The drug-resistance of malaria parasites is the main problem in the disease control. The huge Brazilian biodiversity promotes the search for new compounds, where the animal kingdom is proving to be a promising source of bioactive compounds. The main objective of this study was to evaluate the antiplasmodial and cytotoxic activity of the compounds obtained from the toad venoms of Brazilian Amazon. Toad venoms were collected from the secretion of Rhinella marina and Rhaebo guttatus in Mato Grosso State, Brazil. The powder was extracted at room temperature, yielding 2 extracts (RG and RM) and a substance ('1') identified as a bufadienolide, named telocinobufagin. Growth inhibition, intraerythrocytic development, and parasite morphology were evaluated in culture by microscopic observations of Giemsa-stained thin blood films. Cytotoxicity was determined against HepG2 and BGM cells by MTT and neutral red assays. The 2 extracts and the pure substance ('1') tested were active against chloroquine-resistant Plasmodium falciparum strain, demonstrating lower IC₅₀ values. In cytotoxic tests, the 2 extracts and substance '1' showed pronounced lethal effects on chloroquine-resistant P. faciparum strain and low cytotoxic effect, highlighting toad parotoid gland secretions as a promising source of novel lead antiplasmodial compounds.
Amphibian Venoms* ; Animals ; Biodiversity ; Brazil* ; Bufo marinus ; Malaria ; Neutral Red ; Parasites ; Plasmodium falciparum

BACKGROUND: Microsopic examination of potassium hydroxide(KOH) preparation and fungus culture is required for the diagnosis of fungal infection. Sometimes dermatophytes fail to grow on culture medium, although these are observed on microscopic examination of KOH preparation. Recently this discrepancy between microscopic examination and fungus culture can be explained by the hyphothesis that some of the fungal elements are non-viable. OBJECT: This study was made to evaluate the viability of dermatophytes using neutral red(NR) staining for the explaination of discrepancies between microscopic examination and fungus culture. METHODS: After identification of fungus by culture from dermatophytic lesion the hyphae was collected for this study. In order to confirm whether the NR staining is suitable for check the viability of hypae or not, we designed to prepare the preparations of hyphaes by 2 ways. One was killed hyphae by autoclave, the other was kept as viable hyphae. And then we compared the stainability of NR staining and autoradiographic study using (3)H-thymidine. And we compared the results of NR staining and the subsequent culture using the scales which were collected from the lesions 30 dermatophytic patients. RESULTS: The structure inside of cell wall of hyphae stained red color only in case of viable hyphae preparations, but not stained in killed hyphae preparation. Autoradiographic study using (3)H-thymidine confirmed that grain-positive cells(viable cells) were stained with NR, whereas grain negative cells (non viable cells) were not stained. Among the 30 cases with dermatophytosis 27(90.0%) cases showed NR positive and 14(46.6%) cases showed culture-positive. Except the tinea unguium cases which have shown low culture positive rate, 9(75.0%) cases of the 12 NR positive samples were positive on culture. All 14 cases of the culture positive samples were positive on NR staining. And all 3 cases of the NR negative samples were negative on culture. CONCLUSION: NR staining can be a useful method for the evaluation of viability of the fungal elements.
Arthrodermataceae* ; Cell Wall ; Edible Grain ; Diagnosis ; Fungi ; Humans ; Hyphae ; Neutral Red* ; Onychomycosis ; Potassium ; Tinea ; Weights and Measures

Serial changes in the size of infracted area induced by MCA occlusion(MCAO) were compared with Neutral Red(NR) and 2, 3, 5-Triphenyl tetrazolium chloride(TTC) stains. The differences in size of the infracted area as shown by the 2 stains and its significance were also evaluated. The experimental animals were divided into 7 troups, with each group consisting of rats;these groups were stained at 2, 4, 6, 8, 12, 24 and 48 hours after MCAO. After MCAO, NR was infused into the femoral vein, after which the brain was removed, the fraontal pole of the brain cut into 1.5mm sections, and each section photographed. Then, the NR-stained sections were immersed in TTC solution for 45 minutes and photographed. Results showed that the infracted area progressively increased according to time duration after MCAO(one-way ANOVA, p<0.01). Between 4 and 6 hour groups, the difference of the infracted area was greater than at any other timed groups, this being statistically significant(unpaired t-test, p<0.05). After 6 hours, the infracted area with NR stain became relatively stable. In contrast, however, the infracted area with TTC stain did not stabilize, but continued to increase up to 24 hours. Overall, the infracted area with NR stain was greater than with TTC stain in all the timed groups(paired t-test, p<0.05). As time progressed, the differences tended to decrease 48 hours post occlusion. In our study, serial changes of the ischemic penumbra area were evaluated by staining the ischemic area simultaneously with Neutral red and TTC stain. The results suggest that the ischemic penumbra area may still persist even after 48 hours post-MCAO.
Animals ; Brain ; Cerebral Infarction* ; Coloring Agents ; Femoral Vein ; Infarction ; Neutral Red*

BACKGROUND: Paraquat is a widely used herbicide, known to cause lethal toxicity in humans. Most studies about paraquat have concentrated on systemic toxicity, however several cases of paraquat-induced dermatitis have been reported. OBJECTIVE: The purpose of this study was to confirm the cutaneous toxic effect of paraquat and to select potential antidotes in paraquat-induced dermatitis. METHODS: Keratinocyte toxicity due to paraquat and the toxicity reduction capacity of several drugs were investigated in eitro. Topical effects of these drugs on paraquat-induced dermatitis in guinea pig skin was also investigated. RESULTS: Over 50% of keratinocytes failed to survive at a concentration of 2X10-4M paraquat by a neutral red uptake assay. Skin irritation by paraquat was observed at 2% concentration by non-invasive methods as well as a skin biopsy. Dexamethasone, glutathione and tocopherol showed some capacity to reduce paraquat-induced keratinocyte toxicity in vitro. Only dexamethasone, however, showed a reduction of cutaneous blood flow volume and dermal inflammatory cell infiltration in the guinea pig study. CONCLUSION: This result indicates the possible in eitro protective effect of paraquat toxicity in glutathione and tocopherol. Dexamethasone was capable of reducing paraquat-induced cytotoxicity and dermatitis both in vitro and in vivo.
Animals ; Antidotes* ; Biopsy ; Dermatitis ; Dexamethasone ; Glutathione ; Guinea Pigs ; Humans ; In Vitro Techniques ; Keratinocytes ; Neutral Red ; Paraquat* ; Skin* ; Tocopherols

The current study was performed to investigate the influence of acidosis on focal cerebral ischemia in view of morphometric assay and neuropathological examination. The acidosis was induced by increment of halothane concentration and by decreasing respiratory rate. The mean pH were 7.423+/-0.012 in control group and 7.184+/-0.038 in acidosis group. Twenty-four hours after MCA occlusion(MCAO), neutral red staining and perfusion fixation was performed. The ischemic area was measured and morphometric analysis was undertaken. In acidosis group, the infarct area was 25.23+/-4.78% of the total cerebral area;in control group, the infarct area was 27.69+/-4.05%. The histopathological findings were examined under light microscopy, in which the field scanning was carried out from the midline by 0.5mm interval at cortical and basal ganglia levels. These results indicated that although there was no satistically significant difference in infarct area between acidosis and control group, increased acidosis aggravated the extent of histopathologic ischemic neuronal damage.
Acidosis* ; Animals ; Basal Ganglia ; Brain Ischemia* ; Halothane ; Hydrogen-Ion Concentration ; Microscopy ; Neurons ; Neutral Red ; Perfusion ; Rats* ; Respiratory Rate

PURPOSE: Despite the significance of the p53 adenoviral vector in cancer gene therapy, an advanced strategy for the development of preferential tumor cell-specific delivery and the long-term persistent gene expression control of p53 are required. In this study, the time-course expression patterns of p53 and E6, on cervical cancer cells, were investigated to obtain a molecular level understanding of the cell-dependent tumor growth suppression effects of a recombinant adenovirus expressing p53, both in vitro and in vivo. MATERIALS AND METHODS: The expressions of p53 and E6 in CaSki, SiHa, HeLa, HeLaS3, C33A and HT3 cervical cancer cell lines were examined. After infection with AdCMVp53, the cell growth inhibition was studied via cell count, MTT and Neutral red assays. After transfecting the AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, the anti-tumor effects were investigated for one month. RESULTS: The p53 protein levels were more notably expressed in the CaSki and HeLa than in the SiHa and HeLaS3 On day 6, the p53 was only detected in the HeLaS3. In contrast, the p53 expression was highly maintained in the C33A and HT3. The E6 mRNA levels gradually decreased in only the CaSki and HeLa. The growth suppression effects also showed cell-dependent patterns, which were consistent with the reciprocal expression patterns of p53 and E6. After transfection of the AdCMVp53, into the CaSki- and SiHa-xenografted nude mice, the tumor size was remarkably decreased in the SiHa cells as compared to that in the AdCMVLacZ transfected mice, indicating cell-specific growth inhibition patterns. CONCLUSION: The adenovirus-mediated p53 gene transfection was very effective both in vitro and in vivo. Also, the anti-tumor effects were accomplished via the differential role of p53-specific apoptotic cell death, which was dependent on the cervical cancer cell line.
Adenoviridae* ; Animals ; Cell Count ; Cell Death ; Cell Line ; Gene Expression ; Genes, Neoplasm ; Genes, p53 ; Genetic Therapy ; Humans* ; Mice ; Mice, Nude ; Neutral Red ; RNA, Messenger ; Transfection ; Uterine Cervical Neoplasms*

To overcome the limitations of clonogenic assay (low plating efficiencies, clumping artifacts, long assay duration), we performed chemosensitivity test using MTT and neutral red biologic assay on SC-115 (androgen stimulated mouse tumor) and TCC-SuP (human bladder tumor) cell lines. The anticancer agents tested were adriamycin, cisplatin, methotrexate and combination of these drugs. And the following results were obtained ; 1) The growth of two cell lines were inhibited dose-dependently by application of adriamycin, cisplatin and combination of drugs. But methotrexate was not effective on TCC-SuP cell line. 2) The neutral red biologic assay is more convenient and rapid method than MTT assay but staining ability is superior in MTT assay. 3) The MTT and neutral red biologic assay offer advantages in speed, simplicity, cost & safety for the chemosensitivity test and for determining the new regimen.
Animals ; Antineoplastic Agents ; Artifacts ; Biological Assay* ; Cell Line* ; Cisplatin ; Doxorubicin ; Humans* ; Methotrexate ; Mice* ; Neutral Red* ; Staining and Labeling ; Urinary Bladder Neoplasms* ; Urinary Bladder*

OBJECTIVES: This study was carried out to survey the prevalence of Helicobacter pylori infection and the incidence of vacuolating toxin producing H. pylori. A further aim of this study was to evaluate the quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by vaculoes of the cells. METHODS: We studied the gastric biopsy specimens of patients with 154 cases of gastritis, 74 cases of gastric ulcer, and 167 cases of gastric cancer and in 44 cases of healthy persons. One of the biopsy specimen was placed into a CLOtest plate for rapid urease test and the other one of the biopsy spcimen was inoculated on Brain Heart Infusion blood agar for culture. The culture supernatant of isolated H. pylori was serially diluted with BHI broth. After 24 hour incubation of cultured RK-13 cells treated with the culture supernatant of H. pylori, cytoplasmic vacuolation of the cells were observed microscopically. RESULTS: The positivity of urease test and the rate of isolation of H. pylori from urease positive gastric biopsy materials were 34.1% and 93.3% in healthy person, 55.8% and 70.9% in gastritis, 60.8% and 71.1% in gastric ulcer, and 56.3% and 96.8% in gastric cancer. The isolation rate of H. pylori from patients between 20 and 39 years old was 16.8%, for patients between 40 and 59 years old it was 51.9%, and for patients above 60 years old it was 31.2%. The isolation rate of the vacuolating toxin producing H. pylori from gastric biopsy specimens was 66.7% in a healthy person, 76.6% in gastritis, 79.4% in gastric ulcer, and 80% in gastric cancer. CONCLUSIONS: The isolation rate of H. pylori from the patients with gastric diseases is higher than the rate of H. pylori from healthy persons, but the isolation rate of the vacuolating toxin producing H. pylori is not different between the patients with gastric diseases and healthy persons. The titers of vacuolating toxin produced by some H. pylori isolated from the patients with gastric diseases are higher than those from healthy persons.
Adult ; Agar ; Biopsy ; Brain ; Cytoplasm ; Gastritis ; Heart ; Helicobacter pylori* ; Helicobacter* ; Humans ; Incidence* ; Middle Aged ; Neutral Red ; Prevalence ; Stomach Diseases* ; Stomach Neoplasms ; Stomach Ulcer ; Urease

OBJECTIVE: The purpose of this article is to estimate the anti-cancer effects of the major components of the green tea (polyphenols, catechin and EGCG) and the mechanism of EGCG on different cervical cancer cell lines. METHODS: Six cervical cancer cell lines (HeLa, HeLaS3, Caski, SiHa, HT3 and C33A) were treated with 20 microgram/ml green tea polyphenols (GTPs), 50 micrometer catechin and various concentrations of (-)-epigallo- catechin-3-gallate (EGCG). The viabilities were determined by trypan blue exclusion assay, neutral red assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. DNA fragmentation and nuclear condensation were used to see whether EGCG-induced anti-proliferation effect was due to apoptosis. RESULTS: Both GTPs, catechin and EGCG had growth inhibition effects on cervical cancer cell lines, but EGCG appeared to be the most effective. What's more, the sensitivity of each cell lines to EGCG was different. HT3 cells (HPV negative, mutant type p53) were most sensitive to EGCG (estimate IC50: 10 micrometer). Caski (HPV-16 positive, wild type p53) and HeLaS3 cells (HPV-18 positive, wild type p53) were less sensitive (estimate IC50: 35 and 70 micrometer respectively). EGCG-induced apoptosis can be seen in all the cell lines and it happened as early as 8 hours after EGCG treatment. CONCLUSION: Green tea or EGCG alone will be beneficial to the cervical cancer patients.
Apoptosis* ; Catechin ; Cell Line* ; Chemoprevention ; DNA Fragmentation ; Guanosine Triphosphate ; Humans ; Inhibitory Concentration 50 ; Neutral Red ; Polyphenols* ; Tea* ; Trypan Blue ; Uterine Cervical Neoplasms*

BACKGROUND: The extract and hemiterpene glycosides of Ilex Rotunda Thunb exert antioxidant and anti-inflammatory effects. The effect of rotundarpene on apoptosis in neuronal cells caused by the 1-methyl-4-phenylpyridinium (MPP⁺) has not been reported previously. METHODS: Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on MPP⁺-caused apoptosis in relation to the cell death process. RESULTS: MPP⁺-induced cell death was identified using the MTT and neutral red uptake tests. Apoptosis was induced by eliciting decreases in the cytosolic levels of Bid and Bcl-2 proteins, increases in the cytosolic levels of Bax and p53, disruption of the mitochondrial transmembrane potential, and the release of cytochrome c and the activation of caspase-8, -9, and -3 in differentiated PC12 cells and SH-SY5Y cells. Treatment with rotundarpene reduced the MPP⁺-induced changes in the levels of apoptosis-regulated proteins, formation of reactive oxygen species, depletion and oxidation of glutathione, and cell death in both PC12 and SH-SY5Y cells. CONCLUSIONS: Rotundarpene may reduce MPP⁺-induced apoptosis in neuronal cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8 and Bid pathways. Rotundarpene appears to act by inhibiting the production of reactive oxygen species and by the depletion and oxidation of glutathione.
1-Methyl-4-phenylpyridinium ; Animals ; Apoptosis ; Caspase 8 ; Cell Death* ; Cytochromes c ; Cytosol ; Glutathione ; Glycosides ; Humans ; Ilex ; Membrane Potentials ; Neuroblastoma ; Neurons ; Neutral Red ; PC12 Cells ; Reactive Oxygen Species