RESUMO
Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)-amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH2 (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Corantes Fluorescentes/química , Lipopeptídeos/química , Lipopeptídeos/imunologia , Mycobacterium avium subsp. paratuberculosis/química , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Bovinos , Corantes Fluorescentes/síntese química , Mycobacterium avium subsp. paratuberculosis/imunologia , Conformação ProteicaRESUMO
BACKGROUND: The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. METHODS: By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. RESULTS: Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. CONCLUSION: The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.
Assuntos
Anticorpos Antibacterianos/sangue , Cervos/microbiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Etanol/química , Mycobacterium bovis/isolamento & purificação , Tuberculose/veterinária , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Reações Falso-Positivas , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Tuberculose/sangue , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63-346, Xrcc3(63-346), is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3-Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.
Assuntos
Aminoácidos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Aminoácidos/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Mutação , Fenilalanina/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Tirosina/genéticaRESUMO
Drastic changes in the plasma membrane of Plasmodium falciparum-infected red blood cells (iRBCs) make the surface of iRBCs distinct from that of the uninfected erythrocyte. To identify small peptides that would specifically recognize the altered surface of iRBCs, we screened a phage display peptide library (PDL) on the surface of iRBCs. After the sixth panning of the PDL, eight phage clones of 18 sequenced clones had the same sequence, LVDAAAL (named P1) and specific binding of P1 to the surface of iRBCs was confirmed using phage expressing P1 peptides and synthetic P1 peptide. When P1 peptide was conjugated with a peptide having moderate hemolytic activity, the peptide conjugate inhibited the growth of intracellular parasites in a dose-dependent manner, whereas control peptides were without effect. Our results demonstrate that the P1 peptide may be a lead compound for the development of anti-malarial agents targeting the surface of iRBCs.