In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper ... more In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma membrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of membrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association of the actin-binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with trans-Golgi-derived vesicles. Modifications of the ring were induced subjecting the cells to external Ca2+ depletion and restoration (Ca2+ switch), or by treatment with drugs known to depolymerize actin filaments (cytochalasin D, CD). The distr...
Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of... more Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of the actin cytoskeleton and an increase in proteolytic activities that results in the degradation of the bound protein. The binding is mediated by a 37-kDa FN "receptor" localized in the trophozoite surface and associated to the cytoskeleton. The intracellular signals triggered by the ligand-receptor interaction are not well understood but it is plausible that they drive the observed responses. To address this issue, the activation of protein kinase C (PKC) pathways by FN binding was explored. Stimulation with phorbol myristate acetate (PMA) or FN produced a rapid increase in the amebas adhesion to the substrate and local release of proteases. Two PKC inhibitors, H7 and staurosporine, reverted the PMA stimulus and inhibited the response induced by FN. Interaction with FN as well as treatment with PMA produced transient changes of F-actin levels susceptible to inhibition by H7. Furthermore, phosphorylation of amebic proteins was enhanced in response to FN binding and PMA, while the presence of the PKC inhibitor diminished their phosphorylation. Inositol triphosphate production was stimulated by the FN binding, and PKC activation and translation was registered in cell extracts obtained from the stimulated amebas. Our results suggest that PKC pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.
Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4... more Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4B chromatography of Entamoeba histolytica strain HM38:IMSS homogenates, and the protein was found to have lectin properties. The antibodies specifically recognized this protein in trophozoite homogenates. Immunologically related molecules with the same molecular weight were identified by polyclonal antibodies in strains HM1:IMSS, Entamoeba invadens, and Entamoeba histolytica Laredo. Six monoclonal antibodies recognized only the 220-kDa protein present in strains HM38 and HM1, a result indicating the presence of similar epitopes in the proteins from virulent strains isolated from humans. All the antibodies against the 220-kDa protein have the following properties: (1) they bind to the plasma membrane of live or fixed trophozoites, (2) they partially inhibit the adhesion of trophozoites to erythrocytes and cultured cells, and (3) they inhibit erythrophagocytosis.
Molecular and Biochemical Parasitology, Aug 1, 2016
Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still... more Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.
Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to ob... more Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.
In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the ... more In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconflu...
Comparison of 16S rRNA sequences alone positions E. histolytica in rRNA-based phylogenies branchi... more Comparison of 16S rRNA sequences alone positions E. histolytica in rRNA-based phylogenies branching after flagellates such as Euglenoids and Kinetoplastids, whereas morphological and functional features had suggested an earlier branching and different relationships. As several characteristics of this parasite do not precisely adjust to the phylogenetic frame obtained by comparison of ribosomal sequences, and the inferences from this approach could be skewed because of the high A+T content of Entamoeba rDNA, a re-evaluation of E. histolytica phylogeny seems convenient at this point. The data about genomic and protein sequences could provide bases to complement or expand the rRNA-based phylogeny.
The study of the organization of the genome in diverse organisms has revealed the presence of seq... more The study of the organization of the genome in diverse organisms has revealed the presence of sequences ofvariable length localized between the genes. These sequences have been named intergenic regions (IG). It has been proposed that IG regions contain ...
In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper ... more In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma membrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of membrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association of the actin-binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with trans-Golgi-derived vesicles. Modifications of the ring were induced subjecting the cells to external Ca2+ depletion and restoration (Ca2+ switch), or by treatment with drugs known to depolymerize actin filaments (cytochalasin D, CD). The distr...
Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of... more Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of the actin cytoskeleton and an increase in proteolytic activities that results in the degradation of the bound protein. The binding is mediated by a 37-kDa FN "receptor" localized in the trophozoite surface and associated to the cytoskeleton. The intracellular signals triggered by the ligand-receptor interaction are not well understood but it is plausible that they drive the observed responses. To address this issue, the activation of protein kinase C (PKC) pathways by FN binding was explored. Stimulation with phorbol myristate acetate (PMA) or FN produced a rapid increase in the amebas adhesion to the substrate and local release of proteases. Two PKC inhibitors, H7 and staurosporine, reverted the PMA stimulus and inhibited the response induced by FN. Interaction with FN as well as treatment with PMA produced transient changes of F-actin levels susceptible to inhibition by H7. Furthermore, phosphorylation of amebic proteins was enhanced in response to FN binding and PMA, while the presence of the PKC inhibitor diminished their phosphorylation. Inositol triphosphate production was stimulated by the FN binding, and PKC activation and translation was registered in cell extracts obtained from the stimulated amebas. Our results suggest that PKC pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.
Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4... more Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4B chromatography of Entamoeba histolytica strain HM38:IMSS homogenates, and the protein was found to have lectin properties. The antibodies specifically recognized this protein in trophozoite homogenates. Immunologically related molecules with the same molecular weight were identified by polyclonal antibodies in strains HM1:IMSS, Entamoeba invadens, and Entamoeba histolytica Laredo. Six monoclonal antibodies recognized only the 220-kDa protein present in strains HM38 and HM1, a result indicating the presence of similar epitopes in the proteins from virulent strains isolated from humans. All the antibodies against the 220-kDa protein have the following properties: (1) they bind to the plasma membrane of live or fixed trophozoites, (2) they partially inhibit the adhesion of trophozoites to erythrocytes and cultured cells, and (3) they inhibit erythrophagocytosis.
Molecular and Biochemical Parasitology, Aug 1, 2016
Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still... more Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.
Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to ob... more Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.
In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the ... more In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconflu...
Comparison of 16S rRNA sequences alone positions E. histolytica in rRNA-based phylogenies branchi... more Comparison of 16S rRNA sequences alone positions E. histolytica in rRNA-based phylogenies branching after flagellates such as Euglenoids and Kinetoplastids, whereas morphological and functional features had suggested an earlier branching and different relationships. As several characteristics of this parasite do not precisely adjust to the phylogenetic frame obtained by comparison of ribosomal sequences, and the inferences from this approach could be skewed because of the high A+T content of Entamoeba rDNA, a re-evaluation of E. histolytica phylogeny seems convenient at this point. The data about genomic and protein sequences could provide bases to complement or expand the rRNA-based phylogeny.
The study of the organization of the genome in diverse organisms has revealed the presence of seq... more The study of the organization of the genome in diverse organisms has revealed the presence of sequences ofvariable length localized between the genes. These sequences have been named intergenic regions (IG). It has been proposed that IG regions contain ...
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