This year saw the continuation of some on-going projects, conclusion and publication of the results of some projects and the initiation of new projects. We have expanded our studies of changes in the distribution of erythrocyte surface components during the intraerythrocytic cycle of Plasmodium falciparum. We compared the total level of fluorescently-labeled band 3 molecules using a new erythrocyte preparation technique that minimizes artifacts resulting from damaged erythorcyte membranes, an anti-band 3 monoclonal antibody, and a quantum dot imaging protocol involving the construction of 3-dimensional maximum projections of z-stack sections consisting of 70 or more focal planes through infected and uninfected erythrocytes. The integral of total fluorescence was used as a measure of the number of band 3 molecules. Our new quantum-dot-based procedure demonstrated a non-homogeneous, raft-like distribution of band 3 protein in the erythrocyte membrane. We demonstrated that total fluorescence intensity decreased as the intraerythrocytic development of a P. falciparum infection proceeds. The fluorescence intensity of schizont stage erythrocytes is only ~50 % of that of uninfected erythrocytes. These data indicate that either the accessibility of antibody to band 3 decreases as a consequence of membrane modifications by malaria parasites, or that the amount of band 3 protein on the erythrocyte membrane decreased in later stages of malaria infection. Although there have been many studies of malaria sporozoites, how sporozoites know where the salivary glands are and the mechanism of invasion are not known. When a Matrigel environment is used, sporozoite motility can be plotted 3-dimensionally using a custom-built chamber and low-light-level video microscopy. We found that: 1. Sporozoite motility decays with time 2. Initiation of sporozoite motility is inhibited by lowering the temperature 3. Matrigel promotes the net forward motion of sporozoites 4. Medium 199 plus BSA with 3X glucose and Matrigel improves sporozoite longevity (optimization of nutrients?). 5. Sporozoites are attracted to salivary glands Upon invasion of human erythrocytes by Plasmodium falciparum, the erythrocyte is morphologically and functionally modified. Several proteins are synthesized at the early intra-erythrocytic stage of infection, and these components are transported to the erythrocyte membrane. Consequently, the surface structure of the infected erythrocytes is modified and protrusions or knobs begin to appear on the surface. These knobs play an important role in the disease process. Individuals with the Hemoglobin C (HbC) genotype have a survival advantage against severe malaria. Although the HbC genotype does not protect against a P. falciparum infection, the sickle cell trait does provide a survival advantage against the severe malaria over people with normal hemoglobin. In normal (AA) genotype erythrocytes, parasite-induced knobs are not present during the ring stage. However, as the intracellular cycle progresses, knobs appear uniformly distributed over the surface of the erythrocyte and the total number of knobs increases as the intracellular cycle progresses as we reported previously. In contrast, in the same malaria strain, the knobs of HbCC cells are morphologically more heterogeneous, larger in width, fewer in number and non-uniformly distributed. Furthermore, the knobs do not appear to vary structurally between early and late intracellular stages. Preliminary AFM data of 'hybrid' AC erythrocytes is indistinguishable from AA cells. We have completed NSOM-based studies of lipid membrane bilayers, containing two phosphocholines (dilauroyl-sn-glycero-3-phosphocholine, DLPC and dipalmitoyl-sn-glycero-3-phosphocholine) and cholesterol. The distributions of the two phospholipids are not uniform throughout the membrane, and there is a marked phase separation between them. Autocorrelation analyses on images both with and without cholesterol demonstrated that there is a mix mode phase separation consisting of short range (nanoscopic domains) and long range(microscopic domains). Three component mixtures have a nanoscopic domain size of ~80-100 nm as well as large microscopic domains which are greater than 2mm. Using NSOM we could directly visualize both the nanoscopic and microscopic domains. The rapid and reliable estimation of the status of an erythrocyte culture infected with malaria parasites, specifically Plasmodium falciparum, is critical to the successful utilization of the culture for both basic and applied research on this medically important parasite. Although the Giemsa reaction is the classical method to diagnose a malaria infection, it is less useful for estimating the relative health of malaria-infected erythrocyte cultures. We have developed a rapid computer-assisted, microscope-based assay method utilizing fresh wet mount preparations of malaria-infected erythrocyte cultures reacted with both Hoechst 33258 and ethidium homodimer which overcomes the subjectivity and variability problems associated with the Giemsa-based assay method. Only a 5 ul portion of a malaria-infected erythrocyte culture is required to estimate the proportion of live and dead malaria parasites present in the culture. Comparative studies of the live dead malaria culture assay method versus the conventional Giemsa assay method highlight several advantages. For example, the live dead assay is capable of assaying the status of merozoites which is not practical using the Giemsa method. In addition, the identification of the percentages of live vs dead parasites in unambiguous and rapid.
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