ABSTRACT
The present work describes the isolation and purification of two Leishmania chagasi (= syn. Leishmania infantum) recombinant proteins, rLci2B and rLci1A, and their use in the development of an immunoassay for the diagnostic of canine leishmaniasis. After protein expression and cell disruption, rLci2B was purified by immobilized metal affinity chromatography followed by size exclusion chromatography, whereas rLci1A, expressed as an inclusion body, was treated with urea and purified by anion-exchange chromatography. Homogeneities were ascertained by denaturing gel electrophoresis (MW (rLci2B) = 46,370; MW(rLci1A) = 88,400), isoelectric focusing (pI (rLci2B) = 5·91; pI (rLci1A) = 6·01) and Western blot. An indirect ELISA was developed using the purified antigens rLci2B and rLci1A and a leishmaniasis canine serum panel (n = 256). The ELISA showed 100% sensitivity and 95% specificity for rLci2B and 96% sensitivity and 92% specificity for rLci1A. The purified proteins did not present cross-reactivity with sera from dogs infected with Trypanosoma caninum, Babesia canis and Ehrlichia canis. Cross-reaction was verified with sera from dogs infected with Leishmania brasiliensis (11·7% for rLci2B and 2·9% for rLci1A). Based on ELISA results, it is suggested the use of rLci2B and rLci1A as antigens in an alternative serological assay for diagnostic of canine leishmania.
Subject(s)
Antibodies, Protozoan/blood , Antigens, Protozoan , Dog Diseases/diagnosis , Leishmania infantum/immunology , Leishmaniasis/veterinary , Animals , Antigens, Protozoan/genetics , Antigens, Protozoan/isolation & purification , Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Cross Reactions , Dogs , Enzyme-Linked Immunosorbent Assay/methods , Leishmania infantum/genetics , Leishmaniasis/diagnosis , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
Peroxide gels are effective in changing tooth colour but their effect on restorative materials has been poorly studied. The purpose of this investigation was to assess the impact of a commercially available whitening gel containing hydrogen peroxide and a sodium percarbonate formulation on the surface of restorative materials. A total of 12 subjects participated in a double-blinded crossover study. Each wore an intra-oral appliance containing five bovine enamel blocks restored with amalgam, posterior composite, microfilled composite, glass ionomer cement and porcelain. Appliances were worn continuously for 14 days and whitening products were applied twice daily. After 14 days the appliances were removed and values for roughness (R(a)) were obtained using atomic force microscopy. Mean values of R(a) were assessed between baseline and 14 days, and although minor variations were seen, there were no statistically significant differences detected for any material or any whitening product.
Subject(s)
Carbonates , Dental Materials , Hydrogen Peroxide , Oxidants , Tooth Bleaching , Analysis of Variance , Animals , Cattle , Composite Resins , Cross-Over Studies , Dental Amalgam , Dental Porcelain , Dental Restoration, Permanent , Double-Blind Method , Glass Ionomer Cements , Humans , Surface PropertiesABSTRACT
Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H2O2. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylene blue and azure B (AB) in reaction mixtures containing different dye:H2O2 stoichiometric relations aiming at the selective formation of N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1:1 and 1:2 MB:H2O2, respectively. Using azure B as substrate in reaction mixtures containing 1:1 AB:H2O2, a yield of 70% was also observed in azure A. Reaction mixtures containing 1:3 MB:H2O2 and 1:2 AB:H2O2, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH3)2 oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye:H2O2 relation of 1:10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500-700 nm. The product identifications were performed using a reverse-phase high-performance liquid chromatography (HPLC) C-18 column and thin-layer chromatography.
