Trypanosoma cruzi: cell surface dynamics in trypomastigotes of different strains

Capping and shedding of ectodomains in Trypanosoma cruzi may be triggered by different ligands. Here, we analysed the mobility and shedding of cell surface components of living trypomastigotes of the Y strain and the CL Brener clone in the presence of poly-L-lysine, cationized ferritin (CF) and Concanavalin A (Con A). Poly-L-lysine and CF caused intense shedding in Y strain parasites. Shedding was less intense in CL Brener trypomastigotes, and approximately 10% of these parasites did not show any decrease in poly L-lysine or CF labelling. Binding of Con A induced low-intensity shedding in Y strain and redistribution of Con A-binding sites in CL Brener parasites. Trypomastigotes of the Y strain showed intense labelling with anti-〈-galactosyl antibodies, resulting in the lysis of approximately 30% of their population, in contrast with what was observed in CL Brener parasites. Incubation with Con A and CF protected trypomastigotes of the Y strain from lysis by anti-αGal. The last treatment did not interfere with the survival of the CL Brener parasites. This study corroborates with the idea that a ligand can differentially modulate the cell surface of T. cruzi, depending on the strain used, resulting in variable immune system responses and recognition by host cells.

Expression of endothelial and inducible nitric oxide synthases is modulated in the endometrium of cyclic and early pregnant mares

The expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS, respectively) was examined in the endometrium of cyclic and pregnant mares by real-time polymerase chain reaction and immunohistology. The concentration of eNOS mRNA varied throughout the oestrous cycle, with significantly higher transcripts on Day 5 of the oestrous cycle (P < 0.05), whereas iNOS transcription did not change significantly over time (P > 0.05). In early pregnant mares both eNOS and iNOS mRNA increased between Days 12 and 15 (P < 0.05). In cyclic mares, eNOS protein was detected immunocytochemically in endometrial epithelia, the basement membrane, the endothelial layer and smooth muscle cells of the vasculature. Using immunocytochemical methods, iNOS protein was undetectable in the endometrium of cyclic mares but could be demonstrated in pregnant mares. Endometrial epithelia of pregnant mares were immunopositive for both proteins with a more intense labelling for iNOS. Thus, the present study describes for the first time the modulation and spatial distribution of eNOS and iNOS expression during the oestrous cycle and early pregnancy, suggesting that ovarian steroids are differently involved in the regulation of each NOS. Localisation of eNOS protein in endometrial epithelia and various vascular components indicates that this isoform may be involved in the regulation of endometrial cyclicity. The presence and increase of both forms of NOS during early gestation suggest a role for them in the control of endometrial vascular bed and glandular activity to provide a suitable microenvironment for successful pregnancy.

Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.

Sperm passage through the zona pellucida

Sperm passage through the zona pellucida requires the occurrence of the acrosome reaction. This latter reaction, in turn, is supposedly needed to release the acrosomal enzymes, particularly acrosin which has been involved in the sperm binding to and penetration through the zona pellucida. The application of the immunogold technique using the monoclonal antiacrosin antibody ACR-C2E5 allowed us to identify acrosin in hamster, human and guinea-pig spermatozoa at the optic and scanning electron microscope, in which the immunogold labelling was silver enhanced. At the transmition electron microscope the monoclonal antibody was indirectly labelled with 10nm gold particles. In hamster spermatozoa there were three patterns of labelling: a) either no labelling, or an intense one over acrosomal region of apparently acrosome intact spermatozoa (Figures la,b, 2a & 3a); b) an intense labelling over the inner acrosomal surface and on the acrosomal cap of acrosome reacted spermatozoa (Figures lc,d, 2b & 3b); and c) either a slight labelling over the equatorial region or no labelling on acrosome reacted spermatozoa (Figure le,f,2c).

Prekeratin biosynthesis in human scalp epidermis

Analysis of human scalp epidermal prekeratin polypeptides by two-dimensional gel electrophoresis revealed that each of the bands observed in one-dimensional electrophoresis consisted of three to five polypeptides of the same molecular weight but differing in isoelectric points. It was possible to divide the polypeptides into two families, with isoelectric points in the ranges pH 6.0-8.0 and pH 5.0-5.5 respectively. Incorporation of radiolabelled amino acids into freshly excised pieces of scalp epidermis showed that some of the polypeptides had relatively greater contents of glycine and serine than others. Radiolabelled methionine and leucine were, in contrast, incorporated more or less uniformly into all the polypeptides. After incubation with 32P-labelled orthophosphate, relatively more intense labelling by 32P was observed in the higher molecular weight bands of each family. The most basic of the isoelectric variants in each case did not take up phosphate, implying that at least some of the variation in charge was due to different degrees of phosphorylation. Polyadenylated RNA isolated from scalp epidermis was translated in an RNA-dependent reticulocyte haemolysate system followed by immunoprecipitation and electrophoresis. The polypeptides isolated by using anti-(human scalp prekeratin) immunoglobulin G had similar electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels to authentic prekeratin polypeptides, but had different isoelectric properties. This suggested that the products of keratin gene expression undergo post-translational modification.

Autoradiographic studies on the uptake of [14C]-Glucose by Bithynia tentaculata (Mollusca: Gastropoda) and its larval digeneans

Autoradiographs revealed that sporocysts of Cercaria helvetica XII and rediae of Sphaeridiotrema globulus, incubated in a medium containing [14C]-glucose, exhibited slight radioactivity in their body wall after only 30 seconds incubation. Activity in sporocysts and rcdiae gradually increased as the incubation period was extended until after 4 minutes, a heavy reaction was observed in the parasite body wall as well as in the germ balls and developing cercariae within the parasite brood chambers. The redial caecum appeared to incorporate the labelled compound much more intensely than did the redial body wall.When uninfected Bithynia tentaculata, as well as B. tentaculata infected with either S. globulus or C. helvetica XII, were maintained in pond water containing [14C]-glucose, activity could be detected in the gills of both infected and uninfected snails after 5 hours incubation. After 12 hours a much heavier labelling was apparent in the gills, together with moderate activity in the haemocoelic spaces, foot musculature, stomach wall, and gut contents. After 20 hours a more intense labelling was apparent in these regions of the snail body and, in addition, some activity could be detected in the digestive gjand tubules of the host and, in infected snails, in the bodies of developing parasites. After 30 hours incubation the labelled compound had become incorporated into most of the host tissues but was most intense in the digestive gland tubules. Heavy activity was also evident in the parasites at this stage although certain cystogenous gland cells of the cercariae of both parasite species did not incorporate the labelled compound.