We have expanded our biophysical/biochemical studies of parasitized erythrocytes to include: (1) detergent-resistant membrane studies and lipid analyses of human erythrocytes containing hemoglobin C, a mutant hemoglobin known to protect children against severe forms of falciparum malaria in West Africa; (2) atomic force microscopy of knob formation on P. gallinaceum-infected erythrocytes; (3) assessment of possible nitric oxide production in intra-erythrocytic stages of P. falciparum; and (4) characterization of Zeta potentials associated with the host erythrocyte surface in malaria infection. [unreadable] [unreadable] (1) A major transport molecule of human erythrocytes (Band 3) shows clustering in the membrane of human erythrocytes homozygous for hemoglobin C (CC erythrocytes). Erythrocytes that are normal and are homozygous for human hemoglobin (AA) show no such clustering. Our studies have also shown that CC erythrocytes have a clear difference in surface Zeta potential relative to AA erythrocytes. This difference may be a consequence of factors including: (a) serum protein binding to the erythrocyte surface; (b) protein clustering partially induced by hemichrome aggregation; and/or (c) lipid modification due to hemoglobin denaturation and induced oxidation effects. By examining detergent-resistant fractions of membranes, we have found that the distribution of molecules including flotillin-1 (a raft marker), band 3 and CD47 are shifted to denser fractions of CC erythrocytes separated by centrifugation. In contrast, a major structural protein of human erythrocytes (spectrin) was not detected in raft fractions from either AA or CC erythrocytes. [unreadable] [unreadable] We have also shown that the lipid compositions of CC and AA erythrocytes carry characteristically different levels of phosphatidylinositol, and phosphatidylserine, all of which are major components of the erythrocyte membranes inner leaflet. Slight changes in lipid composition modulate phase behavior and domain formation in cellular membranes, resulting in a variety of effects on membrane protein clustering and signal transduction. These effects may help account for band 3 clustering and modified PfEMP1 display in human erythrocytes carrying hemoglobin C.[unreadable] [unreadable] (2) In atomic force microscopy (AFM) studies of parasitized erythrocytes, we have observed characteristic membrane furrow-like structures on the surface of chicken erythrocytes infected with Plasmodium gallinaceum parasites. The correlation of the formation of furrows with parasite developmental stages is similar that of knobs with stages of P. falciparum in human erythrocytes. Transmission electron microscopy has revealed vesicle-like structures in the cytosol of P. gallinaceum-infected erythrocytes, suggesting a mechanism of parasite-derived protein transport that may be lead to furrow formation. [unreadable] [unreadable] (3) We have sought to answer if nitric oxide (NO) is produced and has a significant function in P. falciparum. In 2006 we initiated experiments to look for NO in intra-erythrocytic stages of the parasite. Fluorescence microscopy data with a reporting dye yielded signals in the food vacuole region of intra-erythrocytic parasites, suggestive of NO production. We have therefore been working to identify a biosynthetic pathway of the parasite for NO production. Our data suggest that NO production in P. falciparum may not depend on L-arginine, a classic substrate for NO production in eukaryotic cells. We are searching for an alternative pathway in P. falciparum for nitrogen incorporation and NO synthesis. Using the P. falciparum genomic database, we have found two possible genes that could be involved in NO synthesis by P. falciparum. We have confirmed their expression in our P. falciparum cultures by RT-PCR and have generated antibodies by DNA vaccine technology. Current focus is on two main efforts: a) identification of a protein responsible for NO synthesis and its sub-cellular localization in P. falciparum; and b) understanding the role of NO production in or around the parasitic food vacuole. This latter effort will involve electron paramagnetic resonance (EPR) to search for a possible association of nitric oxide to hematin sub-units of the hemozoin crystal. [unreadable] [unreadable] (4) Zeta potential is a measure of cell-surface electrochemical potential. Preliminary data indicate that this potential changes at the surface of the erythrocyte during parasite growth, and that these changes are associated with the deposition of knob-associated proteins and cytoadherence properties of the infected cell: knobless cell line of P. falciparum has higher z.p. than that of knob expressing cell lines. [unreadable] [unreadable] Collaborative Projects[unreadable] [unreadable] In this fiscal year, we have completed two collaborative projects. With Dr. Eri Hayakawa at Tokyo Womens Medical University we have studied a survival mechanism of bacillus Mycobacterium tuberculosis (MTB) that involves release of a lipid, Lipoarabinomannan (LAM), in host macrophages. MTB is able to reside inside macrophages by preventing phagosome-lysosome fusion, thereby escaping host attack and destruction. However, the mechanism of this fusion inhibition has not been understood. We focused the physicochemical effects of LAM on the phagosome membrane and studied structural modifications of lipid membrane domains. By applying AFM to artificial membranes mimicking phagosome membranes, we found characteristic modulations of membrane domains and phase behaviors. Membrane domains in LAM-containing lipid bilayers were significantly more scattered and not well isolated compared to normal membrane. Responses to temperature change were also reduced compared to control. Fluorescence Energy Transfer was significant reduced in LAM-containing vesicles. The results suggest that LAM induces a dramatic membrane reorganization in vitro, resulting in abnormal membrane fusion. Taken together, these findings help explain how MTB stay in the macrophage and escape destruction to establish persistent infection.[unreadable] In another collaborative study, we have worked with Drs. Francischetti and Calvo to study the functionality of Aegyptin, a mosquito salivary gland protein that binds to collagen to prevent blood coagulation. Using differential interference contrast microscopy and flow chamber preparations, we demonstrated and quantified a significant reduction in platelet binding to collagen. We also helped show that this new protein interacts with glycoprotein VI, integrin alpha2beta1, and von Willebrand factor.