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PubMed1981

Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.

The choice, basis and calculation of certain numerical coefficients for describing chemical structures of such staining reagents as dyes, enzyme substrates and visualising agents as described. The general value of electric charge, conjugated bond number, Hansch pi values, and molecular or ionic weights is emphasised. Hansch pi values as indicators of hydrophobic character, are then used to analyse reagents giving rise to the staining of neutral lipids. Dyestuffs used to stain lipids all have high Hansch pi values (greater than or equal to +1.0), with superior stains, e.g. Sudan Black B and Oil Red O, having values of greater than or equal to +7.0. In keeping with this, conversion of the basic dyes Nile Blue and Brilliant Cresyl Blue, with Hansch pi values of -2.4 and -3.6, into their oxazone derivatives, with Hansch pi values of +4.4, and 3.6, generates lipid staining compounds. Also in keeping with this correlation of lipophilia with Hansch pi values greater than +1.0 is the occurrence of artifactual lipid staining in enzyme histochemistry. Such artifacts can arise for instance when using certain naphthyl substrates which give rise to naphthoic intermediate reaction products, or when tetrazolium salts as used as visualising agents, yielding formazans as final reaction products. The Hansch pi values of the naphthols and formazans generated histochemically typically fall into the lipophilic range. Another artifact of lipid staining is the staining of basophilic entities, such as cell nuclei, by fat stains which carry amino substituents. Calculation of the Hansch pi values for protonated (and hence cationic) derivatives of such dyes yields values typical of basic dyes.

Animals↗
PubMed1984

A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages.

Acridine orange and ethidium bromide and a combination of fluorescent and transmitted light microscopy used in conjunction with the qualitative nitroblue tetrazolium assay for superoxide anion (O2-) release demonstrated dramatic differences in the binding of and respiratory burst (RB) activity elicited by promastigotes and amastigotes of Leishmania donovani in resident peritoneal macrophages (M phi) from C57BL/10ScSn mice. When amastigotes were incubated with M phi for 30 min the number of parasites per 100 M phi was 2-4-fold higher, a higher proportion of M phi became infected and the mean number of parasites per infected M phi was higher than in promastigote infections. RB activity was higher for promastigotes than amastigotes both in terms of the percentage of infected M phi containing formazan positive parasites and the percentage of individual formazan positive parasites. In an attempt to explain the differential response to promastigotes and amastigotes, RB activity was examined for sodium azide-treated, glutaraldehyde-fixed and heat-killed parasites and for various transformation intermediates between amastigotes and promastigotes. Binding and RB activity were also examined in conjunction with competitive binding assays designed to determine the specific receptors involved in ligand binding of both forms of the parasite to the M phi. The results indicate that, while amastigotes may possess an azide-sensitive mechanism which either competes for O2- produced or causes localized inactivation of RB activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. The transformation and competitive binding studies suggest that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the M phi plasma membrane and that the well characterized mannose/fucose receptor may be important in promastigote, but not amastigote, binding and RB activity.

Animals↗
PubMedDate not supplied

[Determination of the phagocytic ability of leukocytes in patients with chronic polyarthritis].

The phagocytic function of leukocytes from peripheral blood of patients with classical rheumatoid arthritis was investigated using nitroblue-tetrazolium reduction of formazan. One control group (A) and two groups of RA-patients were compared. Group B was treated with nonsteroidal antirheumatics, the third group C additionally with low dose steroids (5,0-7,5 mg/d). Reduction to formazan was increased in group B in relation to the control, whereas treatment with cortico-steroids produced decreased phagocytic function of peripheral leukocytes.

Adrenal Cortex Hormones↗
PubMed1994

Nitric oxide synthase in the brain: light and electron microscopical findings based on the NADPH-diaphorase reaction.

The recent discovery of the identify of nitric oxide synthase with the reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) has powerfully stimulated the anatomical localization of sites of nitric oxide synthesis in the nervous system. In the present study the widely used light microscopical technique for NADPH-d staining was adapted to the electron microscopical level by applying the tetrazolium salt 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride (BSPT) which produces an electron-dense reaction product, BSPT-formazan. Predominantly membranes of the endoplasmic reticulum were stained. Apart from singular heavily labeled neurons, a majority of nerve cells, light microscopically "unstained", shows sporadically formazan deposits, and, likewise, but regionally different, a few astroglial cells. Lesions induced by the glutamate agonists quinolinic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) display surviving neurons, which are predominantly stained for NADPH-d. Astroglial cells within lesioned areas exhibit increased amounts of reaction product, apparently as a consequence of enzyme induction.

Amino Acid Oxidoreductases↗
PubMed1976

In vitro quantitation of cell-mediated immunity in guinea-pigs by macrophage reduction of nitro-blue tetrazolium.

In the cell-mediated immune (CMI) system lymphocytes from sensitized animals incubated with antigen manufacture and release lymphokines which activate the hexose-monophosphate shunt in macrophages. The rate-limiting enzyme of this activation is NADPH oxidase, the activity of which can be quantitated by the amount of nitro-blue tetrazolium reduced to formazan, a blue precipitate. Data is presented which demonstrates that lymphokine-activated macrophages can be microscopically quantitated, both in the direct and indirect assays, by counting the number of macrophages containing formazan precipitate. The indirect component of this assay correlates directly to the skin test diameter. Further, it correlates better to the skin test than another assay for CMI, the macrophages aggregation factor assay. The simplicity and reproducibility of this assay provides another method whereby lymphokine activation of physiological events in macrophages can be determined.

Animals↗
PubMedDate not supplied

MCF-7 cell cycle arrested at G1 through ursolic acid, and increased reduction of tetrazolium salts.

The effect of ursolic acid on the proliferation of MCF-7 human breast tumor cells was studied. During investigations of the anti-proliferative effects of this triterpene, we observed a clear difference between MTT colorimetric assay and direct cell counting, particularly 24 h after drug treatment. The MTT assay showed a stimulation of formazan production in the first 24 h exposure of cells to drug. The maximum stimulation was obtained with 15 and 20 microM of ursolic acid (about 30 - 40% of increase with respect to control); however, the number of cells was not increased as revealed by direct cell counting. Ursolic acid is a potent inhibitor of MCF-7 cell proliferation. This triterpene exhibits both cytostatic and cytotoxic activity. It exerts an early cytostatic effect at G1 followed by cell death. Cell cycle analysis is performed by propidium iodide staining and flow cytometry technique. These results suggest that alterations in cell cycle phase redistribution of MCF-7 human breast cancer, by ursolic acid, may significantly influence MTT reduction to formazan.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Unpredictability of triphenyltetrazolium chloride in staining irreversible ischaemia-reperfusion injury in the skeletal muscle of rats.

OBJECTIVE: To find out whether pale staining with triphenyltetrazolium chloride (TTC) in skeletal muscle of rat hindlimbs which had been subjected to ischaemia-reperfusion definitely indicated irreversible tissue damage. DESIGN: Laboratory study. SETTING: University hospital, Taiwan. MATERIAL: 77 Female Wistar rats. INTERVENTIONS: Ischaemia of one hindlimb was caused by wrapping of a tourniquet above knee joint for 1.5 (n = 14). 2 (n = 15), 2.5 (n = 17), 3 (n = 17) or 4 (n = 14) hours. Each ischaemic group was divided into three subgroups to receive nil, 1 or 1.5 hours reperfusion, respectively. Gastrocnemius and soleus muscles were excised bilaterally. MAIN OUTCOME MEASURES: TTC reduction in the ischaemic limbs presented as a percentage of the opposite control limb measured by a spectrophotometric assay. Density of red formazan deposition in the ischaemic limbs assessed by microscopic examination of the TTC histochemical stain in the 3 hour ischaemic group. RESULTS: In the 2, 2.5, and 3 hour ischaemic groups the TTC reduction in the ischaemic limbs after 1.5 hours reperfusion increased significantly as compared with that with one hour reperfusion (p < 0.01 in each case). The density of red formazan deposition in the muscle after 3 hours ischaemia and 1.5 hours reperfusion was significantly higher than that of only 1 hour of reperfusion. CONCLUSION: Lack of TTC staining does not necessarily represent irreversible ischaemia-reperfusion injury of the skeletal muscle in rats.

Animals↗
PubMed1996

[Assessment of the toxicity of TAH on the cell lines of SO-Rb50 and SO-Rb70].

PURPOSE: MTT assay was evaluated on cytoxicity for suspension growing cell lines of SO-Rb50 and SO-Rb70, and the toxicity of TAH (the total alkaloid of peqanum harmala L) on the above cell lines was assessed. METHODS: The relationships between cell number and optical density, between optical density and exposure time of MTT, and the stability of formazan crystal solution in MDSO were determined. And the toxicity of TAH on the cell lines of SO-Rb50 and SO-Rb70 in vitro with MTT assay was assessed. RESULTS: There was a direct proportional relationship between the amount of cell number and its optical density; The optical density increased gradully within 12 hours of the MTT incubation time; The stable time of the formazan crystal solved in DMSO was 11 hours. The IC50 values (micrograms/ml) of TAH on SO-Rb50 were 10.66, 4.82 respectively for 48 and 72 hours; and on SO-Rb70 were 6.38, 4.2 respectively for 48 and 72 hours. CONCLUSION: MTT assay can be used for suspension growing cell lines of SO-Rb50 and SO-Rb70: TAH has obvious toxicity to these two cell lines.

Alkaloids↗
PubMed2001

Charge-coupled device camera-based detection of fluorescence-labeled proteins immobilized on nitrocellulose membranes.

Proteins dotted on nitrocellulose membranes are biotinylated by reaction with a biotinyl-succinimide ester. The resulting biotinyl residues serve as specific binding sites for a subsequent streptavidin-based detection system. Using streptavidin-peroxidase, the proteins are visualized either by deposition of a colored formazan dye or by enhanced chemiluminescence the latter being twofold more sensitive. Alternatively, streptavidin-fluorescein isothiocyanate (FITC) is substituted for the peroxidase conjugate as tool for protein staining. The sensitivity of both staining variants is dramatically improved by the inclusion of the reporter deposition technique. The fluorescence-labeled proteins are visualized on a visible blue light emitting illuminator preventing the bothering effect of photobleaching. In combination with a charge-coupled device (CCD) camera-based image analyzing system the established stain with streptavidin-FITC detects about 10 pg of protein dot blotted on nitrocellulose membranes.

Biotinylation↗
PubMedDate not supplied

Cardioteratogenic dose of ethanol reduces both lactic dehydrogenase and succinic dehydrogenase activity in the bulbar ridges of the embryonic chick heart.

It has been established previously that the chick embryo is an appropriate model for studying the pathogenesis of ethanol-induced ventricular septal defect and that the growth and fusion of the proximal bulbar ridges are critical to ventricular septation within the embryonic heart. To determine whether enzyme activity was altered significantly in bulbar ridges in response to a cardioteratogenic dose of ethanol, we injected 0.32 ml of 50% ethanol in chick Ringer's saline (CRS) into the air cells of 3-day-old chick embryos (72-84 h incubation, Hamburger-Hamilton stages 18-19, Dekalb Delta strain). Embryos were harvested 48-60 h after treatment (stages 26-27). Hearts were removed, frozen and sectioned, and the tissues were processed for enzyme histochemical demonstrations of lactic and succinic dehydrogenase. Twelve ethanol-treated embryos and 12 CRS-treated controls were compared for lactic dehydrogenase (LDH) levels. Fourteen ethanol-treated embryos and 14 controls were compared for succinic dehydrogenase (SDH) levels. Formazan deposits were quantified by means of computer-assisted microdensitometry. Our results showed that ethanol decreased the mean LDH activity level by 36.3% (P = 0.005) and the mean SDH level by 40.8% (P < 0.02). Furthermore, ethanol significantly reduced LDH activity in 41.7% of the matched embryo pairs and significantly reduced SDH activity in 57.1% of the matched embryo pairs. Although some investigators have proposed that a disturbance in neural crest cell migration plays a major role in the pathogenesis of ethanol-induced cardiac malformations, this study provides evidence that the bulbar ridges of the embryonic heart are also potential target tissues for ethanol.

Animals↗
PubMed1996

Evaluation of in vitro chemosensitivity using human lung cancer cell lines.

The use of well-characterized human lung cancer cell lines has allowed for new opportunities in preclinical and clinical drug evaluation. Development of semiautomated tests of in vitro cytotoxicity such as the MTT assay, which utilizes the formazan salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), has allowed for preclinical evaluation of novel chemotherapeutic agents and drug combinations. In addition, techniques such as this make possible the testing of sufficient data sets to allow determination of true biochemical drug synergy. Assessment of drug combinations which possess in vitro synergy or supraadditive effects can suggest chemotherapeutic regimens for further clinical testing. Using the MTT assay in conjunction with isobolographic analysis, it is possible to test commonly used regimens which are based on presumed or apparent in vivo drug synergy, such as the combination of etoposide and cis-platinum. This frequently prescribed combination was found to lack in vitro biochemical synergy when tested with human lung cancer cell lines, indicating that the observed clinical benefits of this drug combination may be due to factors in the tumor microenvironment, drug metabolism, or non-overlapping toxicities. Finally, although it remains to be determined if a significant role for in vitro drug testing will be found in direct clinical applications, preclinical drug evaluation during the drug development process using cultured tumor cell lines may ultimately allow for disease or patient specific therapies for testing.

Animals↗
PubMed2002

Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications.

Spectrophotometric methods for determining the activity of cytokinin oxidase/cytokinin dehydrogenase (EC 1.5.99.12) were developed and optimized. A sensitive end-point method based on a combination of the electron acceptor 2,6-dichlorophenolindophenol and Schiff base formation of the reaction product with 4-aminophenol under acidic conditions can be applied to crude cell and tissue extracts. The assay was also adapted for other substrates than N6-(2-isopentenyl)adenine, such as zeatin and the aromatic cytokinins, although an enzyme which degrades the latter compounds has not yet been identified. The second novel method is an initial rate method based on the coupled redox reaction of phenazine methosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide resulting in the formation of a formazan dye. This method can be used for kinetic studies with purified enzyme and is entirely substrate independent.

2,6-Dichloroindophenol↗
PubMed2000

Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation.

Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe). For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C). Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19). The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils. Such granules stained vitally with Neutral Red, and correspond to lysosomes. Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells. Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase. Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect. Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium. Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells. Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.

5'-Nucleotidase↗
PubMed1995

Anabolic-androgenic steroid-induced toxicity in primary neonatal rat myocardial cell cultures.

Recent literature reports of myocardial infarction in athletes who self-administer anabolic-androgenic steroid (AAS) and previous animal studies of the effects of AASs on the heart suggest that these drugs may be directly injurious to the myocardium. We have previously demonstrated that 100 microM testosterone cypionate (TC) inhibits all beating activity of primary neonatal rat myocardial cell cultures within 1 hr of exposure and causes significant LDH release by 4 hr of exposure, indicating a direct toxic effect of TC. The purpose of this investigation was to evaluate the effects of commonly abused AASs on primary neonatal rat myocardial cell cultures and to provide insight into early cellular changes that may lead to TC-induced toxicity. Significant LDH release was observed in 5-day-old primary myocardial cell cultures (obtained from 3-to-5-day-old Sprague-Dawley rats) exposed to 100 microM testosterone enanthate (TE), testosterone propionate (TP), and oxymetholone (O) for 4 and 24 hr and in cultures exposed to 100 microM testosterone (T) for 24 hr. Neutral red retention and MTT formazan production were significantly decreased in cell cultures exposed to 100 microM TE, TP, and O after only 4 hr of exposure, indicating a loss of viability and mitochondrial activity. However, there was no effect on viability of cell cultures exposed for 24 hr to 100 microM of a variety of other commonly abused AASs. Phase-contrast microscopy revealed complete disruption of the monolayer in cell cultures treated with 100 microM TE, TP, and O for 4 hr. Treatment of fura-2-loaded myocardial cell cultures with 100 microM TC produced no significant changes in calcium transients or baseline calcium levels for up to 13 min of exposure. These results indicate that O, T, TC, TE, and TP produce a direct toxic effect in heart cell cultures and that early (< 13 min) changes in calcium homeostasis are unlikely to participate in the mechanism of toxicity.

Anabolic Agents↗
PubMed1998

Toxicity of atractyloside in precision-cut rat and porcine renal and hepatic tissue slices.

Atractyloside (ATR) causes acute fatal renal and hepatic necrosis in animals and humans. Precision-cut renal cortical and hepatic slices (200 +/- 15 microns) from adult male Wistar rat and domestic pigs, incubated with ATR (0.2-2.0 mM) for 3 h at 37 degrees C, inhibited pyruvate-stimulated gluconeogenesis in a concentration- and time-dependent manner. p-Aminohippurate accumulation was significantly inhibited in both rat and pig renal cortical slices from 0.2 mM ATR (p < 0.05). There was a small decrease in mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium to formazan in both rat and pig kidney slices, which was significant at > or = 2 mM, but no changes in liver slices from either species. However, cellular ATP was significantly depleted at > or = 0.2 mM ATR in kidney and in liver slices from both species. ATR also caused a marked leakage of lactate dehydrogenase and alkaline phosphatase from both pig and rat kidney slices at all concentrations, but only lactate dehydrogenase was significantly elevated in liver slices from both species. ATR > or = 0.5 mM caused a significant increase in lipid peroxidation, but only in liver slices of both species, and > or = 0.2 mM ATR caused a marked depletion of reduced glutathione and significant increase in oxidized glutathione in both kidney and liver slices of both species. However, GSH to GSSG ratio was only significantly altered in the liver slices, indicating that oxidative stress may be the cause of toxicity in this organ. Both rat and pig tissue slices from the same organ responded similarly to ATR, although their basal biochemistry was different. ATR toxicity to both kidney and liver showed similar patterns but it appears that the mechanisms of toxicity are different. While cytotoxicity of ATR in kidney is only accompanied with GSH depletion, that of the liver is linked to both lipid peroxidation and GSH depletion. Striated muscle slices from both species were not affected by the highest ATR concentration. This further strengthens the argument that the molecular basis of ATR, target selective toxicity, is not a measure of the interaction between ATR and mitochondria and that other factors such as selective uptake are involved. Precision-cut tissue slices show organ-specific toxicity in kidney and liver from both rat and pig and suggest different mechanisms of injury for each organ.

Adenosine Triphosphate↗
PubMed1995

Ultrastructural localisation of NADPH-diaphorase in the chick thymic medulla.

Nicotinamide-adenine-dinucleotide phosphate-diaphorase positive cells in the chick thymus were studied at the electron-microscopic level. The formazan, a marker for the enzyme nitric oxide synthase, labelled cystic, undifferentiated, endocrine-like and myoid cells in the medulla. Some lymphoid and reticulo-epithelial cells were also lightly labelled. The reaction product was predominantly bound to the membranes of the endoplasmic reticulum in all the cells labelled and also to the nuclear envelope and outer membrane of mitochondria. The Golgi apparatus and the plasma membrane were free of the reaction product.

Amino Acid Oxidoreductases↗
PubMed1977

Chromosomal location of soluble glutamic-pyruvic transaminase-1 (Gpt-1) in the mouse.

Three alleles at the Gpt-1 (glutamic-pyruvic transaminase-1) locus in the mouse, as identified by electrophoresis on cellulose acetate, and their distribution among inbred mouse strains and wild stocks are described. The Gpt-1 locus was shown to control the soluble form of the enzyme. Three-point linkage analysis established th location of Gpt-1 on chromosome 15 between uw and bt. In addition, a new staining procedure is described that allows the visualization of GPT activity on gels by the deposition of formazan. This is an improvement over previous methods that produced bands of nonfluorescence against a fluorescent background.

Alanine Transaminase↗
PubMed1976

Electron cytochemical localization of gamma-aminobutyric acid catabolism in rat cerebellar cortex.

An electron cytochemical technique is described for the localization of GABA-T, the enzyme which degrades the neurotransmitter GABA, in rat cerebellar cortex. The technique allows ultrastructural demonstration of GABA-T activity by the final deposition of an electron dense formazan precipitate at reaction sites, whilst maintaining adequate ultrastructural preservation for recognition of cellular and subcellular structures. Numerous electron dense precipitates are evident as discrete punctate deposits situated mainly in mitochondria of stellate cells, basket cells and astrocytic glial cells; they are also seen in axonal or dendritic profiles at some synaptic junctions. The technique enables the first cytochemical demonstration of the mitochondrial localization of GABA-T activity in nervous tissue to be presented. It establishes that GABA-T is present in supposed GABA neurones, in pre- or post-synaptic endings, or both, of presumed inhibitory synapses and in glial cells which may be associated with these synapses. From this seemingly ubiquitous distribution, functional aspects of GABA-T in these cells is considered.

4-Aminobutyrate Transaminase↗