Colorimetric enzymatic assay of L-malic acid using dehydrogenase from baker's yeast

A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH) from dry baker's yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In the present study, the method exhibited sensitivity in the range of 500-4000 mu M of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7-10(-2) g/L, the upper limit of 53.6.10(-2) g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker's yeast was also optimized, the enzyme showed a high stability at pH=8.0-9.0 and the activity was maintained completely at temperatures up to 40 degrees C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker's yeast is a simple and low-cost method with possibility of wide application.

Analysis of systemic inflammatory response in the carcinogenic process of uterine cervical neoplasia

The inflammatory response is an active process in cervical cancer and may act in the progression and/or regression of the lesion. At the site of inflammation, macrophages and neutrophils are present as well as cytokines such as TNF-alpha and IFN-gamma. This study aims to evaluate the inflammatory response levels in women with cervical intraepithelial lesions (CIN) and with squamous cell carcinoma (SCC) of the cervix. Serum samples obtained from women without evidence of disease (n = 30), with CIN (n = 30) and with SCC of the cervix (n = 30) were analyzed for the activities of N-acetylglucosaminidase (NAG) and myeloperoxidase (MPO) by enzymatic assay and the serum levels of TNF-alpha and IFN-gamma by ELISA assay. The activities of NAG and MPO and the level of TNF-alpha were higher in women with CIN compared to the women with SCC. The levels of IFN-gamma were lower in the group of women with CIN compared to the group with SCC. There was not a significant association between the degree of the CIN and the staging of the SCC of the cervix and the degree of inflammation as assessed by the levels of inflammatory markers. The inflammatory response was inversely correlated with the progression of the carcinogenic process. In the three groups, the control group, women with CIN and women with invasive SCC, there was no association between the degree of preinvasive lesions and staging of the SCC of the cervix. (C) 2011 Elsevier Masson SAS. All rights reserved.

Cytotoxicity of Portland Cement with Different Radiopacifying Agents: A Cell Death Study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tillage and phosphorus management effects on enzyme-labile bioactive phosphorus availability in Cerrado Oxisols

Phosphorus (P) is an essential element in crop nutrition, which can be growth limiting or an environmental contaminant, if present in excess. Tillage practices have a direct effect on the behavior and availability of soil P. Sorption and availability of various P forms were evaluated in an incubation-fractionation study of three soils, a Typic Paleudults (CR soil) and two Cerrado Oxisols (Latossolo Vermelho-Amarelo [LVA] and Latossolo Vermelho [LV]) with distinct biogeochemical characteristics and tillage management history. Phosphate and myo-inositol hexakisphosphate (mIPH) were strongly sorbed by the soils. Maximum adsorption capacities (S(max)) were 2.2-6.9, 3.3-7.8, and 1.6-19.8 mmol kg(-1) for phosphate in the 0-40 cm depths of the CR, LV, and LVA soils, respectively. For mIPH, S. were 1.2-3.7, 3.7-5.5, and 4.6-5.2 mmol kg(-1). Saturation indices reflected the long-term effect of repeated manure applications on the Paleudults and the near saturation of its P holding capacity, in contrast to the recently cultivated Cerrado soils. Tillage method appeared to have altered P retention characteristics of the near-surface zone very slightly, while increases in ligand-exchangeable (EEP;) and enzyme-labile organic P (EDTA-PHP) forms were observed in no-till Oxisols. In the Paleudults, added manure P increased bioactive P fractions and P saturation of no-till near-surface soil zone. Estimates of all bioactive P fractions using the ligand-based enzymatic assay showed it to be an effective method for assessing P availability in soil and developing sustainable P management strategies, particularly in Cerrado Oxisols that were low in organic matter while having an extensive P-fixing capacity. Published by Elsevier B.V.

Capillary blood glucose and venous blood glucose measured with portable digital glucometer in diabetic dogs

This study aimed to compare the glycemic values obtained with a glucometer with those determined by a colorimetric enzymatic assay in venous blood as well as to evaluate the possibility of using capillary blood samples of dogs with diabetes mellitus. A group with 30 diabetic dogs was formed and from each dog three blood samples were obtained for glycemic evaluations by different methods and blood collection sites. The mean glycemic values showed no significant difference between the different sites of blood collection and methods (P=0.90). Venous, pinna and carpal pad blood glucose showed excellent correlation with the colorimetric enzymatic assay (r=0.98; r=0.95 and r=0.96 respectively) and the obtained values fit properly the clinically acceptable intervals in the error grid analysis. The present study revealed that carpal pad, venous and pinna glucose measurements are clinically acceptable and this method is feasible for use in hospitalized diabetic dogs. The sample attainment of carpal pad proved to be effective and a viable alternative. Further work is necessary to assess the utility of this technique in a home environment.

Avaliação da resposta inflamatória em mulheres com lesões pré-invasoras e invasoras de colo uterino

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

N-acetylglucosaminidase, myeloperoxidase and vascular endothelial growth factor serum levels in breast cancer patients

Inflammatory cells surround breast carcinomas and may act promoting tumor development or stimulating anti-tumor immunity. N-acetylglucosaminidase (NAG) has been employed to detect macrophage accumulation/activation. Myeloperoxidase (MPO) is considered a marker for neutrophils activity/accumulation. Vascular Endothelial Growth Factor (VEGF) is as strong pro-angiogenic cytokine. The aim of this study was to measure the systemic inflammatory response by measuring serum levels of NAG, MPO and VEGF in women diagnosed with breast cancer and associate this response to the peritumoral inflammatory infiltrate and to prognostic factors. Serum samples obtained from women with no evidence of disease (n = 31) and with breast cancer (n = 68) were analyzed for the activities of NAG, MPO and VEGF by enzymatic assay. Serum levels of NAG and VEGF were higher in healthy volunteers (P < 0.0001) and serum levels of MPO were higher in patients with breast cancer (P = 0.002). Serum levels of NAG were positively correlated to serum levels of MPO and VEGF (P < 0.0001 and P = 0.0012, respectively) and MPO and VEGF serum levels had also a positive correlation (P = 0.0018). The inflammatory infiltrate was not associated to serum levels of the inflammatory markers, and higher levels of MPO were associated to lymphovascular invasion negativity (P = 0.0175). (C) 2013 Elsevier Masson SAS. All rights reserved.

Prostate hyperplasia caused by long-term obesity is characterized by high deposition of extracellular matrix and increased content of MMP-9 and VEGF

Recent studies have shown a positive association of cancer and obesity, but the morphological and molecular mechanisms involved in this relationship are still unknown. This study analysed the impact of long-term obesity on rat prostate, focusing on stromal changes. Male adult Wistar rats were treated with high-fat diet to induce obesity, while the control group received a balanced diet. After 30 weeks of feeding, the ventral prostate was analysed by immunohistochemistry for cell proliferation, smooth muscle α-actin, vimentin, chondroitin sulphate and metalloproteinases (MMP-2 and 9). The content of androgen receptor (AR), oestrogen receptors (ERs) and vascular endothelial growth factor (VEGF) was measured by Western blotting, and activity of catalase and Glutathione-S-Transferase (GST) were quantified by enzymatic assay. Long-term obesity decreased testosterone plasma levels by 70% and resulted in stromal prostate hyperplasia, as evidenced by increased collagen fibres. Such stromal hyperplasia was associated with increased number of blood vessels and raised VEGF content, and increased expression of chondroitin sulphate, vimentin, α-actin and MMP-9. In spite of the high cell density in prostate, the proliferative activity was lower in the prostates of obese rats, indicating that hyperplasia was established during the early phases in this obesity model. AR levels increased significantly, whereas the ERα decreased in this group. Moreover, the levels of catalase and GST were changed considerably. These findings indicate that long-term obesity, besides disturbing the antioxidant control, causes intense stromal remodelling and release of factors that create an environment that can promote proliferative disorders in the gland, culminating with diffuse hyperplasia.

Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC (R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Protein phosphatase activities in the serum and saliva of healthy children

The purpose of this study was to investigate the activities of the total acid phosphatase (TAP), tartrate-resistant acid phosphatase (TRAP), low molecular weight protein tyrosine phosphatase (LMW-PTP) and alkaline phosphatase (ALP) enzymes, as well as the possible correlation in the serum and in unstimulated whole saliva of children. Enzymatic activities were measured in pairs of concurrently obtained serum and salivary samples from 32 children in good oral and systemic health (16 of each sex) with a median age of 6.4 ± 3.3 years (range 1.08 – 12.92 years). All collections were made between the hours of 08:00 – 10:00 a.m. We used p-nitrophenyl phosphate as the substrate in the enzymatic assay for TAP, TRAP and LMW-PTP, and thymolphthalein monophosphate as the substrate for ALP. The enzymatic activities of all the studied enzymes were higher in serum than in saliva. The mean of enzymatic activities of serum TAP, TRAP, LMW-PTP and ALP were 36.51 ± 8.21, 23.99 ± 5.73, 11.16 ± 5.65 and 76.50 ± 17.32 U/L, respectively, while the mean salivary TAP, TRAP, LMW-PTP and ALP enzymatic activities were 9.60 ± 5.04, 1.36 ± 0.87, 5.65 ± 3.07 and 4.08 ± 1.83 U/L in this order. The TRAP revealed a positive linear correlation between its activity in the serum and saliva (Spearman r = 0,4685, p < 0,05). We concluded that the salivary TRAP has a potential to be use as biomarkers of pathologies and states that modify its activity in the serum.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Enzymatic Hydrolysis of Sweet Lupin, Chickpea, and Lentil 11S Globulins Decreases their Antigenic Activity

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11 S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11 S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation

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

Viable human buccal mucosa cells do not yield typical nucleoids: Impacts on the single-cell gel electrophoresis/comet assay

Buccal mucosa (BM) cells have been used in human biomonitoring studies for detecting DNA adducts and chromosomal damage in an epithelial cell population. In the present study, we have investigated if human BM cells are suitable for use in the single-cell gel electrophoresis (SCGE)/Comet assay as an approach for estimating the exposure of epithelial cells to DNA-damaging agents. Our results indicate that only a few cells from BM cell samples yield comets that can be analyzed by current methods, and that the yield of cells with comets is independent of the percentage of viable BM cells in the sample. Data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggest that most of the BM cells that do form comets are probably leukocytes. Moreover, by reevaluating specific cells after running the Comet assay, we found that viable epithelial BM cells give rise to atypical comets that are not included in the analysis. Comparing DNA migration patterns between small groups of smokers and nonsmokers indicated that long-term smoking had no effect on the subpopulation of cells that yield typical comets. Our results indicate that the SCGE assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human epithelial BM cells.

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained from Streptomyces aureofaciens

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.