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

BACKGROUND: There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies. Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models. OBJECTIVE: In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants. METHODS: The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants RESULTS: The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration. CONCLUSION: The new human skin recombinant can be used as an alternative to animals for skin irritancy screening.
Animals ; Cell Culture Techniques ; Collagen ; Dermis ; Epidermis ; Healthy Volunteers ; Humans* ; Interleukin-1alpha ; Irritants ; Keratinocytes ; Mass Screening ; Neutral Red ; Organ Culture Techniques ; Patch Tests ; Polyethylene ; Skin*

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

OBJECTIVES: The aim of this study is to find out the activity of autoproliferation of ratfibroblast exposed to crystalline silica and the role of mediators secreted from rat fibroblast. METHODS: The effect of alpha-quartz on production of growth factor (platelet-derived growth factor-AA and transforming growth factor beta)from rat fibroblasts were evaluated by ELISA and immunocytochemical analysis. Gene expression of these growth factors in rat fibrobast exposed to crystalline silica was evaluated by RT-PCR. Furthermore, fibroblast proliferation by culture supernatant of rat fibroblast was assayed by the neutral red test. RESULTS: The amounts of H2O2 and growth factors synthesized in rat fibroblasts were significantly increased by the stimulation of crystalline silica(alpha-quartz), which showed the dose-dependent manner to the concentration of alpha-quartz with the maximum response at the dosage of 100 microgram/cm2. The result of RT-PCR demonstrated that alpha-quartz induced gene expression of PDGF-AA and TGFbeta in rat fibroblast. We also found that supernatant of alpha-quartz-cocultured rat fibroblast induced a significant proliferation of fibroblast. CONCLUSION: Crystalline silica directly induce functional change in fibroblast such as increased release of reactive oxygen species and growth factors. The products of these functional change promote fibroblast proliferation via autocrine loop.
Animals ; Crystallins* ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Gene Expression ; Intercellular Signaling Peptides and Proteins ; Neutral Red ; Rats ; Reactive Oxygen Species ; Silicon Dioxide* ; Transforming Growth Factor beta ; Transforming Growth Factors

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*

The present study was undertaken to investigate the influence of hyperglycemia on focal cerebral ischemia in view of morphometric assay and neuropathological examination. Forty Sprague-Dawley rats were divided into two groups of 20 each. Rat MCA occlusion model was used for induction of focal ischemia. Hyperglycemia(20 rats, mean(SEM plasma glucose concentration 378(97.6 mg/dl) was established 30 minutes before MCA occlusion by intraperitoneal injection of 50% dextrose in water;the control group(20 rats, mean(SEM plasma glucose concentration 121(24.9 mg/dl) received normal saline only. Twenty-four hours after MCA occlusion neutral red staining and perfusion fixation was performed and ischemic area were measured using computerized image analysis on cortical surface and coronal cut surface. There was no significant difference on coronal cut surface, but on cortical surface showed increase of non-stained area(infarct core) and decrease of lightly stained area(transitional zone) in hyperglycemic rats(p<0.05) and the sum of two area was not different between two groups. Pathological findings were evaluated under light microscopy, in which the field scanning was carried out from the midline by 0.5 mm interval at cortical and basal ganglia level. There showed no significant difference at basal ganglia level, but at cortical level ischemic transitional zone was decreased in hyperglycemic rats(p<0.05). We conclude that hyperglycemia may worsen the brain from severe, focal ischemic neuronal damage.
Animals ; Basal Ganglia ; Blood Glucose ; Brain ; Brain Ischemia* ; Glucose ; Hyperglycemia* ; Injections, Intraperitoneal ; Ischemia ; Microscopy ; Neurons ; Neutral Red ; Perfusion ; Rats ; Rats, Sprague-Dawley