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Disruption of lipid rafts by lidocaine inhibits erythrocyte invasion by Plasmodium falciparum
Experimental Parasitology 123 (2009) 381–383
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
Disruption of lipid rafts by lidocaine inhibits erythrocyte invasion by Plasmodium falciparum Ichiro Koshino, Yuichi Takakuwa * Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
a r t i c l e
i n f o
Article history: Received 1 April 2009 Received in revised form 19 August 2009 Accepted 21 August 2009 Available online 4 September 2009 Keywords: Plasmodium falciparum Apicomplexan Lipid rafts Lidocaine Parasite invasion Erythrocytes
a b s t r a c t Membrane lipid rafts have been implicated in erythrocyte invasion process by Plasmodium falciparum. In this study, we examined the effect of lidocaine, a local anesthetic, which disrupts lipid rafts reversibly without affecting membrane cholesterol content on parasite invasion. In the presence of increasing concentrations of lidocaine in the culture medium, parasite invasion was progressively decreased with complete inhibition at 2 mM. Decreased invasion was also seen in erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine. This inhibitory effect on parasite invasion was reversed following removal of lidocaine from erythrocyte membranes. Our findings show that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits parasite invasion and confirm an important role for lipid rafts in invasion of erythrocytes by P. falciparum. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Plasmodium falciparum is the causative agent of the most severe form of human malaria, resulting in one million deaths annually. P. falciparum invades human erythrocytes and intra-erythrocytic forms are responsible for all of the clinical symptoms and pathology associated with malaria (Miller et al., 2002). Membrane microdomains enriched with cholesterol and sphingolipids are referred to as ‘‘lipid rafts” (Simons and Ikonen, 1997). Lipid rafts are also rich in several proteins, such as Gsa, which are involved in signaling processes (Murphy et al., 2004). Lipid rafts and cell signaling have been implicated in the invasion of erythrocytes by malarial parasites, since membrane cholesterol depletion resulting in disruption of lipid rafts inhibited erythrocyte invasion by P. falciparum (Samuel et al., 2001; Harrison et al., 2003; Murphy et al., 2006b). However, since a significant amount of cholesterol exists in non-raft membrane regions (Samuel et al., 2001), it can be argued that the observed effects of cholesterol depletion may not be solely due to disruption of lipid rafts. In this study, to determine whether lipid rafts are essential for parasite invasion, we examined the effect on parasite invasion of lidocaine, a local anesthetic which we have recently shown to disrupt lipid rafts without altering membrane cholesterol content (Kamata et al., 2008). Our findings showed that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits * Corresponding author. Fax: +81 3 5269 7416. E-mail address: [email protected] (Y. Takakuwa). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.08.019
parasite invasion, confirming an important role for lipid rafts in invasion of erythrocytes by P. falciparum. 2. Materials and methods 2.1. Parasite culture Plasmodium falciparum (strain F32) was cultured in human erythrocytes by standard methods in RPMI1640 supplemented with 25 mM HEPES, 0.37 mM hypoxanthine, 5% human AB serum, and 0.25% (w/v) Albumax I (Sigma) at a 2% hematocrit under a lowoxygen atmosphere (7.5% O2, 5% CO2, 87.5% N2). 2.2. Parasite invasion assay P. falciparum cultures were synchronized by 5% sorbitol treatment (Fernandez, 2008) and mature stage parasites (trophozoites and schizonts) were purified using VarioMACS CS column (Vogt, 2008). Purified mature stage parasites were used to inoculate cultures of fresh red cells. Cultures were carried out either in the absence or presence of lidocaine (0–2 mM) in the culture medium. In another series of experiments erythrocytes pre-treated with lidocaine were cultured in the absence of lidocaine in the culture medium. For these experiments, 5% human AB serum and 0.25% (w/v) Albumax I in the culture medium was replaced with 10% human AB serum. Parasitemia was determined at 15–18 h after the initiation of cultures by enumerating the newly formed ring-form parasites on thin blood films stained with Giemsa. All experiments
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I. Koshino, Y. Takakuwa / Experimental Parasitology 123 (2009) 381–383
were performed in triplicate and representative data from 2 to 4 independent experiments are shown. 2.3. Pre-treatment of erythrocytes by lidocaine Venous blood was collected from healthy volunteers after obtaining informed consent. White blood cells were removed using poly-urethane porous filters (Immuguard III, Terumo). Erythrocytes were washed in Tris-buffered saline (TBS) and treated with 18.5 mM lidocaine in TBS containing 0.1% (w/v) glucose (TBS-G) at a hematocrit of 16.7% for 20 min at 37 °C. Treated cells were washed once with TBS and used in the invasion assay. In other experiments, lidocaine-treated cells were incubated in 3% (w/v) bovine serum albumin (BSA) in TBS-G at 10% hematocrit for 20 min at 37 °C, followed by four washes in TBS to remove lidocaine incorporated into the erythrocyte membrane. 3. Results and discussion Purified mature stages of P. falciparum parasites were used to quantitate their ability to invade fresh human erythrocytes in the presence of various concentrations of lidocaine in the culture medium. Ring-stage infected erythrocytes, with parasitemias up to 15% were readily observed in control cultures without lidocaine (Fig. 1). In marked contrast, no ring-stage infected erythrocytes were detected in the presence of 2 mM lidocaine, indicating that lidocaine at this concentration completely inhibits parasite invasion (Fig. 1). The inhibitory effect of lidocaine on parasite invasion was dose dependent (Fig. 1). Similar results were obtained in another independent experiment. Erythrocyte morphology was unaltered at the concentrations of lidocaine used in the study. Furthermore, the surface area/volume ratio of lidocaine-treated erythrocytes was comparable to that of untreated cells as quantitated by ektacytometry (Bessis et al., 1980) (data not shown). Thus, the observed inhibitory effect of lidocaine on parasite invasion is not due to either erythrocyte shape changes or altered cellular deformability (Ziegler et al., 2002; Boampong et al., 2007). The documented inhibitory effect of lidocaine on parasite invasion was reversible. The ability of the parasites to invade erythrocytes pre-treated with lidocanie and cultured in the absence of lidocaine as expected was inhibited compared to the untreated control cells (Fig. 2). Importantly, upon removal of 75% of the lidocaine incorporated into the membrane of treated erythrocytes by washing with BSA (Kamata et al., 2008), the parasite invasion
Fig. 1. Effect of lidocaine on the parasite invasion. Dose-dependent decrease in parasitemia in the presence of increasing concentration of lidocaine in the culture medium. Parasitemia is expressed as mean ± SD derived from measurements performed in triplicate.
Fig. 2. Reversibility of lidocaine effect. Untreated (Cont), lidocaine-treated (Lid), and BSA-washed (Lid/BSA) erythrocytes were used for invasion assay. After 15–18 h, parasitemia was determined on thin blood films stained with Giemsa. Parasitemia is expressed as mean ± SD derived from measurements performed in triplicate. Note increased invasion following removal of lidocaine from membranes of lidocaine-treated cells by albumin washing.
was restored to near normal levels. It should be noted that the observed inhibition of invasion for erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine was only 50%. This is most probable because the lidocaine incorporated into the treated cells was partially extracted from the membrane by albumin present in the human serum in the culture medium. In fact our calculations suggest that, under the culture conditions used, the effective concentration of lidocaine associated with erythrocytes will correspond to an effective concentration of 0.37 mM in the culture medium. Thus the observed 50% inhibition agrees well with the inhibition data shown in Fig. 1, where 0.37 mM lidocaine present throughout the culture conditions inhibited parasite invasion by 50%. We repeated these experiments four times and obtained similar results. We have previously shown that lidocaine can be used to reversibly disrupt lipid rafts in erythrocyte membranes (Kamata et al., 2008). Thus the present finding of reversible inhibitory effect of lidocaine on parasite invasion, in conjunction with the previous findings of on inhibition of parasite invasion following raft disruption by cholesterol depletion (Samuel et al., 2001) strongly imply an important role for lipid rafts in parasite invasion of erythrocytes. A distinguishing feature of this study is that the strategy we used for reversible disruption of lipid rafts does not involve depletion of cholesterol and as such we can conclude that the presence of rafts, but not membrane cholesterol content per se, is critical for the parasite invasion. A possible mechanism for the effect of lidocaine on parasite invasion might be raft disruption mediated interruption of Gsamediated signal transduction. It has been previously demonstrated that the agonists of G protein-coupled receptor (GPCR) increases intracellular cyclic AMP (cAMP) with concomitant increase in invasion, and the inverse agonist blocked both increased cAMP levels and efficiency of invasion (Harrison et al., 2003). Furthermore, G protein signaling is also implicated in parasite growth (Murphy et al., 2006a). We have also previously shown that lidocaine treatment abolishes increases in intracellular cAMP level by an agonist of GPCR, as well as PKA-phosphorylation of erythrocyte membrane skeletal proteins (Kamata et al., 2008). Phosphorylation of erythrocyte membrane skeletal proteins has been shown to alter erythrocyte membrane mechanical properties (Manno et al., 1995, 2005). Thus, it is reasonable to assume that PKA-phosphorylation of some membrane skeletal proteins results in modification of membrane
I. Koshino, Y. Takakuwa / Experimental Parasitology 123 (2009) 381–383
mechanical properties that favor membrane invagination, which is a prerequisite for parasite invasion. This study together with the previous findings of Samuel et al. (2001) strongly suggest that the raft-mediated signaling pathway can be a target of new and novel anti-malarial therapeutic agents. Acknowledgments This study was supported in part by Takeda Science Foundation and supported by Grants-in-Aid for Scientific Research (No. 18590408) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Prof. T. Kobayakawa and Dr. A. Kaneko in the Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University for kindly providing P. falciparum (F32). References Bessis, M., Mohandas, N., Feo, C., 1980. Automated ektacytometry: a new method of measuring red cell deformability and red cell indices. Blood Cells 6, 315–327. Boampong, J.N., Manno, S., Koshino, I., Takakuwa, Y., 2007. Erythrocyte shape change prevents Plasmodium falciparum invasion. Membrane 32, 95–102. Fernandez, V., 2008. Sorbitol-synchronization of Plasmodium falciparum-infected erythrocytes. In: Moll, K., Ljungström, I., Perimann, H., Scherf, A., Wahlgren, M. (Eds.), Methods in Malaria Research. MR4/ATCC, Virginia, p. 24. Harrison, T., Samuel, B.U., Aktompong, T., Hamm, H., Mohandas, N., Lomasney, J.W., Haldar, K., 2003. Erythrocyte G protein-coupled receptor signaling in malarial infection. Science 301, 1734–1736.
383
Kamata, K., Manno, S., Ozaki, M., Takakuwa, Y., 2008. Functional evidence for presence of lipid rafts in erythrocyte membranes: Gsa in rafts is essential for signal transduction. American Journal of Hematology 83, 371–375. Manno, S., Takakuwa, Y., Mohandas, N., 2005. Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation. Journal of Biological Chemistry 280, 7581–7587. Manno, S., Takakuwa, Y., Nagao, K., Mohandas, N., 1995. Modulation of erythrocyte membrane mechanical function by b-spectrin phosphorylation and dephosphorylation. Journal of Biological Chemistry 270, 5659–5665. Miller, L.H., Baruch, D.I., Marsh, K., Doumbo, O.K., 2002. The pathogenic basis of malaria. Nature 415, 673–679. Murphy, S.C., Harrison, T., Hamm, H.E., Lomasney, J.W., Mohandas, N., Haldar, K., 2006a. Erythrocyte G protein as a novel target for malarial chemotherapy. PLoS Medicine 3, 2403–2415. Murphy, S.C., Hiller, N.L., Harrison, T., Lomasney, J.W., Mohandas, N., Haldar, K., 2006b. Lipid rafts and malaria parasite infection of erythrocytes. Molecular Membrane Biology 23, 81–88. Murphy, S.C., Samuel, B.U., Harrison, T., Speicher, K.D., Speicher, D.W., Reid, M.E., Prohaska, R.R., Low, P.S., Tanner, M.J., Mohandas, N., Haldar, K., 2004. Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection. Blood 103, 1920–1928. Samuel, B.U., Mohandas, N., Harrison, T., McManus, H., Rosse, W., Reid, M., Haldar, K., 2001. The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection. Journal of Biological Chemistry 276, 29319–29329. Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569– 572. Vogt, A., 2008. Selection of trophozoites by using magnetic cell sorting (MACS). In: Moll, K., Ljungström, I., Perimann, H., Scherf, A., Wahlgren, M. (Eds.), Methods in Malaria Research. MR4/ATCC, Virginia, pp. 31–33. Ziegler, H.L., Staerk, D., Christensen, J., Hviid, L., Hägerstrand, H., Jaroszewski, J.W., 2002. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane. Antimicrobial Agents Chemotherapy 46, 1441–1446.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
Disruption of lipid rafts by lidocaine inhibits erythrocyte invasion by Plasmodium falciparum Ichiro Koshino, Yuichi Takakuwa * Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
a r t i c l e
i n f o
Article history: Received 1 April 2009 Received in revised form 19 August 2009 Accepted 21 August 2009 Available online 4 September 2009 Keywords: Plasmodium falciparum Apicomplexan Lipid rafts Lidocaine Parasite invasion Erythrocytes
a b s t r a c t Membrane lipid rafts have been implicated in erythrocyte invasion process by Plasmodium falciparum. In this study, we examined the effect of lidocaine, a local anesthetic, which disrupts lipid rafts reversibly without affecting membrane cholesterol content on parasite invasion. In the presence of increasing concentrations of lidocaine in the culture medium, parasite invasion was progressively decreased with complete inhibition at 2 mM. Decreased invasion was also seen in erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine. This inhibitory effect on parasite invasion was reversed following removal of lidocaine from erythrocyte membranes. Our findings show that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits parasite invasion and confirm an important role for lipid rafts in invasion of erythrocytes by P. falciparum. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Plasmodium falciparum is the causative agent of the most severe form of human malaria, resulting in one million deaths annually. P. falciparum invades human erythrocytes and intra-erythrocytic forms are responsible for all of the clinical symptoms and pathology associated with malaria (Miller et al., 2002). Membrane microdomains enriched with cholesterol and sphingolipids are referred to as ‘‘lipid rafts” (Simons and Ikonen, 1997). Lipid rafts are also rich in several proteins, such as Gsa, which are involved in signaling processes (Murphy et al., 2004). Lipid rafts and cell signaling have been implicated in the invasion of erythrocytes by malarial parasites, since membrane cholesterol depletion resulting in disruption of lipid rafts inhibited erythrocyte invasion by P. falciparum (Samuel et al., 2001; Harrison et al., 2003; Murphy et al., 2006b). However, since a significant amount of cholesterol exists in non-raft membrane regions (Samuel et al., 2001), it can be argued that the observed effects of cholesterol depletion may not be solely due to disruption of lipid rafts. In this study, to determine whether lipid rafts are essential for parasite invasion, we examined the effect on parasite invasion of lidocaine, a local anesthetic which we have recently shown to disrupt lipid rafts without altering membrane cholesterol content (Kamata et al., 2008). Our findings showed that disruption of lipid rafts in the context of normal cholesterol content markedly inhibits * Corresponding author. Fax: +81 3 5269 7416. E-mail address: [email protected] (Y. Takakuwa). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.08.019
parasite invasion, confirming an important role for lipid rafts in invasion of erythrocytes by P. falciparum. 2. Materials and methods 2.1. Parasite culture Plasmodium falciparum (strain F32) was cultured in human erythrocytes by standard methods in RPMI1640 supplemented with 25 mM HEPES, 0.37 mM hypoxanthine, 5% human AB serum, and 0.25% (w/v) Albumax I (Sigma) at a 2% hematocrit under a lowoxygen atmosphere (7.5% O2, 5% CO2, 87.5% N2). 2.2. Parasite invasion assay P. falciparum cultures were synchronized by 5% sorbitol treatment (Fernandez, 2008) and mature stage parasites (trophozoites and schizonts) were purified using VarioMACS CS column (Vogt, 2008). Purified mature stage parasites were used to inoculate cultures of fresh red cells. Cultures were carried out either in the absence or presence of lidocaine (0–2 mM) in the culture medium. In another series of experiments erythrocytes pre-treated with lidocaine were cultured in the absence of lidocaine in the culture medium. For these experiments, 5% human AB serum and 0.25% (w/v) Albumax I in the culture medium was replaced with 10% human AB serum. Parasitemia was determined at 15–18 h after the initiation of cultures by enumerating the newly formed ring-form parasites on thin blood films stained with Giemsa. All experiments
382
I. Koshino, Y. Takakuwa / Experimental Parasitology 123 (2009) 381–383
were performed in triplicate and representative data from 2 to 4 independent experiments are shown. 2.3. Pre-treatment of erythrocytes by lidocaine Venous blood was collected from healthy volunteers after obtaining informed consent. White blood cells were removed using poly-urethane porous filters (Immuguard III, Terumo). Erythrocytes were washed in Tris-buffered saline (TBS) and treated with 18.5 mM lidocaine in TBS containing 0.1% (w/v) glucose (TBS-G) at a hematocrit of 16.7% for 20 min at 37 °C. Treated cells were washed once with TBS and used in the invasion assay. In other experiments, lidocaine-treated cells were incubated in 3% (w/v) bovine serum albumin (BSA) in TBS-G at 10% hematocrit for 20 min at 37 °C, followed by four washes in TBS to remove lidocaine incorporated into the erythrocyte membrane. 3. Results and discussion Purified mature stages of P. falciparum parasites were used to quantitate their ability to invade fresh human erythrocytes in the presence of various concentrations of lidocaine in the culture medium. Ring-stage infected erythrocytes, with parasitemias up to 15% were readily observed in control cultures without lidocaine (Fig. 1). In marked contrast, no ring-stage infected erythrocytes were detected in the presence of 2 mM lidocaine, indicating that lidocaine at this concentration completely inhibits parasite invasion (Fig. 1). The inhibitory effect of lidocaine on parasite invasion was dose dependent (Fig. 1). Similar results were obtained in another independent experiment. Erythrocyte morphology was unaltered at the concentrations of lidocaine used in the study. Furthermore, the surface area/volume ratio of lidocaine-treated erythrocytes was comparable to that of untreated cells as quantitated by ektacytometry (Bessis et al., 1980) (data not shown). Thus, the observed inhibitory effect of lidocaine on parasite invasion is not due to either erythrocyte shape changes or altered cellular deformability (Ziegler et al., 2002; Boampong et al., 2007). The documented inhibitory effect of lidocaine on parasite invasion was reversible. The ability of the parasites to invade erythrocytes pre-treated with lidocanie and cultured in the absence of lidocaine as expected was inhibited compared to the untreated control cells (Fig. 2). Importantly, upon removal of 75% of the lidocaine incorporated into the membrane of treated erythrocytes by washing with BSA (Kamata et al., 2008), the parasite invasion
Fig. 1. Effect of lidocaine on the parasite invasion. Dose-dependent decrease in parasitemia in the presence of increasing concentration of lidocaine in the culture medium. Parasitemia is expressed as mean ± SD derived from measurements performed in triplicate.
Fig. 2. Reversibility of lidocaine effect. Untreated (Cont), lidocaine-treated (Lid), and BSA-washed (Lid/BSA) erythrocytes were used for invasion assay. After 15–18 h, parasitemia was determined on thin blood films stained with Giemsa. Parasitemia is expressed as mean ± SD derived from measurements performed in triplicate. Note increased invasion following removal of lidocaine from membranes of lidocaine-treated cells by albumin washing.
was restored to near normal levels. It should be noted that the observed inhibition of invasion for erythrocytes pre-treated with lidocaine and cultured in the absence of lidocaine was only 50%. This is most probable because the lidocaine incorporated into the treated cells was partially extracted from the membrane by albumin present in the human serum in the culture medium. In fact our calculations suggest that, under the culture conditions used, the effective concentration of lidocaine associated with erythrocytes will correspond to an effective concentration of 0.37 mM in the culture medium. Thus the observed 50% inhibition agrees well with the inhibition data shown in Fig. 1, where 0.37 mM lidocaine present throughout the culture conditions inhibited parasite invasion by 50%. We repeated these experiments four times and obtained similar results. We have previously shown that lidocaine can be used to reversibly disrupt lipid rafts in erythrocyte membranes (Kamata et al., 2008). Thus the present finding of reversible inhibitory effect of lidocaine on parasite invasion, in conjunction with the previous findings of on inhibition of parasite invasion following raft disruption by cholesterol depletion (Samuel et al., 2001) strongly imply an important role for lipid rafts in parasite invasion of erythrocytes. A distinguishing feature of this study is that the strategy we used for reversible disruption of lipid rafts does not involve depletion of cholesterol and as such we can conclude that the presence of rafts, but not membrane cholesterol content per se, is critical for the parasite invasion. A possible mechanism for the effect of lidocaine on parasite invasion might be raft disruption mediated interruption of Gsamediated signal transduction. It has been previously demonstrated that the agonists of G protein-coupled receptor (GPCR) increases intracellular cyclic AMP (cAMP) with concomitant increase in invasion, and the inverse agonist blocked both increased cAMP levels and efficiency of invasion (Harrison et al., 2003). Furthermore, G protein signaling is also implicated in parasite growth (Murphy et al., 2006a). We have also previously shown that lidocaine treatment abolishes increases in intracellular cAMP level by an agonist of GPCR, as well as PKA-phosphorylation of erythrocyte membrane skeletal proteins (Kamata et al., 2008). Phosphorylation of erythrocyte membrane skeletal proteins has been shown to alter erythrocyte membrane mechanical properties (Manno et al., 1995, 2005). Thus, it is reasonable to assume that PKA-phosphorylation of some membrane skeletal proteins results in modification of membrane
I. Koshino, Y. Takakuwa / Experimental Parasitology 123 (2009) 381–383
mechanical properties that favor membrane invagination, which is a prerequisite for parasite invasion. This study together with the previous findings of Samuel et al. (2001) strongly suggest that the raft-mediated signaling pathway can be a target of new and novel anti-malarial therapeutic agents. Acknowledgments This study was supported in part by Takeda Science Foundation and supported by Grants-in-Aid for Scientific Research (No. 18590408) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Prof. T. Kobayakawa and Dr. A. Kaneko in the Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University for kindly providing P. falciparum (F32). References Bessis, M., Mohandas, N., Feo, C., 1980. Automated ektacytometry: a new method of measuring red cell deformability and red cell indices. Blood Cells 6, 315–327. Boampong, J.N., Manno, S., Koshino, I., Takakuwa, Y., 2007. Erythrocyte shape change prevents Plasmodium falciparum invasion. Membrane 32, 95–102. Fernandez, V., 2008. Sorbitol-synchronization of Plasmodium falciparum-infected erythrocytes. In: Moll, K., Ljungström, I., Perimann, H., Scherf, A., Wahlgren, M. (Eds.), Methods in Malaria Research. MR4/ATCC, Virginia, p. 24. Harrison, T., Samuel, B.U., Aktompong, T., Hamm, H., Mohandas, N., Lomasney, J.W., Haldar, K., 2003. Erythrocyte G protein-coupled receptor signaling in malarial infection. Science 301, 1734–1736.
383
Kamata, K., Manno, S., Ozaki, M., Takakuwa, Y., 2008. Functional evidence for presence of lipid rafts in erythrocyte membranes: Gsa in rafts is essential for signal transduction. American Journal of Hematology 83, 371–375. Manno, S., Takakuwa, Y., Mohandas, N., 2005. Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation. Journal of Biological Chemistry 280, 7581–7587. Manno, S., Takakuwa, Y., Nagao, K., Mohandas, N., 1995. Modulation of erythrocyte membrane mechanical function by b-spectrin phosphorylation and dephosphorylation. Journal of Biological Chemistry 270, 5659–5665. Miller, L.H., Baruch, D.I., Marsh, K., Doumbo, O.K., 2002. The pathogenic basis of malaria. Nature 415, 673–679. Murphy, S.C., Harrison, T., Hamm, H.E., Lomasney, J.W., Mohandas, N., Haldar, K., 2006a. Erythrocyte G protein as a novel target for malarial chemotherapy. PLoS Medicine 3, 2403–2415. Murphy, S.C., Hiller, N.L., Harrison, T., Lomasney, J.W., Mohandas, N., Haldar, K., 2006b. Lipid rafts and malaria parasite infection of erythrocytes. Molecular Membrane Biology 23, 81–88. Murphy, S.C., Samuel, B.U., Harrison, T., Speicher, K.D., Speicher, D.W., Reid, M.E., Prohaska, R.R., Low, P.S., Tanner, M.J., Mohandas, N., Haldar, K., 2004. Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection. Blood 103, 1920–1928. Samuel, B.U., Mohandas, N., Harrison, T., McManus, H., Rosse, W., Reid, M., Haldar, K., 2001. The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection. Journal of Biological Chemistry 276, 29319–29329. Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569– 572. Vogt, A., 2008. Selection of trophozoites by using magnetic cell sorting (MACS). In: Moll, K., Ljungström, I., Perimann, H., Scherf, A., Wahlgren, M. (Eds.), Methods in Malaria Research. MR4/ATCC, Virginia, pp. 31–33. Ziegler, H.L., Staerk, D., Christensen, J., Hviid, L., Hägerstrand, H., Jaroszewski, J.W., 2002. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane. Antimicrobial Agents Chemotherapy 46, 1441–1446.