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PubMed2002

Role of c-Jun concentration in neuronal cell death.

A dimer of the basic region leucine zipper proteins c-Jun and c-Fos constitutes the classical activator protein-1 (AP-1) transcription factor. c-Jun is thought to play essential roles in many important cellular pathways, including the control of proliferation and cell death. To investigate the roles of c-Jun and c-Fos concentrations in the regulation of neuronal cell death, we generated conditional alleles by fusing c-Jun and c-Fos to the ligand binding domain of the murine estrogen receptor (ER), with the aim of controlling the biological activities of c-Jun and c-Fos by the synthetic ligand 4-hydroxytamoxifen (4OHT). Transient transfection experiments revealed an increase in AP-1 activity following transfection of an expression vector encoding a c-Jun/estrogen receptor fusion protein (c-JunER) and stimulation with 4OHT. In contrast, a c-Fos/estrogen receptor fusion protein (c-FosER) was only weakly active in HT22 immortalized hippocampal cells and in PC12 pheochromocytoma cells. Highest levels of AP-1 activity were obtained by cotransfection of c-FosER and c-JunER and stimulation with 4OHT. Using retroviral gene transfer, we generated HT22 and PC12 cells expressing either c-JunER or c-FosER. The AP-1 activity was moderately increased in 4OHT-treated HT22 and PC12 cells expressing c-JunER, whereas no biological activity was observed in cells expressing c-FosER. We tested the influence of 4OHT-activated c-JunER or c-FosER upon cell survival and cell death by quantification of mitochondrial reduction capacity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan dye crystals. We did not observe any 4OHT-dependent decrease in cell survival in cells expressing c-JunER or c-FosER. Likewise, the number of pycnic nuclei did not increase in HT22 or PC12 cells expressing c-JunER or c-FosER. We conclude that an increase in the c-Jun concentration is not sufficient to trigger neuronal cell death. We propose that it is not the concentration of c-Jun that is critical for cell survival but rather the concentration of active, i.e., phosphorylated c-Jun.

Animals↗

PubMed1993

NADPH-diaphorase histochemistry in the postnatal mouse cerebellum suggests specific developmental functions for nitric oxide.

NADPH-diaphorase histochemistry has been applied for the localization of nitric oxide synthase during the postnatal development of the mouse cerebellum. Staining for NADPH-diaphorase during the first week after birth was confined to some but not all laminae of the immature cerebellum: NADPH-diaphorase activity was located in the molecular/Purkinje cell layer and in inner parts of the internal granular layer. The external granular layer and the developing white matter were essentially unstained. Expression was earliest and very strong in parallel fibers and in the internal granule layer of the ventral part of the pyramis and the dorsal part of the uvula. Staining in the Purkinje cell layer was observed throughout the cerebellum. The presence of formazan deposits within Purkinje cells was verified by colocalization with calbindin D-28k immunoreactivity. The distribution of NADPH-diaphorase activity changed into the adult pattern between 8 and 12 days of age: Within the molecular layer, basket cells and their processes became strongly stained. Reaction product within Purkinje cells gradually disappeared. Likewise, strongly stained parallel fibers were no longer detectable. These results suggest that nitric oxide is involved in different processes in cerebellar development.

Amino Acid Oxidoreductases↗

PubMed1978

Analysis of betamethasone and its organic esters in pharmaceutical products.

A rapid, quantitative analysis for betamethasone and its organic esters at room temperature is described. The method is similar to the official blue tetrazolium reaction for corticosteroids, except that methylene chloride is used as the solvent. The reaction is complete in 27-69 min, and the formazans produced are stable for at least 90 min after the addition of tetramethylammonium hydroxide. The results of the analysis of 13 different pharmaceutical formulations by the proposed method are reported. The degradation of betamethasone and its esters caused by strong bases is a pseudo-first-order reaction in methylene chloride. The average half-life of the corticosteroids studied is 56 min under the basic conditions described.

Aerosols↗

PubMed1978

Colorimetric determination of catecholamines by 2,3,5-triphenyltetrazolium chloride.

A convenient spectrophotometric method was developed for the determination of epinephrine, levarterenol, isoproterenol, and methyldopa by reduction of 2,3,5-triphenyltetrazolium chloride and subsequent measurement of the formazan at 485 nm. With absolute alcohol as the solvent, maximum color absorption was attained in 30 min at 25 degrees in the presence of 0.1 N KOH. Evidence is provided to account for the reduction of the tetrazolium salt at the expense of the epinephrine catechol moiety. In addition to the considerably high values of the molar absorptivities of the chromogen formed, ideal adherence of the color absorption to the Beer-Lambert law permitted a sensitive microdetermination of these catecholamines in both pure forms and pharmaceutical formulations. The tetrazolium interaction was selective. No interference was encountered from common catecholamine antioxidants, adjuvants, or noncatechol degradation products.

Catecholamines↗

PubMed1978

Rapid blue tetrazolium procedure for analysis of corticosteroids in pharmaceutical preparations.

A rapid quantitative analysis of nine selected corticosteroids and corticosteroid esters at room temperature is described. The procedure is similar to the official blue tetrazolium reaction for corticosteroids, except that methylene chloride instead of alcohol USP is used as a solvent and the reagents are dissolved in or diluted with nonaqueous solvents. These two modifications reduce the medium polarity, which increases the reaction rate. The reactions are complete in 7-18 min, and the formazans are stable for at least 90 min. The results from 15 different pharmaceutical formulations, 12 containing hydrocortisone and three containing prednisolone acetate, show that the proposed method gives results that compare favorably with those obtained by the official blue tetrazolium, isoniazid, and phenylhydrazine procedures.

Gels↗

PubMed1979

Improved spectrophotometric determination of glycerol and its comparison with an enzymatic method.

A chemical method for the determination of glycerol was developed and compared to an enzymatic assay for sensitivity and reproducibility. The chemical assay is based on glycerol oxidation to formaldehyde and subsequent reaction with chromotropic acid to yield a colored product. With this method, as little as 5 micrograms of glycerol/ml can be detected. The enzymatic assay is based on enzymatic glycerol phosphorylation followed by glycerol phosphate dehydrogenation by nadide (nicotinamide adenine dinucleotide). The reduced nadide is used to reduce iodonitrotetrazolium violet to its colored formazan product. The enzymatic method can be used to deterine 50 micrograms of glycerol/ml in aqueous samples.

Glycerol↗

PubMed1980

Tetrazolium salts in pharmaceutical analysis II: direct assay of diethylstilbestrol and diethylstilbestrol dipropionate.

A convenient spectrophotometric determination of diethylstilbestrol and diethylstilbestrol dipropionate was developed involving their interaction with triphenyltetrazolium chloride at 50 degrees for 45 min and subsequent measurement of the formazan formed. The significance of extended conjugation within t,e 4,4'-stilbenediol molecule to induce the color reaction is documented. Ideal adherence of color absorption to Beer's law permitted accurate and precise determination of diethylstilbestrol and diethylstilbestrol dipropionate pure forms over the range of 2-22 microgram of diethylstilbestrol/ml. Application of the tetrazolium color reaction to the analysis of diethylstilbestrol dipropionate dosage forms was achieved without prior hydrolysis or extraction.

Color↗

PubMed1991

A feasibility study of the SDI test for the evaluation of gastrointestinal cancer sensitivity to anticancer drugs.

The succinic dehydrogenase inhibition (SDI) test has been widely used to evaluate the sensitivity of cancer cells to anticancer agents. Recently, the techniques for tissue culture have progressed; the ability to make formazan has improved with the use of methylthiazol tetrazolium bromide (MTT) instead of triphenyl tetrazolium chloride (TTC) in this assay. Therefore, we modified the SDI test in order to evaluate the drug response to gastrointestinal cancer and applied it in a clinical study. In this assay, the optimal concentration levels of mytomycin-C and Adriamycin for the clinical materials are 10 micrograms/ml, that is, 10 times higher than for the cancer cell line, and that of Cisplatin is 30 micrograms/ml, for Cisplatin is unstable in the culture medium. To evaluate its antitumor effect, it was found necessary to expose the cancer cells to 5-fluorouracil (5-FU) for 3 days. We applied this assay for the clinical materials; the evaluable rate of this assay is 83.3%. The peculiarity of this assay is its high evaluable rate. These results suggest that this test may be useful for clinical application.

Animals↗

PubMed2005

Structural elucidation and analysis of thermal degradation products of the Fusarium mycotoxin nivalenol.

The major class of mycotoxins produced by Fusarium moulds are trichothecenes, a large group of sesquiterpenes sharing the same basic chemical structure, a 12,13-epoxytrichothec-9-ene ring system. Their toxic effects range from causing diarrhoea, vomiting and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. Trichothecenes in general are relatively stable compounds, their degradation is observed only at high temperatures and prolonged heating time. In order to investigate the stability of the trichothecene nivalenol (NIV) under food processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products using gas chromatography-mass spectrometry (GC-MS). Heating of nivalenol, especially under mild alkaline conditions, gave a mixture of four compounds (norNIV A, norNIV B, norNIV C, and NIV lactone), which where isolated and identified by nuclear magnetic resonance (NMR) and MS experiments. Although their formation was also demonstrated in heating experiments with spiked flour samples, only norNIV B was detectable in a screening of several commercially available samples, possibly due to the very low contamination with nivalenol. Furthermore, cell culture experiments using immortalized human kidney epithelial (IHKE) cells showed that the four compounds are less cytotoxic (formazan dye cytotoxicity assay) compared to nivalenol. Whereas nivalenol revealed an EC50 at 0.9 micromol, all other compounds did not show any significant effect up to 100 micromol.

Cell Line, Transformed↗

PubMed1992

Cyanide-resistant reduction of nitroblue tetrazolium and hydrogen peroxide production by the rabbit blastocyst.

The ability of the rabbit blastocyst to reduce nitroblue tetrazolium (NBT) to formazan in the presence of cyanide was assayed as an indicator of extramitochondrial oxidase activity capable of generating the superoxide radical. A cytochemical method initially developed for the detection and localization of hydrogen peroxide production at the ultrastructural level in phagocytosing leukocytes (Briggs et al.: J Cell Biol 67:566, 1975) was also applied to the blastocyst. The results demonstrate that the rabbit blastocyst acquires the ability to reduce NBT by a cyanide-insensitive process and to generate hydrogen peroxide between the fourth and fifth days postcoitum. The enzymatic activity responsible is apparently an NAD(P)H-dependent oxidase in the outer, microvillous plasma membrane of the trophoblast.

Animals↗

PubMedDate not supplied

A rapid TLC autographic method for the detection of xanthine oxidase inhibitors and superoxide scavengers.

A new bioautographic assay suitable for the localization of xanthine oxidase inhibitors and superoxide radical scavengers present in a complex matrix is described. Enzyme activity is detected by reaction of superoxide radicals with nitroblue tetrazolium to form a blue formazan salt. Both activities can be differentiated using a non-enzymatic version of the autographic assay wherein superoxide is chemically generated.

Chromatography, Thin Layer↗

PubMed1988

Application of the MTT assay to human prostate cancer cell lines in vitro: establishment of test conditions and assessment of hormone-stimulated growth and drug-induced cytostatic and cytotoxic effects.

In this study the usefulness of the MTT assay for the quantitation of growth modulating effects on cultured prostate cancer lines (PC-3, PC-93, and LNCaP) was investigated. The MTT test is based on the enzymatic reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide++ +] in living, metabolically active cells but not in dead cells. The reaction is carried out in situ in multiwell plates, and the reaction product, a purple-colored formazan soluble in dimethylsulfoxide, is measured colorimetrically, using a multiwell plate reader. Optimal test conditions were established for each of the cell lines used. With the hormone-sensitive cell line LNCaP, and stimulatory effect of the synthetic androgen R1881 was demonstrable by the MTT test. A sharp optimum occurred at a concentration of 10(-10) M R1881. Treatment of cells of either cell line with antineoplastic agents resulted in a dose-dependent reduction of MTT converting activity, reflecting the impaired survival of the drug-treated cells. Good correlations of the results obtained with the MTT-test, as compared with a thymidine incorporation assay or with direct DNA measurements, were observed. As the MTT test offers a high degree of precision and is easy to do, it is suitable for the purpose of (large-scale) chemosensitivity testing. Moreover, serial measurements might easily be performed in order to provide additional information on the mode of action of the drugs tested, i.e., to discriminate between cytostatic and cytotoxic drug effects.

Androgens↗

PubMed2003

Effects of 17alpha-ethinylestradiol on immune parameters in the lizard Sceloporus occidentalis.

We examined the effect of 17alpha-ethinylestradiol on immunity of the Western fence lizard, Sceloporus occidentalis. Injection of 17alpha-ethinylestradiol resulted in dose-dependent suppression of peripheral blood leukocyte levels as determined by cell counts, whereas total spleen cell levels were decreased only at higher doses of 17alpha-ethinylestradiol. In contrast, spleen cell proliferation was enhanced by 17alpha-ethinylestradiol as measured by reduction of MTT to formazan following a two-way mixed lymphocyte reaction. Antibody responses were unaffected. Effects on peripheral blood leukocyte levels and spleen cell proliferation similar to those observed in response to injection of 17alpha-ethinylestradiol were observed following injection of a single dose of hydrocortisone. However, injection of lizards with 17alpha-ethinylestradiol did not result in a significant increase in serum cortisol. Results of this study suggest that exposure of Western fence lizards to 17alpha-ethinylestradiol leads to decreased numbers of circulating leukocytes and total spleen cell numbers and the enhancement of spleen cell proliferation in a two-way mixed lymphocyte reaction. These effects probably involve mechanisms other than or in addition to the induction of cortisol release.

Animals↗

PubMed1993

Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.

The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.

Animals↗

PubMed1993

Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor.

A spectrophotometric assay method for determining succinate dehydrogenase activity is described in which iodonitrotetrazolium chloride is used as a final electron acceptor. The enzyme activity is determined by measuring the formation of formazan due to the tetrazolium salt reduction. The assay is continuous, rapid, simple, and sensitive, and may be used in the determination of enzyme activity either in tissue homogenates or as a marker of the mitochondrial fraction in cell fractionation procedures.

Animals↗

PubMed1993

RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction.

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.

Acetylation↗

PubMed1996

An in vitro test system for thyroid hormone action.

An in vitro bioassay for the biological action of thyroid hormones has been developed. The test is based on a dose-dependent increase in growth rate and cell activation of the rat pituitary tumor cell line GH3. This cell line is dependent on thyroid hormones for cell division when plated at low density in serum-free medium. The measurement of the cell-stimulating effect of L-triodothyronine (T3) is based on the capacity of mitochondrial enzymes of viable cells to transform the MTT tetrazolium salt into MTT formazan. A dose-dependent response of the MTT signal can be observed within a range from 0.075 to 1 nM T3 in the culture medium. The test is performed in 96-well microplates and allows screening of compounds which may interfere with the action of thyroid hormones.

Animals↗

PubMed1997

A colorimetric assay method for 3beta-hydroxy-delta5-steroid dehydrogenase.

3beta-Hydroxy-Delta5-steroid dehydrogenase is an important enzyme of steroid hormone biosynthesis present in steroidogenic tissues like adrenal, testis, and ovary of vertebrates. The enzyme is assayed mainly by radiochemical substrates. Spectrophotometric assay is not adequately sensitive to detect the enzyme activity since it is often present in low levels. We have developed a simple colorimetric assay based on formazan formation due to the reduction of the tetrazolium salt. The reaction mixture containing the substrate, pregnenolone or dehydroepiandrosterone, NAD and iodonitrotetrazolium in 0.1 M Tris-HCl buffer (pH 7.8), and the enzyme extract is incubated for 1 h at 37 degrees C. Absorbance at 490 nm is read in a spectrophotometer. The enzyme activity was linear with time and protein concentration. The assay works well with adrenal tissue extract, whereas in the case of testis, Leydig cell preparation may be required. We have assayed the enzyme activity in the adrenal of rat, mouse, and gerbil. The method is two- to threefold more sensitive than the spectrophotometric assay.

Adrenal Glands↗