FORMAZAN REACTION AS A CONFIRMATORY TEST FOR ALDEHYDE GROUPS IN HISTOCHEMISTRY.
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The morpho-biochemical characteristics were studied on 115 wild strains of Cryptococcus albidus: 61 isolates of the variety albidus and 54 of the variety diffluens. The mucous aspect of the colony, the pigmentation, the development on liquid medium, the cells' form and the filamentation were the morphological characters observed. As for the physiology, were reported the results concerning: the fermentation, the assimilation of 24 carbohydrates and the potassium nitrate, the development according: the temperature, two rates of actidione (cycloheximide) and four rates of tetrazolium. Were discussed: the atypicity and the intra-specific variations, the differenciation of the varieties and the taxonomic value of the two entities.
Synthesis of novel caffeic acid esters (1 and 2) was accomplished starting from appropriately substituted benzaldehydes (3 and 9). While compound 2 exhibited potent anti-oxidative activity in both the nitroblue tetrazolium and 1,1-diphenyl-2-picrylhydrazyl radical-scavenging models, compound 1 showed moderate 5-lipoxygenase inhibitory activity.
Twenty-seven novel nucleobases and nucleosides were synthesized by structural modification of uracil, and their effects on growth and differentiation of human myeloid leukemia HL-60 cells were examined. Some of the compounds inhibited the growth of HL-60 effectively. The nitroblue tetrazolium (NBT)-reducing activities of cells treated with the concentrations of these compounds for 50% inhibition of growth were compared. TI-66 (2,4-dibenzyl-6-fluoro-7,7,8,8-tetramethyl-cis-2,4-diazabicyclo-[4.2.0] octane-3,5-dione) was the most effective inducer of NBT-reducing activity and morphological differentiation of HL-60 cells into cells of the myelomonocytic lineage. TI-66 was also effective for induction of differentiation of another human myelogenous leukemia cell line, ML-1 cells, but not for differentiation of human erythroid leukemia K562 or HEL cells, or monocytic U937 cells. The effect of TI-66 in inducing differentiation of HL-60 cells was additive or more than additive in combination with retinoic acid or vitamin D3. Adenine or hypoxanthine alone induced NBT-reducing activity of the cells, and at suboptimal concentrations these compounds enhanced the effect of TI-66, but the enhanced NBT-reducing activities did not exceed the maximal activity induced by TI-66 alone. Simultaneous treatment of HL-60 cells with hypoxanthine reduced the growth inhibition by TI-66 alone. TI-66 was about 150 times more potent on a molar basis than adenine in inducing differentiation of HL-60 cells. These results suggest that nucleobase analogs such as TI-66 should be useful for differentiation therapy of some types of myelogenous leukemia.
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INTRODUCTION: The recent outbreak of severe acute respiratory syndrome (SARS) warrants the search for effective antiviral agents to treat the disease. This study describes the assessment of the antiviral potential of nitric oxide (NO) against SARS coronavirus (SARS-CoV) strain Frankfurt-1 replicating in African Green Monkey (Vero E6) cells. RESULTS: Two organic NO donor compounds, S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP), were tested in a broad range of concentrations. The non-nitrosylated form of SNAP, N-acetylpenicillamine (NAP), was included as a control compound in the assay. Antiviral activity was estimated by the inhibition of the SARS-CoV cytopathic effect in Vero E6 cells, determined by a tetrazolium-based colorimetric method. Cytotoxicity of the compounds was tested in parallel. CONCLUSION: The survival rate of SARS-CoV infected cells was greatly increased by the treatment with SNAP, and the concentration of this compound needed to inhibit the viral cytopathic effect to 50% was 222 microM, with a selectivity index of 3. No anti-SARS-CoV effect could be detected for SNP and NAP.
Aeroplysinin-1 (1) and the structurally related dienone 2 were cytotoxic to Ehrlich ascites tumor (EAT) cells and HeLa tumor cells in the microculture tetrazolium (MTT) and clonogenic assays. Both compounds are bromotyrosine derivatives, isolated from the marine spong Aplysina aerophoba. As the effective concentrations in the MTT assay were lower than in the clonogenic assay, 1 and 2 are able to cause growth inhibition as well as actual cell death in these cell lines. With an IC50 value of 8.2 microM (MTT assay, 2-h incubation, EAT cells), 1 was the more toxic compound. When the cells were depleted of glutathione by pretreatment with buthionine sulfoximine, they were significantly more sensitive toward 1 and 2 in the MTT assay. A dose-enhancement factor as high as 11.8 was found in EAT cells after 2-h incubation with 2. Using electron paramagnetic resonance we were able to measure free radical formation of 1 and 2, yielding the semiquinone structures 3 and 4, respectively, in a culture medium with tumor cells. It is concluded that free radicals are, at least in part, responsible for the cytotoxicity of 1 and 2. This conclusion is in line with expectations derived from the chemical structures of both compounds.
3-substituted-2-thiohydantoin derivatives were synthesized and their structures elucidated by IR, 1H-NMR spectroscopy and elemental analysis. The cytotoxicity of the 2-thiohydantoin derivatives to rat embryo fibroblasts (F2408) in vitro was determined, and the effects of these compounds on intracellular free Ca2+, [Ca2+]i, were measured by spectrofluorophotometry. Cytotoxicity was determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay). Compounds 4 and 7 showed cytotoxic activity, with IC50 values in the range of 1-1.2 microM. Introduction of either chlorophenyl, metoxyphenyl, nitrophenyl or benzyl groups at C-3 resulted in concentration-dependent cytotoxic effects. Compounds 1-6 at 1 microM or more significantly increased [Ca2+]i in a dose-dependent manner in the cultured fibroblasts. This action may have been mediated through intracellular calcium stores.
A modification of the MT-4 cell plaque assay for human immunodeficiency virus (HIV) is described, which gave reproducible results with all 4 HIV-1 strains and the two HIV-2 strains that were used. The main feature of this new method is the use of a tetrazolium (MTT) staining procedure. The number of plaques read after 4-6 days was essentially the same as the number of infectious units derived from the 50% cell culture infective dose (CCID50) in MT-4 suspension cultures. For a selected group of antiviral compounds the 50% plaque-inhibitory doses were comparable with the 50% inhibitory doses (ID50) in suspension cultures. In the plaque assay HIV-1 (HTLV-IIIB) and HIV-2 (LAV-2ROD) were equally susceptible to azidothymidine (AZT), and the same was true for didehydrodideoxythymidine (D4T). For these compounds it was irrelevant whether they were already present during the initial HIV adsorption phase or added immediately thereafter. Pentosan polysulfate proved about 20-fold more inhibitory to HIV-2 than HIV-1. There was a 5-fold increase in activity if present during the virus adsorption stage.
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1. A 200 mg. portion of corticosterone was ingested by a healthy man and the urine collected. Part of the urine was treated with the gastric juice of Helix pomatia and extracted with ethyl acetate, and the extract fractionated with Girard T. Paper-chromatographic separation of the non-ketonic fraction in the Bush (1952) system B(5) revealed the presence of two unknown polar components. 2. The unknown compounds did not possess a reducing (blue tetrazolium) or a reducible (potassium borohydride) grouping. Both contained a terminal alpha-glycollic fragment as shown by the formation of formaldehyde, and of a non-volatile aldehyde on oxidation with sodium bismuthate. 3. Unknown compound (I) had paper-chromatographic mobilities identical with those of 5beta-pregnane-3alpha,11beta,20beta,21-tetraol. The oxidation product of compound (I) had a retention time (gas-liquid chromatography) on an SE30 column identical with that of 3alpha,11beta-dihydroxy-21-nor-5beta-pregnan-20-al. The retention times of various derivatives agreed with those produced in an identical manner on the standard, and accordingly compound (I) is formulated as 5beta-pregnane-3alpha,11beta,20xi,21-tetraol. 4. Unknown compound (II) had a higher R(F) than compound (I), and its oxidation product had a longer retention time than that of compound (I). From the group effects observed in paper and gas-liquid chromatography, compound (II) is tentatively formulated as 5alpha-pregnane-3alpha,11beta,20xi,21-tetraol. The 5alpha/5beta ratio found was about 2.0.
BACKGROUND: Cancer cells have alterations in energy metabolism due to defective mitochondrial function. This may be due to generation of excessive free radicals and/or defective antioxidant enzyme systems. The aim of the present study was to assess mitochondrial function and antioxidant defences in the gastric mucosa of patients with gastric carcinoma (CA). METHODS: Gastric mucosal mitochondrial function was assessed by means of the reduction of tetrazolium dye (MTT), and levels of antioxidants such as glutathione S-transferase (GST), catalase, superoxide dismutase (SOD), and thiols were measured in biopsy specimens taken from the tumour mucosa (TM) and tumour-free (TF) mucosa, 2 cm away from the tumour, in 49 patients with gastric CA and compared with that in 54 controls. In a further 10 patients with gastric CA, these studies were done on TM and TF mucosa 2 cm and > or = 5 cm away from the tumour. In 10 patients and 5 controls, specimens were obtained for electron microscopy as well. Helicobacter pylori infection was diagnosed by means of histology. RESULTS: MTT reduction and GST and SOD activities were significantly decreased in TM and TF mucosa in patients with CA compared with controls (P < 0.01). The levels of thiols and catalase activity were significantly increased in CA as compared with controls (P < 0.01). H. pylori positivity did not influence most of these variables but did give significant decrease in MTT reduction in CA (TF) mucosa (P=0.01) and significant increase in thiol levels in CA (TM) mucosa (P=0.04). Electron microscopy showed mitochondrial alterations in tumour cells in all patients and in adjacent mucosa of 10%-50% of the cells. CONCLUSIONS: 1) In gastric CA the cancer mucosal cells and the non-involved cells adjacent to the tumour have defective mitochondrial function, which may be due to altered antioxidant defences and possibly altered free radical formation. 2) Ultrastructural mitochondrial abnormalities are shown to parallel these biochemical abnormalities.
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