14 research outputs found

    Mutations in Transmembrane Domains 1, 4 and 9 of the Plasmodium falciparum Chloroquine Resistance Transporter Alter Susceptibility to Chloroquine, Quinine and Quinidine

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    Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Lt

    Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene

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    <p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum</it>, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP) drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ) exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. <it>Plasmodium falciparum in vitro </it>resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the <it>cytochrome b </it>gene. ATQ -resistant <it>Plasmodium yoelii </it>and <it>Plasmodium berghei </it>lines have been obtained and resistant lines have amino acid mutations in their CYT <it>b </it>protein sequences. <it>Plasmodium chabaudi </it>model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the <it>P. chabaudi </it>clones, to select a resistant parasite line and to perform genotypic characterization of the <it>cytb </it>gene of these clones.</p> <p>Methods</p> <p>To select for ATQ resistance, <it>Plasmodium. chabaudi chabaudi </it>clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. <it>Plasmodium chabaudi cytb </it>gene was amplified and sequenced.</p> <p>Results</p> <p>ATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i) multiple blood passages in the absence of the drug, (ii) freeze/thawing of parasites in liquid nitrogen and (iii) transmission through a mosquito host, <it>Anopheles stephensi</it>. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six-fold increase in MCD to ATQ relative to AS-3CQ.</p> <p>Conclusions</p> <p>A mutation was found on the <it>P. chabaudi cytb </it>gene from the AS-ATQ sample a substitution at the residue Tyr268 for an Asn, this mutation is homologous to the one found in <it>P. falciparum </it>isolates resistant to ATQ.</p

    INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure

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    The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration

    Evolution of Marine Organisms under Climate Change at Different Levels of Biological Organisation

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    Research to date has suggested that both individual marine species and ecological processes are expected to exhibit diverse responses to the environmental effects of climate change. Evolutionary responses can occur on rapid (ecological) timescales, and yet studies typically do not consider the role that adaptive evolution will play in modulating biological responses to climate change. Investigations into such responses have typically been focused at particular biological levels (e.g., cellular, population, community), often lacking interactions among levels. Since all levels of biological organisation are sensitive to global climate change, there is a need to elucidate how different processes and hierarchical interactions will influence species fitness. Therefore, predicting the responses of communities and populations to global change will require multidisciplinary efforts across multiple levels of hierarchy, from the genetic and cellular to communities and ecosystems. Eventually, this may allow us to establish the role that acclimatisation and adaptation will play in determining marine community structures in future scenarios

    A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

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    Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites

    The strike-slip Yussuf fault system (Alboran Sea): Multi-scale seismic imaging and evidences of Quaternary activity

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    Paleoseismology along the North Anatolian Fault (PANAF) & Commemorating the 9 August 1912 MĂĽrefte Earthquake after 100 years, 8-10 October 2012, Ä°stanbul, TurkeyThe NW-SE convergence (4-5 mm/yr) between the African and Eurasian plates controls the present-day crustal deformation along the southern Iberian and northern African margins. The strain due to this convergence is accommodated over a wide deformation zone with significant seismic activity. Although seismicity is mainly characterized by low to moderate magnitude events, large and destructive earthquakes (Intensity > IX) have occurred in the region, as shown by the historical and instrumental earthquake catalogues. The location and characterization of the active structures in the Alboran Sea trough sub-aqueous paleoseismological studies is, therefore, essential to significantly improve the knowledge about earthquake and tsunami hazards along the coasts of Spain and North Africa. During the EVENT-DEEP (May-June 2010) and TOPOMED (September-October 2011) cruises the geomorphology and geometry of some active strike-slip faults identified in the Eastern Alboran Sea were investigated. Along the dextral strike slip Yussuf fault system medium (22) and high (7) penetration multichannel seismic profiles (MCS) and parametric sub-bottom profiler lines were acquired. At the same time, a high resolution bathymetric coverage of the area was collected by means of multibeam echosounder. The energy source used during the EVENT-DEEP cruise to acquire the medium penetration MCS was an 800 c.i. volume airgun array, working at 2000 psi pressure, towed at 2 m depth and triggered every 18 m. The receiver consisted on a 600 m long streamer, composed by 96 channels separated 6.25 m. This configuration allowed us to penetrate an average depth of 1.5 s TWTT. In order to study the deeper part of the margin, the source of the high penetration MCS used during the TOPOMED-GASSIS cruise was an airgun array of 3160 c.i., towed at 7.5 m and triggered every 50 m at 2000 psi of pressure. The receiver consisted on a 5379 m long streamer, composed by 431 channels separated 12.5 m. The average penetration of the seismic profiles reached with this configuration was 4 s TWTT. The preliminary analysis of the acquired data allow us to describe some characteristics of the Yussuf fault. First of all, this is a fault system composed by several strike-slip faults fairly vertical and with WNW-ESE direction. Moreover, the dextral strike-slip movement along these faults has produced the formation of a pull-apart basin. Finally, we have observed some of the faults reaching the seafloor surface and in some places offsetting it, implying the Quaternary activity along the fault system. Much analyses is needed in order to accurately determine the fault seismic parameters of the Yussuf fault system (e.g. geometry, slip rate, maximum event magnitude, recurrence interval and time elapsed since the last earthquake, maximum earthquake they can produce) and to investigate how it accommodates the present-day strain along the plate convergence.Peer Reviewe
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