ABSTRACT
The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3-25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.
Subject(s)
Cysts/parasitology , Cysts/ultrastructure , Giardia/physiology , Giardia/ultrastructure , Animals , Blotting, Western , Cysts/pathology , Microfibrils/pathology , Microfibrils/ultrastructure , Microscopy, Electron , Microscopy, Fluorescence , Peptides/metabolism , Polysaccharides/metabolismSubject(s)
Chromatin/physiology , Entamoeba histolytica/cytology , Acridine Orange , Animals , Cattle , Cell Division , Cell Nucleus/chemistry , Chromatin/chemistry , DNA, Protozoan/analysis , Deoxyribonuclease I , Entamoeba histolytica/chemistry , Entamoeba histolytica/ultrastructure , Fluorescent Dyes , Microscopy, Fluorescence , RNA, Protozoan/analysis , RibonucleasesABSTRACT
The presence of IgA anti-Entamoeba histolytica antibodies has been demonstrates in intestinal secretions and serum of human amebiasis. We investigated which are the cellular components of trophozoites that react with IgA anti-ameba antibodies from immune serum, colostrum and human milk. The cellular localization of such antigens was accomplished by an indirect immunoperoxidase technique using anti-human IgA (alpha chain specific) labeled with peroxidase, both for light and transmission electron microscopy. Intracellular antigens were localized after permeating the parasites with cold acetone (-10 degrees C) for 3 min. and cryosections of 1 micron thick. The antigens that react with IgA antibodies from immune serum, colostrum and human milk were located in the plasma membrane and the internal portion of some cytoplasmic vesicles. So far, it is unknown what is the biological function of IgA in human amebiasis but in other systems it protects against certain parasites.
