23 research outputs found
An in vitro lung model to assess true shunt fraction by multiple inert gas elimination
The Multiple Inert Gas Elimination Technique, based on Micropore Membrane Inlet Mass Spectrometry, (MMIMS-MIGET) has been designed as a rapid and direct method to assess the full range of ventilation-to-perfusion (V/Q) ratios. MMIMS-MIGET distributions have not been assessed in an experimental setup with predefined V/Q-distributions. We aimed (I) to construct a novel in vitro lung model (IVLM) for the simulation of predefined V/Q distributions with five gas exchange compartments and (II) to correlate shunt fractions derived from MMIMS-MIGET with preset reference shunt values of the IVLM. Five hollow-fiber membrane oxygenators switched in parallel within a closed extracorporeal oxygenation circuit were ventilated with sweep gas (V) and perfused with human red cell suspension or saline (Q). Inert gas solution was infused into the perfusion circuit of the gas exchange assembly. Sweep gas flow (V) was kept constant and reference shunt fractions (IVLM-S) were established by bypassing one or more oxygenators with perfusate flow (Q). The derived shunt fractions (MM-S) were determined using MIGET by MMIMS from the retention data. Shunt derived by MMIMS-MIGET correlated well with preset reference shunt fractions. The in vitro lung model is a convenient system for the setup of predefined true shunt fractions in validation of MMIMS-MIGET
Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties
Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni2+-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 x 10(8) min(-1) M-1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors
Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts
T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved
Bland-Altman analysis with saline as priming fluid.
<p>Bland-Altman analysis of MMIMS-MIGET based shunt fraction–with saline as priming fluid (MM-SNS) on predefined in vitro lung model shunt (IVLM-SNS). Bias ± precision (2 SD) was -0.04 00B1 0.12 with 95% limits of agreement (dashed) of -0.154 and 0.082.</p
Linear regression analysis with blood as priming fluid.
<p>Linear regression analysis for MMIMS-MIGET based shunt fraction–with blood as priming fluid (MM-SRBC) on predefined in vitro lung model shunt (IVLM-SRBC): MM-SRBC = 0.87*IVLM-SRBC-0.02 (r2 = 0.96, P< 0.0001). Duplicate data from 0 to 0.8 model shunt fractions included. Solid line = linear regression; Dashed line = line of identity.</p
Linear regression analysis with saline as priming fluid.
<p>Linear regression analysis for MMIMS-MIGET shunt fraction–with saline as priming fluid (MM-SNS) on predefined in vitro lung model shunt (IVLM-SNS): MM-SNS = 0.91*IVLM-SNS +0.005 (r<sup>2</sup> = 0.99, P< 0.0001). Duplicate data from 0 to 0.8 model shunt fractions included. Solid line = linear regression; Dashed line = line of identity.</p
Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection
<div><p>Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme <i>de novo</i>, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of <i>Plasmodium berghei</i> (<i>Pb</i>). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out <i>in vitro</i> radiolabeling of heme in <i>Pb</i>-infected mouse reticulocytes and <i>Plasmodium falciparum</i>-infected human RBCs using [4-<sup>14</sup>C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the <i>de novo</i> pathway is absolutely essential for parasite development in the mosquito and liver stages. <i>Pb</i>KO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in <i>Pb</i>ALASKO parasites recovered when mosquitoes received an ALA supplement. <i>Pb</i>ALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.</p></div
Oocyst and sporozoite formation in <i>P.berghei</i>-infected (WT and KOs) mosquitoes.
<p>(A) Mercurochrome staining of oocysts in the midgut preparations. Arrows indicate oocysts and the magnified images of oocysts are provided in insets. Scale bar: 100 µm. (B) Sporozoites in the salivary glands. Magnified images of sporozoites are provided in insets. Scale bar: 50 µm. (C) Quantification of oocysts. P values for <i>Pb</i>ALASKO and <i>Pb</i>FCKO with respect to WT are <0.02. P value for <i>Pb</i>ALASKO(Mq<sup>+ALA</sup>) with respect to <i>Pb</i>ALASKO is <0.01 and <i>Pb</i>FCKO(Mq<sup>+Blood</sup>) with respect to <i>Pb</i>FCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for <i>Pb</i>ALASKO, <i>Pb</i>FCKO, <i>Pb</i>ALASKO(Mq<sup>+ALA</sup>) and <i>Pb</i>FCKO(Mq<sup>+Blood</sup>) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; <i>Pb</i>ALASKO(Mq<sup>+ALA</sup>) and <i>Pb</i>FCKO(Mq<sup>+Blood</sup>), <i>P. berghei</i> KO parasites from mosquitoes supplemented with ALA and blood feeding, respectively.</p
Growth curves for intraerythrocytic stages of <i>P. berghei</i> WT and KO parasites in mice.
<p>Mice were injected intraperitoneally with 10<sup>5 </sup><i>P. berghei</i> infected-RBCs/reticulocytes and the parasite growth was routinely monitored as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003522#s4" target="_blank">Materials and Methods</a>. Multiple fields were used to quantify the parasite infected cells. The data provided represent the mean ± S.D. obtained from 6 animals.</p