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Maintenance of iron homeostasis must balance the demand for iron due to heme synthesis, which is driven by hematopoiesis, and the restricted intestinal uptake of iron, which otherwise limits absorption of this toxic element. The consequences of perturbed iron homeostasis are witnessed in inherited forms of beta-thalassemia in which erythroid hyperplasia results in enhanced intestinal iron absorption despite tissue iron overload. To gain a better understanding of intestinal factors that are induced when iron homeostasis is disrupted, a panel of monoclonal antibodies that recognize intestinal microvillous membrane proteins of the beta-thalassemic Hbbd(th3)/Hbbd(th3) mouse was established. The monoclonal antibodies were screened by differential Western blotting against normal and beta-thalassemic mouse intestine to identify antigens modulated in the disease state. Here we report the initial characterization of one immunoreactive species that is up-regulated in beta-thalassemic mouse intestine and the tentative identification of this antigen as sucrase-isomaltase. Studies in Caco-2 cells revealed the rather unexpected finding that expression of this intestinal hydrolase is increased in response to iron toxicity. [ABSTRACT FROM AUTHOR]

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.

Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.

The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.

The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.

Our previous studies have shown that riboflavin has activity against Plasmodium falciparum asexual-stage parasites in vitro. In the present study we examine the gametocytocidal activity of riboflavin and the interaction of riboflavin with some commonly used antimalarial drugs against the asexual forms of P. falciparum in vitro. The addition of riboflavin to P. falciparum cultures killed gametocytes at all stages, even those at late stages (III to V), which are not affected by many of the commonly used antimalarials. Combinations of riboflavin with mefloquine, pyrimethamine, and quinine showed a marked potentiation of the activities of these drugs against asexual-stage parasites in vitro. The combination of riboflavin with artemisinin was additive, while that with chloroquine was mildly antagonistic. High doses of riboflavin are used clinically to treat several inborn errors of metabolism with no adverse side effects. Its efficacy in combination with standard antimalarial drugs in treating and preventing the transmission of P. falciparum malaria can therefore be evaluated in humans.

The human malaria parasite Plasmodium falciparum digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% +/- 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% +/- 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by P. falciparum.