ABSTRACT
El desarrollo de mecanismos para evadir la acción de los fármacos y desarrollar resistencia a los mismos es un evento universal en los organismos vivos. Se presenta en virus, bacterias, hongos, plantas y animales. En una población de seres vivos algunos individuos son capaces de encender genes apagados, de mutar la secuencia de otros genes, de sintetizar nuevas moléculas para desarrollar los mecanismos que les permiten sobrevivir y perpetuar la especie en circunstancias adversas. La acumulación de eventos de adaptación y mutagénesis pueden dar lugar a la aparición de organismos con características diferentes a la mayoría de los individuos de su especie, las cuales al acumularse podrían constituir nuevas especies. De manera que los cambios para la adaptación y la sobrevivencia de los individuos constituyen en última instancia las bases de la evolución. Uno de los mecanismos que las células han desarrollado para sobrevivir en presencia de sustancias tóxicas es el llamado fenotipo de resistencia a múltiples drogas o MDR (por sus siglas en inglés, multidrug resistance). Este evento se caracteriza porque las células presentan resistencia a drogas con estructuras químicas y blancos de acción distintos. El fenotipo de MDR se descubrió primeramente en células transformadas de mamífero, las cuales expresan una glicoproteína de aproximadamente 170 kDa en su superficie, denominada Pgp. Posteriormente este mecanismo se ha descubierto en muchas especies, incluyendo a los protozoarios parásitos...
Subject(s)
Animals , Entamoeba histolytica , Antiprotozoal Agents , Drug Resistance, Multiple , Phenotype , Promoter Regions, Genetic , Cloning, Molecular , Genes, Protozoan , Genes, MDR , Emetine , Entamoeba histolytica , Gene Amplification , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Protein Isoforms/genetics , Protein Isoforms/physiology , Protozoan Proteins/genetics , Protozoan Proteins/physiology , Transcription, GeneticABSTRACT
We report a study on the DNA organization in Entamoeba histolytica using a ribosomal DNA probe. The rDNA genes were found forming mers which were separated in a typical ladder pattern by pulse field electrophoresis. DNA rosette structures were visualized through electron microscopy in DNA eluted from bands recognized by the ribosomal probe. The in situ hybridization experiments using a DNA probe suggested that the rDNA genes are portioned between the nucleus and a cytoplasmic structure. These findings provide new data on DNA organization in E. histolytica and open the question concerning the presence of a novel organelle in this eukaryotic parasite
Subject(s)
Animals , DNA, Ribosomal/genetics , DNA, Ribosomal/ultrastructure , Cytoplasm/chemistry , DNA, Protozoan/genetics , DNA, Protozoan/ultrastructure , Entamoeba histolytica/genetics , Entamoeba histolytica/ultrastructure , In Situ HybridizationABSTRACT
In this paper, we present the most relevant facts on multidrug resistance (MDR) in the protozoan parasite Entamoeba histolytica. MDR in E. histolytica presents characteristics similar to transformed mammalian cells. E. histolytica drug resistant mutants show cross-resistance to several drugs, and as in mammalian cells the resistance is reverted by verapamil. Six P-glycoprotein-like genes (EhPgp) have been cloned and characterized. Apparently, four of thses genes are transcribed in drug-resistance mutants (EhPgh1, EhPgp2, EhPgp5 and EhPgp6), although only Egpgp1, EhPgp5 and EhPgp6 transcripts were clearly detected. The open reading frame (ORF) of the four completely full length genes is about 1300 amino acids long. EhPgp1, EhPgp2 and EhPgp5 have between 64 and 67 percent of positional identity among them, while EhPgp6 shows 38 to 46 percent positional identity to the other ameba genes. Insterestingly, the phylogenetic tree suggested that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins. Differential gene expression in drug-resistant mutants was detected when specific probes for each EhPgp gene were used. To understand the differential expression of EhPgp genes we initiated the characterization of the upstream flanking regions of EhPgp1 and EhPgp5 genes. Upstream sequences showed between 53 and 66 percent of positional identity to Dictyostelium discoideum promoters
Subject(s)
Blotting, Northern , Blotting, Southern , Cloning, Molecular , Drug Resistance, Microbial/physiology , Entamoeba histolytica/drug effects , Molecular BiologyABSTRACT
To identify the molecules involved in the adhesion of Entamoeba histolytica trophozoites to target cell we used monoclonal antibodies (MAbs) and adhesion-deficient mutants. Human red blood cell (RBcs) were also used as especific carriers to identify the ameba molecules with affinity to the target cell receptors. MAbs adh-1 and Adh-2 inhibited adhesion of RBCs to the trophozoites and recognized a 112-kDa surface protein that was present in the wild type strain, but was absent or modified in adhesion-deficient mutants. In other experiments, live trophozoites were incubated with fixed-RBCs and after lysis of the trophozoites, proteins attached to the RBCs surface were separated by PAGE, electrotransferred to nitrocellulose membranes and detected by polyclonal antibodies. The 112 kDa protein was found attached to the RBCs. Other molecules identified as proteins involved in the target cell-parasite contact were the 210, 160, 90, 70, 50 and 24 kDa proteins. The 112, 90 and 24kDa polypeptides were functionally altered in adhesion-deficient mutants