A characteristic structural feature of Toxoplasma gondii and Neospora caninum is the presence of a triple-membrane pellicle, on the zoite stages of their complex life-cycle. Here we report the results of electron microscopic studies which show that the pellicle is made of a typical plasmalemma covered on its cytoplasmic side by a system of flattened vesicles named the inner membrane complex. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, we have evaluated the same methodology for the preparation of pellicles and plasmalemma from N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and the proteins and antigens present in them were subsequently studied by SDS-PAGE and Western blotting. Electron microscopy of the pellicle fractions of N. caninum demonstrated preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained 2 abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report, for the first time, a detailed molecular characterization of the pellicle and plasmalemma of N. caninum.

Cryptosporidium parvum (Apicomplexa, formerly Sporozoa) is the causative agent of cryptosporidiosis, an enteric disease of substantial medical and veterinary importance. C. parvum shows a number of unique features that differ from the rest of the class of coccidea in which it is currently grouped taxonomically. Differences occur in the overall structure of the transmission form and the invasive stages of the parasite, its intracellular location, the presence of recently described additional extracellular stages, the host range and target cell tropism, the ability to autoinfection, the nonresponsiveness to anticoccidial drugs, the immune response of the host, and immunochemical and genetic characteristics. These differences have an important impact on the infectivity, the epidemiology, the therapy, and the taxonomy of the parasite. The present article describes the structural analysis of the parasite using light and electron microscopy with an emphasis on structural details unique to C. parvum.

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the β-galactosyltransferase (β-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-α-N-acetyl-glucosaminyltransferase (O-α-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the β-Gal transferase, the O-α-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1Cβ1, the first intron from the constant region of the mouse T-cell receptor-β (TCR-β) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1Cβ1 and its pre-mRNA had half-lives of 6.0 ± 1.4 min and 3.7 ± 1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1Cβ1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1Cβ1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1Cβ1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1Cβ1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1Cβ1 also appeared to be selectively exported to the cytoplasm, whereas TCR-β pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.