ABSTRACT
An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.
Subject(s)
Actinomycetales/genetics , Catalase/genetics , Nocardia/enzymology , Nocardia/genetics , Rhodococcus/genetics , Actinomycetales/enzymology , Amino Acid Sequence , Base Sequence , Catalase/chemistry , Consensus Sequence , DNA Primers , DNA, Fungal/genetics , Genes, Fungal , Molecular Sequence Data , Polymerase Chain Reaction , Rhodococcus/enzymology , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Streptomyces/enzymology , Streptomyces/geneticsABSTRACT
It is known that lead is a metabolic substitute of calcium and recently, in vitro results showed that glycosaminoglycans and proteins are capable of binding lead. Therefore, in this work, the level of lead in aqueous humor, vitreous humor, lens and sclera of bovine eyes was analyzed in order to establish a relationship between lead concentration and the contents of glycosaminoglycans, proteins, water and calcium in those structures. Lead was determined by means of atomic absorption spectrometry after acid digestion. Our results suggest a direct relationship between the contents of proteins and glycosaminoglycans, and the ability of the different eye structures to retain lead. Nevertheless, an inverse relationship between water content and lead concentration was found. No association between calcium and lead concentrations was observed.
