PubMed Health⌕ Search

Biomedical subjects

Abhai K Tripathi

Publications and source records attributed to Abhai K Tripathi.

6 recordsLinked to original sources
PubMed2007

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization.

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

Animals↗
PubMed2006

Plasmodium falciparum-infected erythrocytes increase intercellular adhesion molecule 1 expression on brain endothelium through NF-kappaB.

Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-IRBC) in postcapillary brain endothelium is a hallmark of cerebral malaria (CM) pathogenesis. There is a correlation between adherent Pf-IRBC and increased expression of intercellular cell adhesion molecule 1 (ICAM-1), which is also a receptor for Pf-IRBC on human brain microvascular endothelial cells (HBMEC). The underlying mechanism for the increased ICAM-1 expression has not been clearly defined. Therefore, we investigated the mechanisms of ICAM-1 expression on isolated HBMEC after exposure to Pf-IRBC. Ultrastructural characterization of the model confirmed that there was attachment through both Pf-IRBC knobs and HBMEC microvillus protrusions. Pf-IRBC induced a dose- and time-dependent increase in ICAM-1 expression on HBMEC that was specific for human brain endothelium and was not observed with human umbilical vein endothelium. Involvement of both membrane-associated Pf-IRBC proteins and parasite-derived soluble factors with the increase in ICAM-1 expression was demonstrated by surface trypsinization and fractionation. Pf-IRBC exposure induced nuclear translocation of NF-kappaB in HBMEC, which was linked to ICAM-1 expression, as shown by use of specific inhibitors of the transcription factor NF-kappaB and immunocytochemistry. In addition, inhibition of reactive oxygen species decreased Pf-IRBC-induced ICAM-1 expression on HBMEC. Parasite-induced ICAM-1 expression explains the localization of this molecule on brain endothelium in postmortem CM brain samples. By increasing ICAM-1 expression, Pf-IRBC may increase their sequestration, thereby perpetuating CM.

Active Transport, Cell Nucleus↗
PubMed2004

Spectrophotometric determination of de novo hemozoin/beta-hematin formation in an in vitro assay.

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.

Animals↗
PubMed2004

An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum. Cloning and biochemical characterization of the enzyme.

Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs.

Amino Acid Sequence↗
PubMed2002

A physiochemical mechanism of hemozoin (beta-hematin) synthesis by malaria parasite.

Malaria parasite homogenate, the lipid extracts, and an unsaturated fatty acid, linoleic acid, which have been shown to promote beta-hematin formation in vitro, were used to investigate the mechanism of hemozoin biosynthesis, a distinct metabolic function of the malaria parasite. In vitro beta-hematin formation promoted by Plasmodium yoelii homogenate, the lipid extracts, and linoleic acid were blocked by ascorbic acid, reduced glutathione, sodium dithionite, beta-mercaptoethanol, dithiothreitol, and superoxide dismutase. Oxidized glutathione did not show any effect. Preoxidized preparations of the lipids extracts or the P. yoelii homogenate failed to catalyze beta-hematin formation. Depletion of oxygen in the reaction mixtures also inhibited the lipid-catalyzed beta-hematin formation. Under the reaction conditions similar to those used for the in vitro beta-hematin formation assay, the antioxidants and reducing agents mentioned above, except the DTT and beta-mercaptoethanol, did not cause degradation of heme. beta-Hematin formation was also inhibited by p-aminophenol, a free radical chain reaction breaker. Hemozoin biosynthesis within the digestive vacuoles of the malaria parasite may be a lipid-catalyzed physiochemical reaction. An oxidative mechanism may be proposed for lipid-mediated beta-hematin formation, which may be mediated by generation of some free radical intermediates of heme.

Aminophenols↗