311 research outputs found

    Aspartate 19 and Glutamate 121 Are Critical for Transport Function of the myo-Inositol/H+ symporter from Leishmania donovani

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    The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopusoocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone and dinitrophenol inhibited inositol transport by 50–70% in the wild-type as well as in E121Q, despite its reduced transport activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT

    Kinetics and Stoichiometry of a Proton/myo-Inositol Cotransporter

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    Voltage clamp recording was used to measure steady-state and presteady-state currents mediated by a myo-inositol transporter cloned from Leishmania donovani and expressed in Xenopus oocytes. Application of myo-inositol resulted in inward currents, which did not require external sodium and which were increased by increasing the extracellular proton concentration and by membrane hyperpolarization. Alkalinization of the extracellular space occurred concomitantly with myo-inositol influx. Correlation of membrane currents with radiolabeled myo-inositol flux revealed that one positive charge is translocated with each molecule of myo-inositol, consistent with cotransport of one proton. The transport concentration dependence on both species suggested ordered binding of a proton followed by a molecule of myo-inositol. In the absence of myo-inositol, a voltage-dependent capacitance was observed that correlated with the transporter expression level. This charge movement obeyed a Boltzmann function, which was used to estimate a turnover of 0.70 ± 0.06 s−1 at −60 mV. The pH and voltage dependence of the charge movements were simulated with a model involving alternating access of internal and external protons to sites within an occluded pore

    Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii

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    The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization

    A Family of Putative Receptor-Adenylate Cyclases from Leishmania donovani

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    Leishmania parasites are exposed to pronounced changes in their environment during their life cycle as they migrate from the sandfly midgut to the insect proboscis and then into the phagolysosomes of the vertebrate macrophages. The developmental transformations that produce each life cycle stage of the parasite may be signaled in part by binding of environmental ligands to receptors which mediate transduction of extracellular signals. We have identified a family of five clustered genes in Leishmania donovani which may encode signal transduction receptors. The coding regions of two of these genes, designated rac-A and rac-B, have been sequenced and shown to code for proteins with an NH2-terminal hydrophilic domain, an intervening putative transmembrane segment, and a COOH-terminal domain that has high sequence identity to the catalytic domain from adenylate cyclases in other eukaryotes. We have expressed the receptor-adenylate cyclase protein (RAC)-A protein in Xenopus oocytes and demonstrated that it functions as an adenylate cyclase. Although RAC-B exhibits no catalytic activity when expressed in oocytes, co-expression of RAC-A and RAC-B negatively regulates the adenylate cyclase activity of RAC-A, suggesting that these two proteins interact in the membrane. Furthermore, a truncated version of RAC-A functions as a dominant negative mutant that inhibits the catalytic activity of the wild type receptor. The rac-A and rac-B genes encode developmentally regulated mRNAs which are expressed in the insect stage but not in the mammalian host stage of the parasite life cycle

    Equilibrative Nucleoside Transporter Family Members from Leishmania donovani Are Electrogenic Proton Symporters

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    Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters

    KHARON Is an essential cytoskeletal protein involved in the trafficking of flagellar membrane proteins and cell division in African trypanosomes

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    African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability

    The Ursinus Weekly, November 14, 1974

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    15 U.C. seniors named to Who\u27s who • Puzzle solved • Letter to the Editor • Part two: The Legend of the three doors • Growth of the Greeks: Green and gold - KDK • Library metamorphosis • Rebuilding Bears lose to Dickinson • Only one game lost • Alumna corner: First foreign student • Pages from Ursinus past: Freshmen regulations • Photo reviewhttps://digitalcommons.ursinus.edu/weekly/1025/thumbnail.jp

    Arsenic transport by zebrafish aquaglyceroporins

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    <p>Abstract</p> <p>Background</p> <p>Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (As<sup>III</sup>) is one of the major species that exists environmentally. The transport of As<sup>III </sup>has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct As<sup>III </sup>and its organic metabolite, monomethylarsenite (MAs<sup>III</sup>). However, the transport of As<sup>III </sup>and MAs<sup>III </sup>in in any fish species has not been characterized.</p> <p>Results</p> <p>In this study, five members of the aquaglyceroporin family from zebrafish (<it>Danio rerio</it>) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (As<sup>III </sup>and MAs<sup>III</sup>) and antimonite (Sb<sup>III</sup>) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named <it>aqp3, aqp3l, aqp9a, aqp9b </it>and <it>aqp10 </it>according to their similarities to the corresponding human AQPs. Expression of <it>aqp9a, aqp9b</it>, <it>aqp3, aqp3l </it>and <it>aqp10 </it>in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from <it>Xenopus leavis</it>. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic As<sup>III</sup>, MAs<sup>III </sup>and Sb<sup>III</sup>. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic.</p> <p>Conclusion</p> <p>This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.</p

    Coxiella burnetii and Leishmania Mexicana Residing Within Similar Parasitophorous Vacuoles Elicit Disparate Host Responses

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    Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV) derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage) cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up-regulated and 311 genes downregulated) in C. burnetii infected cells, whereas 698 genes (534 genes up-regulated and 164 genes down-regulated) were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (∼7%) are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs) that were expressed in THP-1 cells, and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections, and our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches

    Operon conservation and the evolution of trans-splicing in the phylum Nematoda

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    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage
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