ABSTRACT
Entamoeba histolytica trophozoites recovered from the host-parasite interface during abscess development obtain different stimuli compared with long-term cultured cells. In order to have a better understanding about the mechanisms in which the 140 kDa fibronectin (FN)-binding molecule (EhFNR) is involved during the invasive process, we decided to compare the regulation process of this molecule among long-term cultured trophozoites, FN-stimulated trophozoites, and trophozoites recently recovered from a liver abscess. A cDNA clone (5A) containing a fragment of the EhFNR that shows identity to the C-terminal region of the intermediate galactose lectin subunit Igl, was selected with a mAb (3C10). Identity of EhFNR with Igl was confirmed by immunoprecipitation with 3C10 and EH3015 (against the Gal/GalNAc intermediate subunit) mAbs. The 3C10 mAb was used as a tool to explore the modulation of the amoebic receptor (EhFNR). Our results showed specific regulation of the EhFNR in FN-interacted amoebas, as well as in trophozoites recovered at different stages of abscess development. This regulation involved mobilization of the receptor molecule from internal vesicles to the plasma membrane. Therefore, we suggest that in the host-parasite interface, the EhFNR (Igl) plays an important role in the adhesion process during abscess development.
Subject(s)
Entamoeba histolytica/metabolism , Fibronectins/metabolism , Host-Parasite Interactions , Liver Abscess, Amebic/parasitology , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cricetinae , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Entamoeba histolytica/chemistry , Fibronectins/chemistry , Immunohistochemistry , Male , Molecular Weight , Protozoan Proteins/chemistry , Sequence AlignmentABSTRACT
Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.
Subject(s)
Bacteriophage lambda/growth & development , Bacteriophage lambda/genetics , Escherichia coli/growth & development , Genes, Viral , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/virology , Genes, Viral/genetics , Genes, Viral/physiology , Mutation , Plasmids , Protein BiosynthesisABSTRACT
1. Calmodulin (CaM) was detected during secretion of electron-dense granules by Entamoeba histolytica trophozoites with immunofluorescence. 2. It was purified to apparent homogeneity by chromatography with a yield of 2.26 micrograms of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis. 3. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes: an apparent molecular weight of 19 or 17 kDa in presence of EGTA or CaCl2, respectively, activation in a calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase, and its UV spectrum.
Subject(s)
Calmodulin/metabolism , Entamoeba histolytica/metabolism , Animals , Calmodulin/isolation & purification , Cytoplasmic Granules/metabolism , Entamoeba histolytica/ultrastructure , Microscopy, Fluorescence , Molecular Weight , Subcellular Fractions/metabolismABSTRACT
Several isolates of pathogenic and non-pathogenic E. histolytica from xenic cultures were tested for their capacity to digest native type I collagen gels. The results demonstrate that the pathogenic isolate HM48:IMSS has a high collagenolytic activity. The non-pathogenic isolates, HM43:IMSS, HM44:IMSS and HM46:IMSS showed significantly lower collagenolytic activity. Six groups showing different degrees of collagenase activity or non-activity were separated from the non-pathogenic isolates by a Percoll gradient. The groups obtained from the pathogenic isolate HM48:IMSS always displayed enzymatic activity.
