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Metabolism of a novel antitumor agent, crisnatol, by a human hepatoma cell line, Hep G2, and hepatic microsomes. Characterization of metabolites.

Metabolism of the anticancer agent crisnatol was investigated using a human hepatoma cell line, Hep G2, and human liver microsomes. Crisnatol was metabolized extensively by both systems. The TLC/autoradiographic analysis showed that the crisnatol metabolite profile was similar for both systems and the major metabolites were shown to have structural characteristics similar to those formed by the rat. The Hep G2 cells formed three isomeric dihydrodiols; one of these has been identified by GC/MS and 1H-NMR as the crisnatol 1,2-dihydrodiol. Human liver microsomes also formed two isomeric dihydrodiols with 1,2-dihydrodiol as the major isomer and, in addition, produced 1-hydroxycrisnatol. Crisnatol concentrations of 1.3 micrograms/mL completely inhibited the replication of Hep G2 cells as measured by thymidine incorporation and cell growth kinetics and, at this concentration, cell viability decreased by only 35% as determined by vital staining of cells using neutral red dye.

Antineoplastic Agents↗

Strong cytotoxicity to human gingival fibroblasts by Porphyromonas gingivalis ATCC 33277.

The aim of the present study was to analyze the cytotoxicity of some bacterial species associated with periodontal diseases. The specificity of cytotoxicity was estimated against cells of various origin and from different individuals. The reference bacteria were Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. These bacteria were cultured for 24 h in liquid media and the supernatants were used in cytotoxicity assays. The target cells used were human gingival fibroblasts (GF), dermal fibroblasts (K4), gingival epithelial cells (E) and HeLa-cells (HeLa). These cells were exposed at 4 h or 24 h, respectively, to various concentrations of culture supernatants from the selected bacteria. The influence on the viability and metabolism of the cells were estimated quantitatively as increase in neutral red uptake and lactic acid production. Growth medium supernatants of P. gingivalis 33277 were strongly cytotoxic to gingival fibroblasts after 24 h incubation, compared to supernatants of P. gingivalis 381 or W 50, A. actinomycetemcomitans or F. nucleatum cultures. The toxic effect of P. gingivalis 33277 decreased drastically after heat inactivation, which indicates effects of proteins. By adding anti-sera the cytotoxicity of P. gingivalis 33277 could be dose dependently neutralized, which was not the case when supernatants of A. actino-mycetemcomitans was tested. Target cells of epithelial origin did not show increased cytotoxicity to P. gingivalis 33277. The results of the present study strengthen the hypothesis that P. gingivalis remains as a suspect causative key component in periodontal diseases.

Aggregatibacter actinomycetemcomitans↗

[Stomach secretion and excretion in mineralocorticoid deficiency].

Experiments on dogs were made to perform bilateral adrenalectomy and substitution therapy with hydrocortisone. Juice and acid secretion and neutral red excretion were found to be lowered after stimulation of secretion by meat and histamine. Excretion of ammonia after meat stimulation decreased, while after histamine administration it remained unchanged.

Adrenalectomy↗

A new fluorescent test for cell vitality using calcofluor white M2R.

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.

Animals↗

[Action of heavy water on actomyosin pellicular filaments].

Deuterium oxide (D2O) prolongates the contractile ability of actomyosin threads by 85%. The contraction of actomyosin threads decreases in the presence of D2O by 28%. D2O decreases neutral red sorption by actomyosin threads. The limiting dye sorption (A infinity) decreases by 45 and 31% within the ranges of weak and high concentrations, resp. Thus, deuterium oxide may affect the conformation of protein molecules.

Absorption↗

[Changes in intercellular exchange intensity in an L cell culture].

Intercellular junctions permeable to fluorescein Na were studied with the aid of intracellular glass microelectrodes in the cultures of transformed mouse-embryo cells (L-strain). The degree of coupling was correlated with the cell culture density. In addition, the degree of coupling increased after short-time incubated of the cells with 1mM lanthanum. Such an incubation did not influence other features of cells: the value of the membrane potential, the character of growth or the inclusion of the vital dye neutral red. Some other factors stimulated cellular aggregation (concanavalin A, protamine, decreasing the pH) but did not influence the degree of coupling.

Animals↗

[Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability].

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.

Animals↗

Non-animal alternative toxicity tests for detergents: genuine replacements or mere prescreens?

Non-animal toxicity tests and testing strategies for use in identifying the potential toxic hazard of chemicals and products, and in providing information for use in risk and safety assessment, are in the course of development, validation and evaluation. Possible replacement alternatives to the rabbit Draize eye irritancy test are discussed, and results obtained for two in-vitro tests developed by FRAME (the kenacid blue test and the neutral red release test), applied to 19 surfactants and 32 formulations, are discussed. It is concluded that there are now legal and moral requirements that relevant and reliable non-animal tests should be developed and accepted for use whenever possible.

Animal Testing Alternatives↗

Photoinactivation of the encephalomyocarditis virus.

Encephalomyocarditis virus was photoinactivated by a dilution of 1:30,000 neutral red and 70 minutes exposure to light. A dye inactivation of the EMC virus is possible without destruction of its antigenicity. The EMC virus vaccine induced a good immunity response in mice; we did not find any infectious virus.

Animals↗

Inorganic thiophosphate effects on chromaffin cell structure and function.

The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3-4 days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radiolabelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97-121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrographs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.

Animals↗

[Chemical modification of RNAse from Bacillus intermedius. Effect on the cytotoxic properties of the enzyme].

Modified derivatives of RNAse from Bacillus intermedius differing in the aggregate enzymatic, physico-chemical and physical properties were prepared. The cytotoxic properties of the compounds were tested by the intravital staining of FL cells with neutral red. It was shown that the native enzyme cytotoxicity correlated with the catalytic activity. However, it was found possible to lower the cytotoxicity of the catalytically active compounds by changing the enzyme aggregate properties.

Bacillus↗

[Gastric excretory function in dogs and its role in radiation lesions].

X-irradiation was shown to cause a 2-3-fold increase in the excretion of intravenously administered 1% neutral red and endogenous ammonia. It is suggested that elimination of excreta from the stomach may change the course of radiation sickness. The postirradiation gastric lavage alleviates the clinical picture of the disease, increases the survival rate and protects the gastric mucosa from "poisoning".

Ammonia↗

[Seasonal changes in lysosomal pH of peripheral blood cells of healthy subjects].

We studied pH values in lysosomes of peripheral blood white cells of 49 blood donors using the two-wavelengths (470 and 450 nm) microspectrophotometry of neutral red after supravital in vitro staining. In polymorphonuclear leucocytes lysosomal pH reaches the highest values in August-September (6.98 +/- 0.04) and the lowest--in February-March (6.23 +/- 0.09), p less than 0.05.

Adolescent↗

Comparison of in vitro antifungal activities of topical antimycotics launched in 1990s in Japan.

In vitro anti-dermatophyte, anti-Candida albicans and anti-Malassezia furfur activities of amorolfine hydrochloride (AMF), terbinafine hydrochloride (TBF), butenafine hydrochloride (BTF), neticonazole hydrochloride (NCZ) and ketoconazole (KCZ), all of which were introduced for the treatment of dermatomycoses in the 1990s in Japan, were compared. Although all of the test drugs are classified as an ergosterol biosynthesis inhibitor, the antifungal properties were found to be different. TBF and BTF exerted extremely potent antifungal activity against Trichophyton spp. but not against C. albicans and M. furfur, whilst KCZ and NCZ showed potent antifungal activity against C. albicans and M. furfur rather than Trichophyton spp. AMF exhibited potent antifungal activity against all of the fungal species tested. Fungicidal activities of these antifungal agents against T. rubrum were determined by using neutral red staining. The fungicidal potentialities correlated with those obtained in the in vitro susceptibility test as determined by MICs against dermatophytes. TBF, BTF and AMF exerted more potent fungicidal action than NCZ and KCZ.

Administration, Topical↗

Vacuolar system of ungerminated Colletotrichum graminicola conidia: convergence of autophagic and endocytic pathways.

Vacuoles of ungerminated Colletotrichum graminicola conidia engulf cytoplasmic structures by a process analogous to microautophagy, demonstrated by using a vacuolar membrane acid phosphatase marker. Fusion of vesicles with vacuoles, without deposition of the acid phosphatase reaction product has been observed, suggesting other pathways of material delivery to vacuoles than microautophagy. Plasma membrane invaginations, multivesicular bodies and retention of neutral red into small vesicles, which were internalized by the vacuole, were verified. These results provided evidence for endocytosis and an active endosomal system. Together, our findings with C. graminicola demonstrated that vacuoles are very dynamic compartments, playing roles in autophagy and endocytic processes.

Acid Phosphatase↗

Mucopoietic activity of zolimidine: is its effect through cellular turnover or by modification of the gastric mucosal blood flow?

Zolimidine 2-(methylsulphonyl-phenyl)-imidazo-(1,2-a)pyridine produces a high percentage of healing of gastro-duodenal ulcers. The action of the drug develops through an increase of protective factors on the gastric mucosal barrier. This study has the purpose of evaluating if the action of the drug is also mediated by microvascular factors. The analysis of the values of BAO, MAO and PAO confirmed that treatment with zolimidine does not modify gastric acid secretion; the values of the Neutral Red clearance at PAO, before and after the treatment, do not show a statistically significant variation of the gastric mucosal blood flow. According to the most recent literature, the increase of the gastric mucopoietic activity is carried out by a direct cellular action.

Adult↗

[The role of the autonomic nervous system in regulating the excretory function of the stomach in irradiated dogs].

Dogs and rats were exposed to gamma/neutron- and X-radiation. The anterior part of dog's stomach was exposed to 10 Gy and 13 Gy respectively; rats were subjected to whole-body irradiation with absolutely lethal doses. Prior to irradiation, various parts of the vegetative nervous system of both types of animals were "switched off" pharmacologically. In addition to clinical investigation of radiation sickness, the excretory function of the stomach was studied by the excretion of intravenously injected neutral red. The "switching-off" of the parasympathetic nervous system prior to irradiation stabilized the excretory processes in the stomach, increased the resistance of animals, and, vice versa, the "switching-off" of the sympathetic nervous system destabilized the excretory processes and decreased the resistance of the organism.

Animals↗

[Lysosomotropic agents as regulators of the activity of lysosomal hydrolases].

Activities of lysosomal enzymes acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-galactosidase, acid lipase and cathepsins B and D were studied after accumulation of neutral red, acridine orange chloroquine and daunorubicin in lysosomes of fibroblasts of the LSM substrain. All the drugs studied proved to be inhibitors of these enzymes except of daunorubicin, which stimulated acid lipase activity. Lysosomotropic drugs are considered as possible regulators of the activity of lysosomes.

Acridine Orange↗