The mitochondrial genome and transcriptome of the basal dinoflagellate Hematodinium sp.: character evolution within the highly derived mitochondrial genomes of dinoflagellates.

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.

Traffic jams: protein transport in Plasmodium falciparum.

Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals. At least a dozen distinct protein destinations are known. The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane. Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system. This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum.

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells.

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway.

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.

Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum.

A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or "apicoplast," is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), beta-ketoacyl-ACP synthase III (FabH), and beta-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.

Plastids in parasites of humans.

It has recently emerged that malarial, toxoplasmodial and related parasites contain a vestigial plastid (the organelle in which photosynthesis occurs in plants and algae). The function of the plastid in these obligate intracellular parasites has not been established. It seems likely that modern apicomplexans derive from photosynthetic predecessors, which perhaps formed associations with protists and invertebrates and abandoned autotrophy in favour of parasitism. Recognition of a third genetic compartment in these parasites proffers alternative strategies for combating a host of important human and animal diseases. It also poses some fascinating questions about the evolutionary biology of this important group of pathogens.

Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress.

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Magnesium sulfate in exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease are commonly seen and difficult to treat. We sought to determine the bronchodilator efficacy of magnesium sulfate in this situation, as this compound is helpful in acute asthma. METHODS: Subjects who came to either of two Veterans Affairs emergency departments were randomized in a double-blind fashion to receive either 1.2 g of magnesium sulfate or placebo over 20 minutes after they first received albuterol, 2.5 mg by nebulization. Peak expiratory flow, dyspnea scores, arterial hemoglobin oxygen saturation by pulse oximetry, maximal inspiratory and expiratory pressures, and vital signs were monitored for 45 minutes after the start of magnesium sulfate or placebo treatment. RESULTS: Seventy-two individuals were studied. The peak expiratory flow increased 16.6% +/- 27.7% (mean +/- SD) in both groups after the initial albuterol treatment, from 121.2 +/- 55.7 L/min to 136.9 +/- 63.9 L/min. The peak expiratory flow increased from 136.7 +/- 69.7 L/min at the start of the infusion to 162.3 +/- 76.6 L/min at 30 minutes and 161.3 +/- 78.7 L/min at 45 minutes with magnesium sulfate treatment. The peak expiratory flow was 137.0 +/- 58.6 L/min on initiation of the intravenous infusion, 143.0 +/- 72.7 L/min at 30 minutes, and 143.3 +/- 70.5 L/min at 45 minutes in the placebo group. The difference in peak expiratory flow from initiation of the infusion to 30 and 45 minutes later (calculated as means of the 30- and 45-minute values) was significantly different for the two groups (25.1 +/- 35.7 L/min vs 7.4 +/- 33.3 L/min; P = 0.3); the difference was also significant when expressed as percentage increase (22.4% +/- 28.5% vs 6.1% +/- 24.4%; P = .01). There was a statistically nonsignificantly trend toward a reduced need for hospitalization in the magnesium sulfate group as compared with the placebo group (28.1% vs 41.9%; P = .25). There were no significant changes in the other parameters with either treatment. CONCLUSION: Magnesium sulfate, 1.2 g over 20 minutes after beta-agonist administration, is safe and modestly efficacious in the treatment of acute exacerbations of chronic obstructive pulmonary disease, and its bronchodilator effect is greater than that of a beta-agonist given alone and lasts beyond the period of magnesium sulfate administration.

Analysis of the inspiratory flow-volume curve. Should it always precede the forced expiratory maneuver?

The inspiratory flow-volume (FV) curve can be used to identify patients with upper airway obstruction, air trapping, and restriction. Current computed pulmonary function testing equipment often mandates a forced expiratory maneuver (FEM) immediately prior to the forced inspiratory maneuver (standard method). We evaluated the inspiratory FV curve with and without an antecedent FEM in 119 subjects referred for pulmonary function testing. The subjects were divided into four groups by grading the degree of airway obstruction using confidence intervals of the FEV1/FVC percent predicted minus the actual FEV1/FVC percent measured from the best FEM according to Intermountain Thoracic Society recommendations. The forced inspiratory vital capacity (FIVC), forced inspiratory flow 50 (FIF50), and peak inspiratory flow (PIF) from the inspiratory FV curve with an antecedent FEM was compared with the FIVC, FIF50, and PIF without an antecedent FEM in each category of obstructive lung disease. The FIVC without the antecedent FEM was significantly larger than that with an antecedent FEM by 170 ml (p < 0.002) in subjects with severe airway obstruction, but was not significantly different in the other groups. The FIF50 was not significantly different in any group, but approached significance in both normal subjects and subjects with severe obstruction. The PIF was not significantly different in any group, but approached significance in the normal subjects, order for patients with severe obstructive airway disease to generate a valid forced inspiratory FV curve, it should be obtained without an antecedent FEM. When a plateau of the inspiratory FV curve is encountered, we suggest that is useful to generate the inspiratory FV curve prior to the FEM and to analyze its flow and volume characteristics independent of the FEM. The "best" inspiratory FV curve should then be displayed with the "best" FEM for proper evaluation of the FV loop.

Can MDIs be used effectively by extubated ICU patients?

Metered-dose inhaler (MDI) therapy can be an effective, cost-efficient means of managing chronic airway obstruction in many patients in medical or surgical intensive care units who have recently been extubated. Because medication is delivered directly to the airways through an MDI, a relatively low dosage may be effective, and few adverse effects may be encountered. Candidates for MDI therapy should have a vital capacity of at least 900 mL, be able to hold their breath for at least 5 seconds, and have a respiratory rate of under 25 breaths per minute. The majority of patients who meet these criteria are able to convert to MDI therapy within 24 hours of extubation.