Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Apoptosis in multiple myeloma: Therapeutic implications

Apoptosis is the primary means by which most radio- and chemotherapy modalities kill cancer cells, and abnormalities in the apoptotic pathways may contribute to disease pathogenesis of cancer. Multiple Myeloma (MM) is a hematological malignancy which will affect 14,000 new individuals in the United States in 2001 and remains irreversibly fatal despite all available therapies. The current review focuses on the studies of apoptotic and survival signaling pathways in MM cells, which have both identified novel apoptotic and anti-apoptotic proteins and provided targets for novel therapeutics.     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

Monoclonal antibodies as therapeutics in oncology

The specificity of antibodies has been harnessed to target cancer cells and the first therapeutic antibodies for use in oncology are now finding application in the clinic. Studies are currently under way to develop new and improved antibodies. Recent developments have been made in the identification of novel targets, including the use of genomic and proteomic technologies. Several methods are also being developed to enhance antibody efficacy.     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.     

Trafficking of intracerebroventricularly injected antisense oligonucleotides in the mouse brain

Intracerebroventricular (icv) delivery of therapeutic molecules directly into the brain parenchyma has attracted considerable attention because of the advantage of bypassing the blood-brain barrier. Exogenous icv administration of antisense oligodeoxynucleotides (AS-ODNs) has been implicated in modifying gene expression within the targeted brain area. The biodistribution, tissue penetration, and stability of exogenously administered AS-ODNs are the major determinants with regard to their potential utility as agents for modifying gene expression. This report examined the distribution and clearance of labeled AS-ODNs with the aim of exploring the feasibility of icv administration of AS-ODNs as a targeted treatment approach to Alzheimer's disease. A single icv injection of fluorescein-labeled 2'-O-(methoxy) ethyl (2'MOE) ribosyl-modified AS-ODNs directed at the beta-secretase cleavage site of beta-amyloid precursor protein (APP) mRNA into the mouse brain showed rapid uptake by 15 minutes, overall gradual spread and retention by 30 minutes to 3 hours, and complete clearance by 8 hours postinjection. Labeled AS-ODNs were observed to penetrate across the cell membrane and accumulate in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cell populations. Current study provides a basic pattern of uptake, distribution, and stability of AS-ODNs in the mouse brain.     

Dehydrophenylalanine zippers: strong helix-helix clamping through a network of weak interactions

A decapeptide Boc-L-Ala-(Delta Delta Phe)(4)-L-Ala-(Delta Delta Phe)3-Gly-OMe (Peptide I) was synthesized to study the preferred screw sense of consecutive alpha,beta-dehydrophenylalanine (Delta Delta Phe) residues. Crystallographic and CD studies suggest that, despite the presence of two L-Ala residues in the sequence, the decapeptide does not have a preferred screw sense. The peptide crystallizes with two conformers per asymmetric unit, one of them a slightly distorted right-handed 3(10)-helix (X) and the other a left-handed 3(10)-helix (Y) with X and Y being antiparallel to each other. An unanticipated and interesting observation is that in the solid state, the two shape-complement molecules self-assemble and interact with an extensive network of C-H...O hydrogen bonds and pi-pi interactions, directed laterally to the helix axis with amazing regularity. Here, we present an atomic resolution picture of the weak interaction mediated mutual recognition of two secondary structural elements and its possible implication in understanding the specific folding of the hydrophobic core of globular proteins and exploitation in future work on de novo design.