Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast

Alcohol dehydrogenases (ADHs) are oxidoreductases present in animal tissues, plants, and microorganisms. These enzymes attract major scientific interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in synthesis, thanks to their broad substrate specificity and stereoselectivity. In the present study, the standardization of the activity of the alcohol dehydrogenase from baker's yeast was accomplished, and the pH and temperature stability showed, that the enzyme presented a high stability to pH 6.0-7.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The assays of ethanol (detection range 1-5 mM or 4.6 x 10(-2) to 23.0 x 10(-2) g/L) in different samples in alcoholic beverages, presented a maximum deviation of only 7.2%. The standard curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable analytical method. (c) 2006 Elsevier B.V. All rights reserved.

Antitumor activity of extracts from Peperomia elongata

The cytotoxic potential of ethanol extracts from Peperomia elongata H. B. & K. (Piperaceae) were evaluated against human cancer cell lines by the MTT method. The samples considered cytotoxic were tested for antimitotic activity with the sea urchin egg development test and for hemolytic activity using mice erythrocytes. The extracts from leaves (hexane), stems (ethanol, hexane, hexane: AcOEt, AcOEt, and MeOH: H2O insoluble), and roots (R4) presented potential cytotoxic action. The stems extracts showed the highest toxicity in all tumor cell lines tested, with an IC50 <= 9.0 mu g/mL for ethanol extract, IC50 <= 11.6 mu g/mL for MeOH:H2O insoluble, IC50 <= 7.3 mu g/mL for hexane extract, IC50 <= 11.4 mu g/mL for hexane: AcOEt, and IC50 <= 16.2 mu g/mL for AcOEt extract. All extracts considered cytotoxic for tumoral cell lines presented antimitotic activity. The samples from roots (R4) and stems (ethanol, MeOH: H2O insoluble, and hexane extract from leaves) were found to possess lytic activity in mice erythrocytes but in higher doses (> 125 mu g/mL). Further studies for the isolation and identification of the active principles of these extracts should be undertaken.

Mutagenic activity in waste from an aluminum products factory in Salmonella/microsome assay

The mutagenic activity of waste material originating from an aluminum products factory was determined by the Salmonella/microsome assay, using the bacterial strains TA100, TA98 and YG1024. The material was obtained by sweeping the factory floor at the end of the work shift. Organic compounds were extracted by ultrasound for 30 min in dichloromethane or 70% ethanol. After evaporation of solvent, these extracts were dissolved in dimethylsulfoxide, and tested for the mutagenic activity at varying concentrations. All the extracts from the factory had mutagenic activity, especially in the YG1024 strain, suggesting the presence of aromatic amines, later confirmed by chemical analysis. The TA98 strain also showed mutagenic activity, though it did not exhibit the highest mutagenicity index observed with the YG1024 strain. In TA100, mutagenic activity was not observed. This study should serve as an alert to management and those who are occupationally exposed, and as a warning that this type of waste should not be discarded in the environment without any control. (C) 2004 Elsevier Ltd. All rights reserved.

Effects of light-curing time on the cytotoxicity of a restorative resin composite applied to an immortalized odontoblast-cell line

This in vitro study evaluated the cytotoxic effects of a restorative resin composite applied to an immortalized odontoblast-cell line (MDPC-23). Seventy-two round resin discs (2-mm thick and 4 mm in diameter) were light-cured for 20 or 40 seconds and rinsed, or not, with PBS and culture medium. The resin discs were divided into four experimental groups: Group 1: Z-100/20 seconds; Group 2: Z-100/20 seconds/rinsed; Group 3: Z100/40 seconds; Group 4: Z-100/40 seconds/rinsed. Circular filter paper was used as a control material (Group 5). The round resin discs and filter papers were placed in the bottom of wells of four 24-well dishes (18 wells for each experimental and control group). MDPC-23 cells (30,000 cells/cm(2)) were plated in the wells and allowed to incubate for 72 hours. The zone of inhibition around the resin discs was measured under inverted light microscopy; the MTT assay was carried out for mitochondrial respiration and cell morphology was measured under SEM. The scores obtained from inhibition zone and MTT assay were analyzed with the Kruskal-Wallis followed by Dunnett tests. In Groups 1, 2, 3 and 4, the thickness of the inhibition zone was 1,593 +/- 12.82 mum, 403 +/- 15.49 mum, 1,516 +/- 9.81 mum and 313 +/- 13.56 mum, respectively. There was statistically significant difference among the experimental and control groups at the 0.05 level of significance. The MTT assay demonstrated that the resin discs of the experimental groups 1, 2, 3 and 4 reduced the cell metabolism by 83%, 40.1%, 75.5% and 24.5%. Only between the Groups 2 and 4 was there no statistically significant difference for mitochondrial respiration. Close to the resin discs, the MDPC-23 cells exhibited rounded shapes, with only a few cellular processes keeping the cells attached to the substrate or, even disruption of plasma membrane. Adjacent to the inhibition zone, the cultured cells exhibited multiple fine cellular processes on the cytoplasmic membrane organized in epithelioid nodules, similar to the morphology observed to the control group. Based on the results, the authors may conclude that the Z-100 resin composite light cured for 20 seconds was more cytopathic to MDPC-23 cells than Z-100 light cured for 40 seconds. The cytotoxic effects of the resin discs decreased after rinsing them with PBS and culture medium. This was confirmed by MTT assay and upon evaluation of the inhibition zone, which was narrower following rinsing of the resin discs.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method, the cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement ( = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3 h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom. (c) 2005 Elsevier Ltd. All rights reserved.

4 '-Aminochalcones As Novel Inhibitors of the Chlorinating Activity of Myeloperoxidase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: A morphological, immunohistochemical, biochemical, and molecular study

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400 mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health. (C) 2011 Elsevier B.V. All rights reserved.

Cellular and tissue effects induced by photogemA (R) and red LED in photodynamic therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation of an immunohistochemistry assay to detect turkey coronavirus: A rapid and simple screening tool for limited resource settings

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytotoxicity of Active Ingredients Extracted from Plants of the Brazilian "Cerrado"

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)