Highly variable upper and abyssal overturning cells in the South Atlantic.

The Meridional Overturning Circulation (MOC) is a primary mechanism driving oceanic heat redistribution on Earth, thereby affecting Earth's climate and weather. However, the full-depth structure and variability of the MOC are still poorly understood, particularly in the South Atlantic. This study presents unique multiyear records of the oceanic volume transport of both the upper (<~3100 meters) and abyssal (>~3100 meters) overturning cells based on daily moored measurements in the South Atlantic at 34.5°S. The vertical structure of the time-mean flows is consistent with the limited historical observations. Both the upper and abyssal cells exhibit a high degree of variability relative to the temporal means at time scales, ranging from a few days to a few weeks. Observed variations in the abyssal flow appear to be largely independent of the flow in the overlying upper cell. No meaningful trends are detected in either cell.

Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro.

We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei which contains C14 acyl and alkyl chains shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.

Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms.

African trypanosomes express a heterodimeric transferrin receptor that mediates iron uptake from the host bloodstream. The genes encoding the receptor, ESAG6 and ESAG7, are found at the beginning of VSG expression sites: these are telomeric, polycistronic transcription units that each terminate with a gene encoding a trypanosome variant surface glycoprotein, VSG. Approximately 20 of these VSG expression sites are found in the trypanosome genome, but only one VSG is expressed at a time. The conventional view is that one expression site promoter is extremely active whereas the others are either inactive or show very low, poorly processive activity, and that all transferrin receptor molecules are encoded by the active expression site. The 3'-end of the ESAG6 gene is more than 5 kb from the promoter. We show here that 20% of ESAG6 mRNA originates from the 'inactive' expression sites. We suggest that many expression site promoters in trypanosomes show low-level activity throughout the life cycle, and that transcription proceeds for at least 5 kb. This suggests a simplified model of VSG expression site control, whereby the only regulated event is the strong activation of a single expression site promoter in bloodstream forms.

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O.

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii) an assessment of the molecular requirements for the process at the erythrocytic side of the vacuolar membrane, we permeabilized infected RBCs with the pore-forming protein streptolysin O using conditions which left the vacuole intact. The distribution of two parasite proteins which served as markers for the vacuolar space and the RBC cytosol respectively was analysed morphologically and biochemically. In permeabilized RBCs the two marker proteins were sorted to the same compartments as in intact RBCs. The protein which was destined for the RBC cytosol traversed the vacuolar space before it was translocated across the vacuolar membrane. Protein transport could be arrested in the vacuole by removing the RBC cytosol. Translocation across the vacuolar membrane required ATP and a protein source at the erythrocytic face of the membrane, but it was independent of the intracellular ionic milieu of the RBC.

Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin.

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.

Chemical and thermal inhibition of protein secretion have stage specific effects on the intraerythrocytic development of Plasmodium falciparum in vitro.

The intraerythrocytic stages of the human malaria parasite Plasmodium falciparum induce a variety of physiological changes of the host erythrocyte. Many proteins are secreted from the parasite and are subsequently found at specific locations within the host cell. To elucidate the importance of protein secretion for parasite survival, infected red blood cells (IRBC) were subjected to the fungal metabolite brefeldin A (BFA) and to incubation at 15 degrees C, treatments that inhibit protein secretion and parasite development. Evidence is provided that retardation of parasite development in the presence of BFA correlates with an inhibition of protein secretion. Incubation at 15 degrees C and BFA reversibly arrest parasite development at the ring stage. Arrested ring stages loose 50% of their competence to develop to trophozoites after 1.5 days of treatment with BFA and after approximately 4 days at 15 degrees C. BFA affects development of trophozoites at concentrations similar to those required to arrest rings. In contrast to rings, the viability of trophozoites cultured at 15 degrees C or in the presence of BFA is completely abolished within 24 h.

Characterization of membrane proteins exported from Plasmodium falciparum into the host erythrocyte.

Plasmodium falciparum is an intracellular parasite of the red blood cell. During development it exports proteins which are transported to specific locations within the host erythrocyte. We have begun to identify and characterize exported membrane proteins of P. falciparum in order to obtain specific marker molecules for the study of the mechanisms involved in the distribution of parasite-derived proteins within the host cell. In this report we describe the characterization of a 35 kDa protein which is recognized by a monoclonal antibody. The protein is tightly associated with membranes isolated from infected erythrocytes; it is resistant to extraction with alkali and soluble after treatment with detergents. It is located at the membrane of the parasitophorous vacuole and in membrane-bound compartments which appear in the cytoplasm of the infected erythrocyte. The protein co-localizes with the previously described exported protein-1 (exp-1). Considering its localization and physical similarities to exp-1, we name the 35 kDa protein the exported protein-2 (exp-2).