Evaluation of an ELISA for canine leishmaniasis immunodiagnostic using recombinant proteins.

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.

Effect of whitening gels on the surface roughness of restorative materials in situ.

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.

An allergenic 2S storage protein from Ricinus communis seeds which is a part of the 2S albumin precursor predicted by c-DNA data.

1. A 1.9S albumin having allergenic activity and denoted Ric c III was isolated from an alcohol extract of defatted Ricinus communis seeds, CB-1A, as a homogeneous protein by ion-exchange chromatography on SP-Sephadex, gel filtration on Sephadex G-75 and preparative polyacrylamide gel electrophoresis (6 mg Ric c III/g CB-1A). 2. The protein contained approximately 98 amino acid residues distributed in 2 chains of 67 and 34 residues, a molecular weight of 11,239 based on amino acid composition and pI = 4.9 Ric c III can be aligned, on the basis of amino acid composition and partial amino acid sequence data, with residues 18 to 50 (51) and 66 to 130 of the 2S albumin precursor predicted by the cDNA data of S.D. Irwin, J.N. Keen, J.B.C. Findlay and J.M. Lord (Molecular and General Genetics, 222: 400-408, 1990). 3. The present data identify Ric c III as the second allergenic 2S storage albumin coded by this DNA.

Isolation and partial characterization of antigen 5.1 from the seeds of Ricinus communis, L. (Var. Amarelo de Irece).

Antigen 5.1 was isolated from the most acidic fraction of castor bean allergens (CB-1A) by gel filtration on Sephadex G-75 followed by polyacrylamide gel electrophoresis (PAGE) (yield: 6.2 mg antigen 5.1/g CB-1A). This antigen was homogeneous by the criteria of PAGE, isoelectric focusing, SDS-PAGE, immunoelectrophoresis and gel filtration. The antigen has an apparent molecular mass of 12 +/- 2 kDa and an isoelectric point of pH 5.1. Antigen 5.1 lacks proline, phenylalanine, threonine and tryptophan. It was immunochemically identical to one of the three immunoprecipitation lines presented by CB-1A by the Ouchterlony technique, and was positive when tested (250 micrograms) by IgE-mediated passive cutaneous anaphylaxis in LOU.M rats.

N-demethylation of methylene blue by lignin peroxidase from Phanerochaete chrysosporium. Stoichiometric relation for H2O2 consumption.

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.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Partial characterization of RC-13, a homogeneous fraction isolated from castor bean allergens (CB-1A).

Allergen RC-13's amino acid composition was determined-Lys3, His1, Arg7, Asp6, Thr2, Ser11, Glu29, Pro2, Gly7, Ala4, Cys6, Val5, Met1, Ile3, Leu3, Tyr1; 91 residues. Partial specific volume and minimum molecular weight evaluated from this composition were, respectively, 0.694 cm3/g and 10,194. SDS-polyacrylamide gel electrophoresis performed in the reduced and alkylated form of the allergen revealed a single band, suggesting the existence of only one polypeptide chain in the molecule. Results obtained for carbohydrate analysis by gas-liquid chromatography were: 22.2% galactose and 16.2% mannose--approximately 38% of neutral sugars. Employment of the amino acid methodology for analysis of amino sugars revealed two residues of glucosamine per molecule of the allergen. Hydrolyses with trypsin, alpha-chymotrypsin and type VII protease were tested for sequence studies. Trypsin was considered a suitable enzyme for such purpose.

Chemical and physicochemical characterization of CB-1a, an allergenic fraction isolated from castor bean (Ricinus communis L.).

CB-1A chemical characterization comprised the determination of nitrogen (Kjeldhal: 15.5%: amino acid composition: 17.5%), total (6.8%) and reducing (1.6%) carbohydrates, amino acid composition (Lys4, His1, Arg11, Asp3, Thr1, Ser7, Glu16, Pro2, Gly5, Ala3, Cys6, Val3, Met1, Ile3, Leu4, Tyr1, Phe1; 82 residues). Its behaviour under several TCA concentrations, ultraviolet absorption of native and oxidized CB-1A under several solvents as well as its enzymatic susceptibility were explored. Physicochemical parameters such as MW (protein moiety - composition: 9.642 Da, column: 9.431 Da: glycoprotein-10.345) Da; E1%1cm,220mm = 49.7 E1%1cm,595mm = 29.7: partial specific volume (anhydrous: 0.703 cm3/g; hydrated; 0.924 cm3/g); Stokes radius (15.1 A); hydration water (22%) and frictional ratio (1:10) were determined. Calculated limit molecular dimensions (semi-axis of revolution: 11.4 to 19.8 A) and equatorial radius (13.2 to 17.5 A suggest dendency to sphericity. CB-1A showed allergenicity by PCA. Fourteen components were detected by IEF.

Structural studies on allergen RC-13 from Ricinus communis L.: isolation and characterization of a major glycopeptide.

A glycoprotein, RC-13, isolated from Ricinus communis seeds was reduced, S-alkylated and cleaved by trypsin. The tryptic digest was fractionated by ion-exchange chromatography and a glycopeptide was isolated and purified by high-voltage paper electrophoresis. When submitted to amino acid and carbohydrate analyses this major glycopeptide showed the following chemical composition: Lys1, Asp1, Thr2, Ser4, Glu1, Pro2, Gly2, Ala2, Val2, GlcN6, Man6 and Gal8. Hydrazynolysis positioned Ser as the C-terminal residue. It is postulated that this glycopeptide belongs to the C-terminal region of the allergen.

An allergenic 2S storage protein from ricinus communis seeds which is a part of the 2s albumin precursor predicted by C-DNA data

A 1.9s albumin having allergenic activity and denoted Ric c III was isolated from an alcohol extract of defatted Ricinus communis seeds CB-1A, as a homogeneous protein by ion -exchange chromatography on SP-Sephadex, gel filtration on Sephadex G-75 and preparative polyacrylamide gel electrophoresis (6 mg Ric cIII/g CB-1A). The protein contained approximately 98 amino acid residues distributed in 2 chains of 67 anmd 34 residues, a molecular weight of 11.239 based on amino acid composition and pI=4.9 Ric c III can be aligned, on the basis of amino acid composition and partial amino acid sequence data, with residues 18 to 50 (51) and 66 to 130 of the 2S albumin precursor predicted by the cDNA data of S. D. Irwin, J. N. Ken, J. B. C. Findlary and J. M. Lord (Molecular and General Genetics, 222:400-408, 1990). The present data identify Ric c III as the second allergenic 2S storage albumin coded by this DNA

Isolation and partial characterization of antigen 5.1 from the seeds of Ricinus communis, L. (var. amarelo de irece)

Antigen 5.1 was isolated from the most acidic fraction of castor bean allergens (CB-1A) by gel filtration on Sephadex G-75 followed by polyacrylamide gel electrophoresis (PAGE) (yield: 6.2 mg antigen 5.1/g CB-1A). This antigen was homogeneous by the criteria of PAGE, isoelectric focusing, SDS-PAGE, immunoelectrophoresis and gel filtration. The antigen has an apparent molecular mass of 12 ñ 2 kDa and an isoelectric point of pH 5.1. Antigen 5.1 lacks proline, phenylalanine, threonine and tryptophan. It was immunochemically identical to one of the three immunoprecipitation lines presented by CB-1A by the Ouchterlony technique, and was positive when tested (250 *g) by IgE-mediated passive cutaneous anaphylaxis in LOU.M rats

Structural studies on allergen RC-13 from Ricinus communis L.: isolation and characterization of a major glycopeptide

A glycoprotein, RC-13, isolated from Ricinus communis seeds was reduced, S-alkylated and cleaved by trypsin. The tryptic digest was fractionated by ion-exchange chromatography and a glycopeptide was isolated and purified by high-voltage paper electrophoresis. When submitted to amino acid and carbohydrate analyses this major glycopeptide showed the following chemical composition: Lys1, Asp1, Thr2, Ser4, Glu1, Pro2, Gly2, Ala2, Val2, GlcN6, Man6 and Gal8. Hydrazynolysis positioned Ser as the C-terminal residue. It is postulated that this glycopeptide belongs to the C-terminal region of the allergen