have very low aqueous solubilities. In cutaneous melanomas, their administration might, however, be very easy, since both compounds are soluble in dimethyl sulfoxide and could thus easily be administered topically in this solvent whose ability to be absorbed through the skin is well known. CONCLUSION: The present results on human tumor cell lines are in line with previous observations that CuSAO2 and CuRES2 essentially totally inhibit the proliferation of leukemia L1210 and Ehrlich ascites carcinoma cells in vitro in a concentration as low as approximately 5 microg/ml.

Animals↗

PubMedDate not supplied

Different regulation of the formation of intra- and extracellular oxygen radicals in macrophages.

The formation and accumulation of reactive oxygen species within liver macrophages and their release into the medium were determined by lucigenin-enhanced chemiluminescence, the reduction of cytochrome c, the formation of formazan from nitroblue tetrazolium and the fluorescence of 5- (and 6)-carboxy-2',7'-dichlorodihydrofluorescein. Zymosan, phorbol ester and fluoride induced the formation and accumulation of oxygen radicals intra- and extracellularly, ionomycin and lipopolysaccharide led to an intracellular accumulation of oxygen radicals, while after arachidonic acid and tumor necrosis factor-alpha, no reactive oxygen species were formed. While zymosan and phorbol ester predominantly induced the formation and release of superoxide, hydroperoxide was the main form released by fluoride. These results indicate that agents of different biological potencies induce reactive oxygen species within and/or outside liver macrophages and that different techniques must be used to detect different oxygen species within and outside cells.

Arachidonic Acid↗

PubMed1989

Anti-inflammatory and analgesic activities of 5-(4-[aryl(aryl azo)methyleneamino]phenyl)-1,3,4-oxadiazole-2(3H)-thiones.

Various new formazans of substituted oxadiazole 2(3H)-thiones were tested for their anti-inflammatory activity against carrageenin-induced paw oedema in albino rats. Among these, two most potent derivatives were evaluated in detail using cotton pellet implantation methods in albino rats of either sex. These two active analogues were also tested for their analgesic activity in albino mice and ulcerogenic liability in albino rats. The toxicity of the compounds was assessed by determination of their approximate LD50 value in albino mice. An attempt has also been made to establish the structure-activity relationship.

Aconitine↗

PubMed1985

NADH-dehydrogenase reaction in combination with immunoperoxidase (PAP) staining for light microscopic observation on the interneuronal relations of the enteric nervous system of the pig.

A novel procedure for a simultaneous demonstration of particular enteric nerve cell types and peptidergic nerve fibres has been developed by combining the histochemical reaction for NADH-dependent dehydrogenase and the unlabelled antibody peroxidase-antiperoxidase (PAP) method described by Sternberger. Whole-mount spreads were successively incubated in a NADH: nitroblue tetrazolium solution, fixed with a picric acid/formaldehyde mixture, dehydrated, cleared and rehydrated before processing for immunocytochemical localization of the neuropeptide by the PAP method. The nerve cells appear heavily stained by deposits of dark blue formazan, whereas the peptide-containing nerves appear bright brown. In the myenteric and submucous plexuses of the porcine small intestine the devised method allows an appropriate identification of Dogiel's type I, type II and type III neurons surrounded by varicose enkephalin-like immunoreactive fibre baskets with button-like twigs to the very surface of the ganglionic cells, suggestive of synaptic connections.

Animals↗

PubMed1988

Growth factor effects on small cell lung cancer cells using a colorimetric assay: can a transferrin-like factor mediate autocrine growth?

A semiautomated colorimetric assay (MTT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a purplish colored formazan product that can be measured spectrophotometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of human small cell lung cancer (SCLC) cell lines. Specifically, the growth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transferrin, 17 beta-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, transferrin, and selenium derived in the previously reported experiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82, NCI-N417, NCI-H526) using the MTT assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 17 beta-estradiol were negligible. Using the MTT we have shown that a SCLC cell line, NCI-H345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an anti-transferrin receptor antibody, when grown in transferrin-free media. The conditioned media from this cell line is stimulatory to other transferrin-sensitive cell lines, suggesting the possibility of an autocrine role for this transferrin-like molecule at least in that cell line. With carefully defined conditions for a given cell line in which cell density and other parameters are within a range of constant MTT metabolism, the assay is well suited for precise analysis of growth factor effects.

Carcinoma, Small Cell↗

PubMed1995

Neutrophil NADPH oxidase activity in chronic myeloproliferative and myelodysplastic diseases by microscopic and photometric assays.

Neutrophil NADPH oxidase is a membrane-bound enzyme complex responsible for the reduction of oxygen to superoxide anion in the respiratory burst. Impaired neutrophil function has often been reported in myeloproliferative diseases and myelodysplastic syndromes (MDSs), but its laboratory features have not been well characterized. Traditionally, NADPH oxidase activity has been evaluated with a microscopic method by nitroblue tetrazolium salt reduction to blue-black insoluble formazan granules identified in positive neutrophils by microscopy. We investigated neutrophil NADPH oxidase in 22 patients with chronic myeloproliferative disorders (cMPDs) and 15 patients with MDSs, using the microscopic method as well as a photometric microplate assay, which monitored the cytochemical reaction for 30 min. The relationship between cMPD and patient susceptibility to infections was also investigated. In the photometric assay, the mean enzyme activity in MDSs and cMPDs was lower than in normal subjects. NADPH oxidase activity was greater in cMPDs (except myelofibrosis) than in MDSs (except chronic myelomonocytic leukemia), and these differences were less evident with microscopy. Moreover, for cMPDs, patients with susceptibility to infections showed a lower NADPH oxidase activity than patients without infections.

Adult↗

PubMed1996

Comparative study of classical, colorimetric and immunologic staining methods for the assessment of tumor cell viability.

The trypan blue exclusion test, the MTT test and an immunostaining test for apoptosis were performed before and after incubation of SW620 human colonic carcinoma cells with different cytotoxic agents (CTAs) in order to assess tumor cell viability and CTA efficacy in vitro. A modified MTT test using light microscopy was also performed. A good correlation was found between the trypan blue assay and the MTT test, as determined by spectrophotometry. There was no 'overestimation' of cell viability as measured by the trypan blue test. The monitoring of formazan formation by light microscopy was feasible, but not very reliable since it did not show a good correlation with findings determined by spectrophotometry. The apoptosis test failed to show good correlation with other tests. Distilled water had no relevant cytotoxic effect, while chlorhexidine cetrimide (HAC 3.5%), chloramine 0.5% and polyvinylpyrrolidone iodine (PVP-I) > or = 0.05% damaged a large majority of cells. PVP-I at a concentration of > or = 5% was found to be the most effective CTA.

Antineoplastic Agents↗

PubMedDate not supplied

High-sensitivity antitumor drug sensitivity testing.

For antitumor drug sensitivity testing we have been performing the succinic dehydrogenase inhibition test using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A new tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino) carbonyl]-2H-tetrazolium hydroxide (XTT) has been synthesized recently, and we have been conducting basic studies using it. We were able to obtain a high degree of sensitivity by adding phenazine methosulfate as a promoter in this assay. In these assays, there was a positive correlation between absorbance and cell count and XTT assay was more sensitive than MTT assay. In XTT assay, the production of formazan increases with reaction time over a protracted period of time, we assessed the possibility of performing assays with fewer cells than MTT was used. The results using cancer cell lines showed that when reacted for a longer time (6 h), it was possible to perform adequate assays using this method with 5,000 cells per well, and it will be useful when the amount of biopsy specimen is limited. We also evaluated the inhibition index of each of the drugs, comparing it with the MTT assay.

Cell Count↗

PubMed1998

Superoxide anion from the adventitia of the rat thoracic aorta inactivates nitric oxide.

The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30+/-2 pmol x min(-1) x mg(-1) and was significantly decreased by superoxide dismutase to 19+/-2 pmol x min(-1) x mg(-1) and by a synthetic superoxide dismutase mimic, Euk-8, to 11+/-2 pmol x min(-1) x mg(-1). The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9+/-1 pmol x min(-1) x mg(-1), whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91phox, p22phox, p47phox, and p67phox almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall.

Acridines↗

PubMed1994

Superoxide dismutase ameliorates neuronal death from hypoxia in culture.

BACKGROUND AND PURPOSE: Studies showing efficacy with free radical scavengers have been conflicting, and when protection was demonstrated it was attributed to action at the level of the vascular endothelium. The purpose of this study was to test the hypotheses that neuronal free radical formation plays a role in the ischemic cascade and occurs intracellularly and that free radical scavengers, if taken up intracellularly, will protect against hypoxic damage. METHODS: A tissue culture model of hypoxia followed by restoration of oxygen was employed, using both superior cervical ganglia and hippocampal neurons. Cells were exposed to an anoxic atmosphere of 95% N2 and 5% CO2 and examined 2 to 24 hours later after restoration of oxygen. Determination of survival was measured by trypan blue exclusion. Nitroblue tetrazolium stain was used to assess free radical formation. RESULTS: Pretreatment with free superoxide dismutase did not decrease cell death after hypoxia as measured by trypan blue exclusion. However, when superoxide dismutase was taken up intracellularly under depolarizing conditions (55 mmol/L KCl in the medium), cell death was decreased significantly compared with hypoxic controls (28.7 +/- 4.34 versus 40.3 +/- 4.33; P < .03). During hypoxia neurons reduced nitroblue tetrazolium to form the blue precipitate formazan, and the color change was blocked in neurons pretreated with superoxide dismutase in depolarizing medium. Similar findings occurred in both superior cervical ganglia and hippocampal neurons. CONCLUSIONS: These findings provide evidence to support the role of neuronal free radical formation in cell death secondary to hypoxia. In addition, free radical scavengers, if taken up intracellularly, may partially ameliorate their deleterious effect.

Animals↗