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121.
A first approach to discover new antimalarials has been recently performed in a combined approach with data from GlaxoSmithKline Tres Cantos Antimalarial Set, Novartis-GNF Malaria Box Data set and St. Jude Children’s Research Hospital. These data are assembled in the Malaria Box. In a first phenotypic forward chemical genetic approach, 400 chemicals were employed to eradicate the parasite in the erythrocytic stages. The advantage of phenotypic screens for the identification of novel chemotypes is that no a priori assumptions are made concerning a fixed target and that active compounds inherently have cellular bioavailability. In a first screen 40 mostly heterocyclic, highly active compounds (in nmol range of growth inhibition) were identified with EC50 values ≤2 μM against chloroquine-resistant Plasmodium falciparum strains and a therapeutic window ≥10 against two mammalian cell lines. 78 % of the compounds had no violations with the Lipinski Rule of 5 and only 1 % of the compounds showed cytotoxicity when applied at concentrations of 10 μM. This pre-selective step of parasitic eradication will be used further for a test of the Malaria Box with a potential in iron chelating capacity to inhibit deoxyhypusine hydroxylase (DOHH) from P. falciparum and vivax. DOHH, a metalloprotein which consists of ferrous iron and catalyzes the second step of the posttranslational modification at a specific lysine in eukaryotic initiation factor 5A (EIF-5A) to hypusine. Hypusine is a novel, non-proteinogenic amino acid, which is essential in eukaryotes and for parasitic proliferation. DOHH seems to be a “druggable” target, since it has only 26 % amino acid identity to its human orthologue. For a High-throughput Screening (HTS) of DOOH inhibitors, rapid and robust analytical tools are a prerequisite. A proteomic platform for the detection of hypusine metabolites is currently established. Ultra performance Liquid Chromatography enables the detection of hypusine metabolites with retention times of 7.4 min for deoxyhypusine and 7.3 min for hypusine. Alternatively, the analytes can be detected by their masses with gas chromatography/mass spectrometry or one-dimensional chromatography coupled to mass spectrometry. Moreover, the identified hits will be tracked further to test their efficacy in novel “in vitro assays”. Subsequently in vivo inhibition in a humanized mouse model will be tested. 相似文献
122.
Jens T. Kaiser Douglas C. Rees 《Protein science : a publication of the Protein Society》2013,22(4):502-509
The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582–1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179–1183). Both structures used protein solubilized in the detergent fos‐choline‐14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent β‐dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, β‐dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos‐choline‐14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent, crystallization conditions, and protein sequence. 相似文献
123.
Remco Stam Julietta Jupe Andrew J. M. Howden Jenny A. Morris Petra C. Boevink Pete E. Hedley Edgar Huitema 《PloS one》2013,8(3)
Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions. 相似文献
124.
Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation
Jue Wang Lisa Wagner-Britz Anna Bogdanova Sandra Ruppenthal Kathrina Wiesen Elisabeth Kaiser Qinghai Tian Elmar Krause Ingolf Bernhardt Peter Lipp Stephan E. Philipp Lars Kaestner 《PloS one》2013,8(6)
Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca2+ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca2+ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca2+ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca2+ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca2+ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca2+ influx and the associated pro-thrombotic activity. 相似文献
125.
126.
Potential impacts on ecosystem services of land use transitions to second‐generation bioenergy crops in GB
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Suzanne Milner Robert A. Holland Andrew Lovett Gilla Sunnenberg Astley Hastings Pete Smith Shifeng Wang Gail Taylor 《Global Change Biology Bioenergy》2016,8(2):317-333
We present the first assessment of the impact of land use change (LUC) to second‐generation (2G) bioenergy crops on ecosystem services (ES) resolved spatially for Great Britain (GB). A systematic approach was used to assess available evidence on the impacts of LUC from arable, semi‐improved grassland or woodland/forest, to 2G bioenergy crops, for which a quantitative ‘threat matrix’ was developed. The threat matrix was used to estimate potential impacts of transitions to either Miscanthus, short‐rotation coppice (SRC, willow and poplar) or short‐rotation forestry (SRF). The ES effects were found to be largely dependent on previous land uses rather than the choice of 2G crop when assessing the technical potential of available biomass with a transition from arable crops resulting in the most positive effect on ES. Combining these data with constraint masks and available land for SRC and Miscanthus (SRF omitted from this stage due to lack of data), south‐west and north‐west England were identified as areas where Miscanthus and SRC could be grown, respectively, with favourable combinations of economic viability, carbon sequestration, high yield and positive ES benefits. This study also suggests that not all prospective planting of Miscanthus and SRC can be allocated to agricultural land class (ALC) ALC 3 and ALC 4 and suitable areas of ALC 5 are only minimally available. Beneficial impacts were found on 146 583 and 71 890 ha when planting Miscanthus or SRC, respectively, under baseline planting conditions rising to 293 247 and 91 318 ha, respectively, under 2020 planting scenarios. The results provide an insight into the interplay between land availability, original land uses, bioenergy crop type and yield in determining overall positive or negative impacts of bioenergy cropping on ecosystems services and go some way towards developing a framework for quantifying wider ES impacts of this important LUC. 相似文献
127.
A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease
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J Carlos Villaescusa Bingsi Li Enrique M Toledo Pia Rivetti di Val Cervo Shanzheng Yang Simon RW Stott Karol Kaiser Saiful Islam Daniel Gyllborg Rocio Laguna‐Goya Michael Landreh Peter Lönnerberg Anna Falk Tomas Bergman Roger A Barker Sten Linnarsson Licia Selleri Ernest Arenas 《The EMBO journal》2016,35(18):1963-1978
128.
Melanie Kaiser Anja Penk Heike Franke Ute Krügel Wolfgang Nörenberg Daniel Huster Michael Schaefer 《Purinergic signalling》2016,12(3):453-463
Effective therapeutic measures against the development of brain edema, a life-threatening complication of cerebral ischemia, are necessary to improve the functional outcome for the patient. Here, we identified a beneficial role of purinergic receptor P2X7 activation in acute ischemic stroke. Involvement of P2X7 in the development of neurological deficits, infarct size, brain edema, and glial responses after ischemic cerebral infarction has been analyzed. Neurologic evaluation, magnetic resonance imaging, and immunofluorescence assays were used to characterize the receptor’s effect on the disease progress during 72 h after transient middle cerebral artery occlusion (tMCAO). Sham-operated animals were included in all experiments for control purposes. We found P2X7-deficient mice to develop a more prominent brain edema with a trend towards more severe neurological deficits 24 h after tMCAO. Infarct sizes, T2 times, and apparent diffusion coefficients did not differ significantly between wild-type and P2X7?/? animals. Our results show a characteristic spatial distribution of reactive glia cells with strongly attenuated microglia activation in P2X7?/? mice 72 h after tMCAO. Our data indicate that P2X7 exerts a role in limiting the early edema formation, possibly by modulating glial responses, and supports later microglia activation. 相似文献
129.
130.
Menthol is known for its analgesic properties, but relatively little information is available on its potential as an anaesthetic on fish. The purpose of this study was to assess anaesthetic and sedative effectiveness of menthol and its safety in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout 180 ± 28 g (mean ± SD) within a range of 152–208 g fish?1 were individually exposed to menthol concentrations between 10 and 150 mg l?1 and observed for behavioural responses, induction time to anaesthesia and recovery time. Menthol concentrations of 40–150 mg l?1 induced anaesthesia with varying exposure times. There was an exponential relationship (p < 0.001) between induction time and menthol concentration. Menthol concentrations of 80–150 mg l?1 induced anaesthesia within three minutes of exposure and fish recovered within three minutes. Induction and recovery data showed that 80 mg l?1 was most suitable for anaesthesia in juveniles of this species. Concentrations of 10–20 mg l?1 had sedative effects. Menthol stock solutions prepared using ethanol and acetone and storage time of stock solutions at room temperature for up to 48 h showed no significant differences in anaesthetic efficiency. When exposure time to menthol was kept constant at three minutes, 22% of fish had temporary cessation of gill ventilation. These fish had longer recovery times than those that did not show that response. Menthol was an effective anaesthetic and could be tested as a sedative for trout. 相似文献