Identification and localization of rab6, separation of rab6 from ERD2 and implications for an 'unstacked' Golgi, in Plasmodium falciparum.

The rab6 gene product in mammalian cells and yeast is localized to and regulates protein transport in the medial and trans Golgi cisternae, as well as the trans Golgi network. We have identified a homologue in the malaria parasite Plasmodium falciparum which displays a rab-like sequence that is 62.4% identical to mammalian rab6. In addition the parasite gene (Pfrab6 gene) contains an N-terminal hydrophobic domain, unique to P. falciparum. Antibodies developed to Pfrab6 localize protein in 4-7 well-resolved sites in a ring-stage parasite, as detected by high resolution fluorescence microscopy. This suggests that there are multiple, distinct foci of medial/trans Golgi membranes in a ring. ERD2 is a cis Golgi marker in mammalian cells. The plasmodial homologue of ERD2 (PfERD2) is concentrated in a single perinuclear region in a ring-stage parasite. This site is distinct from the Pfrab6 membranes, indicating that early and late Golgi markers can be segregated in P. falciparum. Mammalian cells contain a single Golgi complex where cis medial and trans markers are tightly stacked in closely apposed cisternae. In P. falciparum-rings however, rab6-associated membranes are not invariably 'stacked' with an ERD2 structure. In immunoelectron microscopy studies, both the PfERD2- and Pfrab6-associated membranes appear tubulovesicular in nature, devoid of cisternal morphology. Hence the Golgi of ring stage parasites may comprise of multiple, 'unstacked' tubulovesicular clusters, suggesting a primitive organization of the organelle in Plasmodia.

Macrophage receptors for lumican. A corneal keratan sulfate proteoglycan.

PURPOSE: Keratan sulfate proteoglycans (KSPGs) of the cornea exhibit a characteristic change in glycosylation resulting from stromal inflammation and scarring. To examine potential roles for these molecules in the pathobiology of the cornea, the authors investigated interaction of inflammatory macrophages with KSPGs in vitro. METHODS: Attachment and spreading of mouse peritoneal macrophages were examined on surfaces coated with corneal proteoglycans, intact or with modified glycosylation. Solution-phase interactions were demonstrated using soluble proteoglycans labeled with 125I-Iodine or with fluorescein. The affinity and specificity of these interactions were determined by competitive inhibition with unlabeled proteoglycans. RESULTS: Macrophages did not adhere to intact corneal KSPGs but did attach and spread rapidly on the lumican core protein after the removal of keratan sulfate chains. Arterial lumican, a nonsulfated form of this proteoglycan, also stimulated macrophage attachment. Labeled arterial lumican specifically bound to macrophages with high affinity. Flow cytometry demonstrated a high proportion of macrophages binding lumican. Lumican binding was inhibited by divalent cation-chelators and by polyanions. Inhibition and kinetics of lumican binding were distinct from interaction of macrophages with maleated bovine serum albumin, collagen, laminin, and fibronectin. CONCLUSIONS: The highly sulfated KSPGs of cornea do not promote macrophage adhesion; however, the low-sulfate lumican present in pathologic corneas may act to localize macrophages in regions of inflammation. The lumican receptor differs from macrophage scavenger receptors and from receptors for several other extracellular matrix molecules.

Association of host cell mitochondria with developing Toxoplasma gondii tissue cysts.

Ultrastructure of the interactions of host cell mitochondria with developing Toxoplasma gondii tissue cysts was examined in cultured cells, using transmission electron microscopy of infected cells and rhodamine 123 (a mitochondria-specific vital fluorescent dye) staining of isolated tissue cysts. Structurally mature T gondii tissue cysts were observed as early as 2 days after inoculation of cultured cells. During development of T gondii, host cell mitochondria were observed surrounding the parasitophorous vacuole membrane. Mitochondria became flat and elongated in the vicinity of the parasitophorous vacuole membrane. These mitochondria were also closely associated with T gondii tissue cysts. Incubation of tissue cysts from cultured cells and tissue cysts from mouse brains with rhodamine 123 revealed fluorescence of the tissue cyst wall in living specimens. Incubation of tissue cysts with 10 microM rotenone caused diminished fluorescence of the tissue cyst walls, and 100 microM rotenone caused complete inhibition. Mouse RBC, and tissue cysts fixed in 100% methanol did not fluoresce after exposure to rhodamine. Tissue cysts in 9 isolates of T gondii from mouse brains were examined, using rhodamine 123, and the tissue cysts walls of all isolates fluoresced, indicating no isolate effects. Our results indicate that host cell mitochondria may be closely associated with the tissue cysts of T gondii in cell cultures and in mice.

A comparative study of lipid compositions of Cryptosporidium parvum (Apicomplexa) and Madin-Darby bovine kidney cells.

Membrane lipid compositions of Cryptosporidium parvum and Madin-Darby bovine kidney cells, an epithelial-like cell line commonly used to study coccidia in vitro, were analyzed using both thin-layer chromatography and gas-liquid chromatography. Phosphatidylcholine was the predominant lipid in both C. parvum and Madin-Darby bovine kidney cells, comprising 65% and 41% of the total phospholipids, respectively. Phospholipids of C. parvum contained twice the level of 16:0 and twenty-fold more 18:2 than the Madin-Darby bovine kidney cell line. We suggest that the parasite may be capable of sequestering specific complex membrane lipids at concentrations greater than those in the host cells. This study constitutes the first report of the lipid composition of C. parvum.

Incorporation of exogenous uracil by Cryptosporidium parvum in vitro.

Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method.