RESUMO
[11C]Methionine was supplied through barley roots and the 11C signal was followed for 90 min using a real-time imaging system (PETIS), with subsequent development of autoradiographic images of the whole plant. In all cases, [11C]methionine was first translocated to the 'discrimination center', the basal part of the shoot, and this part was most strongly labeled. Methionine absorbed by the roots of the plants was subsequently translocated to other parts of the plant. In Fe-deficient barley plants, a drastic reduction in [11C]methionine translocation from the roots to the shoot was observed, while a greater amount of 11C was found in the leaves of Fe-sufficient or methionine-pretreated Fe-deficient plants. Treatment of Fe-deficient plants with aminooxyacetic acid, an inhibitor of nicotianamine aminotransferase, increased the translocation of [11C]methionine to the shoot. The retention of exogenously supplied [11C]methionine in the roots of Fe-deficient barley indicates that the methionine is used in the biosynthesis of mugineic acid phytosiderophores in barley roots. This and the absence of methionine movement from shoots to the roots suggest that the mugineic acid precursor methionine originates in the roots of plants.
Assuntos
Ácido Azetidinocarboxílico/análogos & derivados , Ácido Azetidinocarboxílico/metabolismo , Hordeum/metabolismo , Metionina/metabolismo , Sideróforos/biossíntese , Ácido Amino-Oxiacético/farmacologia , Transporte Biológico/efeitos dos fármacos , Radioisótopos de Carbono , Inibidores Enzimáticos/farmacologia , Deficiências de Ferro , Proteínas de Plantas/antagonistas & inibidores , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Sideróforos/química , Enxofre/deficiência , Transaminases/antagonistas & inibidoresRESUMO
The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of Leptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against L. interrogans serovar pomona and L. bergpetersenii serovars hardjo and sejroe to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83. 7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-Leptospira antibodies.
Assuntos
Flagelina , Polarização de Fluorescência , Leptospirose/diagnóstico , Animais , Antígenos de Bactérias , Bovinos , Cromatografia em Gel , Fluoresceína-5-Isotiocianato , Polarização de Fluorescência/métodos , Leptospirose/etiologia , Leptospirose/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The binding of tritiated mebendazole, a benzimidazole anthelmintic, to tubulin derived from intestine, body wall muscle, and reproductive system of adult Ascaris suum was examined and compared. Mebendazole binding was resolved into specific and nonspecific binding and the binding affinity (Ka) and maximum binding at infinite ligand concentration (Bmax) determined. Electron microscopy was performed to assess the tubulin in various tissues of A. suum quantitatively by observing the presence of microtubules. Total binding was highest in intestine followed by body wall muscle. It was least in the reproductive system. The intestine demonstrated greater specific binding per milligram of protein than the body wall muscle. However, in the reproductive system extract, high affinity binding was not detected. After correction for nonspecific binding of ligand, the results indicated that the Bmax of mebendazole for the tubulin of A. suum intestine was about 3-fold higher than for that of body wall muscle. The Ka of mebendazole for intestinal tubulin was similar to that for body wall muscle. Electron microscopy of A. suum tissues demonstrated that the tubulin content decreased from the intestine through the body wall muscle to the reproductive system. Differences in tubulin content from different tissues may determine the selective sensitivity of these tissues to benzimidazole attack.
Assuntos
Ascaris suum/metabolismo , Mebendazol/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/ultraestrutura , Feminino , Genitália/efeitos dos fármacos , Genitália/metabolismo , Genitália/ultraestrutura , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/ultraestrutura , Mebendazol/farmacologia , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Músculos/efeitos dos fármacos , Músculos/metabolismo , Músculos/ultraestrutura , Ligação Proteica , SuínosRESUMO
Three monoclonal antibodies (mAb) specific to beta-tubulin were used to investigate the heterogeneity of tubulins from nematodes and mammals. Western blot analysis of one-dimensional SDS-PAGE showed that anti-Brugia pahangi tubulin mAb 1B6 and P3D react with epitope(s) specific to nematode beta-tubulin and recognize tubulin from adults and microfilariae of B. pahangi, adult B. malayi and Dirofilaria immitis, eggs of Haemonchus contortus and adult Ascaris suum. However, the same mAb did not recognize tubulin from trophozoites of Giardia lamblia, pig brain or 3T3 mouse fibroblast cells. In two-dimensional SDS-PAGE, mAb 1B6 recognized one isoform of beta-tubulin and mAb P3D recognized two beta-tubulin isoforms. Limited proteolysis showed that mAb 1B6 reacted with the amino-terminal fragments of beta-tubulin. In contrast, mAb P3D recognized the carboxy-terminal fragments of beta-tubulin. In ELISA, mAb P3D reacted with an 18 amino acid peptide corresponding to residues 430-448 of B. pahangi beta-tubulin. These observations confirm that the epitope of mAb P3D is located on the extreme carboxy-terminal region. Immunogold labelling of adult B. pahangi sections with mAb P3D revealed the presence of beta-tubulin isoforms in the cuticle, hypodermal layer and somatic muscle blocks of B. pahangi. Under in vitro conditions, mAb P3D caused 80% reduction in worm viability, during exposure over 48 h.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Brugia pahangi/imunologia , Tubulina (Proteína)/imunologia , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Feminino , Gerbillinae , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Three monoclonal antibodies specific to alpha- and beta-tubulin were used to examine the expression of tubulin isoforms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of alpha-, beta 1- and beta 2-tubulin. Commercial cross-reactive anti-alpha and anti-beta MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum beta-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum beta-tubulin MAb P3D6 and cross-reactive beta-tubulin MAb 357 recognizing 2-4 beta-tubulin isoforms and anti-alpha-tubulin MAb 356 recognizing 1-6 alpha-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.