Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa)

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and β-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying “doughnut-shaped” vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity.Keywords: Colpodella · Rhoptries · Trichocysts · Apical complex · Plasmodium RhopH

Molecular Factors Responsible for Host Cell Recognition and Invasion in Plasmodium falciparum1\u3c/sup\u3e

ABSTRACT. In Plasmodium falciparum. the rhoptries involved in the invasion process are a pair of flask‐shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225‐, 140/130/110‐, 80/60/40‐, RAP‐1 80‐, AMA‐1 80‐, QF3 80‐, and 55‐kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110‐kDa complex are transferred to the erythrocyte membrane during invasion. The ring‐infected surface antigen (RESA). a 155‐kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte‐binding studies have demonstrated that both the 140/130/110‐kDa rhoptry complex and RESA bind to inside‐out‐vesicles (IOVs) prepared from human erythrocytes. The 140/130/110‐kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110‐kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome‐binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes. The rhoptry complex also binds to membrane and inside‐out‐vesicles prepared from human erythrocytes and erythrocytes from other species. The rhoptry complex associated with the erythrocyte membrane in ring‐infected erythrocytes is accessible to cleavage by phospholipase A. Studies are in progress to identify the molecular epitopes on the individual proteins within the complex responsible for lipid interaction in the erythrocyte bilayer and to determine the specificity of the phospholipid interaction using erythrocyte phospholipids. Copyright © 1992, Wiley Blackwell. All rights reserve

The Role of the Maurer’s Clefts in Protein Transport in Plasmodium falciparum

Maurer\u27s clefts (MCs) are membranous structures that are formed by Plasmodium falciparum and used by the parasite for protein sorting and protein export. Virulence proteins, as well as other proteins used to remodel the erythrocyte, are exported. Discontinuity between major membrane compartments within the infected erythrocyte cytoplasm suggests multiple traffic routes for exported proteins. The sequences of the conserved Plasmodium export element seem insufficient for export of all parasite proteins. The parasite displays remarkable versatility in the types of proteins exported to the MCs and in the functions of the proteins within the MCs. In this Review, protein export to the MCs and the role of the MCs in the transport of proteins to the erythrocyte membrane are summarized. © 2009 Elsevier Ltd. All rights reserved

Nutritional Barriers to the Adherence to the Mediterranean Diet in Non-Mediterranean Populations

Adherence to the Mediterranean diet has been shown to lower the risk of developing chronic non-communicable diseases like cardiovascular and neurodegenerative diseases and cancer. Improvements in depression, participation in daily activities in older individuals, weight loss and a reduction in adverse pregnancy outcomes are associated with adherence to the Mediterranean diet. The number of studies that have evaluated barriers to adherence to the Mediterranean diet in the US and, in particular, in racial and ethnic minority populations within the US are few. Among Native American and Alaskan Native populations, studies evaluating traditional or alternative Mediterranean diet adherence for chronic non-infectious diseases is unavailable. Mediterranean diet scoring instruments used in studies in European and Mediterranean countries and among white participants in the US fail to capture the dietary patterns of racial and ethnic minority populations. In this narrative review, the food components of the traditional Mediterranean diet are discussed, adherence to the Mediterranean diet is examined in Mediterranean and non-Mediterranean countries and barriers preventing adherence to the Mediterranean diet in the US and among racial and ethnic minority populations is reviewed. Recommendations for improving nutrition education and intervention and for increasing adherence and cultural adaptions to the Mediterranean diet are provided

Plasmodium falciparum: Analysis of Protein-Protein Interactions of the 140/130/110-kDa Rhoptry Protein Complex Using Antibody and Mouse Erythrocyte Binding Assays

The high-molecular-weight rhoptry proteins of Plasmodium falciparum exist in a multiprotein complex consisting of proteins of 140, 130, and 110 kDa. The complex of rhoptry proteins binds to human and mouse erythrocyte membranes in association with a 120-kDa SERA protein. These proteins are believed to participate in the process of erythrocyte invasion. We have used six different antibodies (polyclonal and monoclonal) known to precipitate the high-molecular-weight rhoptry protein complex (HMWC) to analyze the structural relationship of proteins within the complex. Limited proteolysis of immune complexes (IC) immobilized on Sepharose beads (protein \u27footprinting\u27) and binding of SV8 protease generated peptides to intact mouse erythrocytes was performed. The 140-kDa polypeptide was more susceptible to protease digestion followed by the 130- and 110-kDa polypeptides. The susceptibility of the 140-kDa polypeptide to protease digestion was independent of the type of precipitating antibody. We identified a 120-kDa protein as the major proteolytic fragment of the 140-kDa protein. SV8 protease generated peptide fragments derived from the 110- and 130-kDa proteins contained putative mouse erythrocyte binding domains. Immunoprecipitation of SV8-generated peptides gave peptide profiles similar to those obtained with protein \u27footprinting\u27. Additional experiments performed to investigate the stability of the HMWC using chaotropic and lyotropic agents demonstrated that the HMWC was stable to perturbatory reagents known to disaggregate macromolecular complexes. Solubilization of schizonts with 6 M urea and 4 M MgCl followed by IC formation led to differential precipitation of the 110-kDa polypeptide, while solubilization with 3 M KCl resulted in the differential precipitation of the 140- and 130-kDa polypeptides, suggesting that both proteins may be in direct association. Treatment of immobilized IC with different perturbatory agents including 6 M urea, 3 M KCl, 4 M MgCl , or 2% SDS from an insoluble matrix resulted in the elution of the intact complex. The mouse erythrocyte binding property of the HMWC is conserved among different geographical isolates of P. falciparum. The results provide insights concerning the mechanism of protein-protein interaction within the complex. © 1993 Academic Press, Inc. 2

Rhoptry Organelles of the Apicomplexa: Their Role in Host Cell Invasion and Intracellular Survival

Members of the phylum Apicomplexa are obligate intracellular parasites that invade erythrocytes, lymphocytes, macrophages or cells of the alimentary canal in various vertebrate species. Organelles within the apical complex of invasive stages facilitate host cell invasion. Parasites in this phylum cause some of the most debilitating diseases of medical and veterinary importance. These include malaria, toxoplasmosis, babesiosis, theileriosis (East Coast fever), and coccidiosis in poultry and livestock. In recent years, opportunistic infections caused by Cryptosporidium parvum, and recrudescent Toxoplasma gondii infections in AIDS patients have prompted intensified efforts in understanding the biology of these parasites. In this review, Tobili Sam-Yellowe examines the unifying and variant molecular features of rhoptry proteins, and addresses the role of multigene families in organelle function: the biogenesis of the rhoptries will also be examined, in an attempt to understand the sequence of events leading to successful packaging, modification and processing of proteins within the organelle