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61.
Marcin Wolny Melanie Colegrave Lucy Colman Ed White Peter J. Knight Michelle Peckham 《The Journal of biological chemistry》2013,288(44):31952-31962
It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301–1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy. 相似文献
62.
Shivani Ahuja Nicole Jahr Sang-Choul Im Subramanian Vivekanandan Nataliya Popovych Stéphanie V. Le Clair Rui Huang Ronald Soong Jiadi Xu Kazutoshi Yamamoto Ravi P. Nanga Angela Bridges Lucy Waskell Ayyalusamy Ramamoorthy 《The Journal of biological chemistry》2013,288(30):22080-22095
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. 相似文献
63.
Seraphine N. Esemu Willington O. Besong Roland N. Ndip Lucy M. Ndip 《Experimental & applied acarology》2013,59(3):377-387
Ehrlichia ruminantium, the etiologic agent of the economically important disease heartwater, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, particularly A. hebraeum and A. variegatum. Although serologic and microscopic evidence of the presence of heartwater have been reported in ruminants in Cameroon, knowledge of E. ruminantium infection in the tick vector, A. variegatum, is lacking. In order to determine the infectivity of A. variegatum ticks by E. ruminantium, we analysed 500 un-engorged A. variegatum ticks collected by hand-picking from predilection sites from 182 cattle [115 ticks from 82 cattle at Société de Développement et d’Exploitation des Productions Animales (SODEPA) Dumbo ranch (SDR) and 385 ticks from 100 cattle at the Upper Farms ranch (UFR)] by amplification of the open reading frame (ORF) 2 of the pCS20 region of E. ruminantium. PCR amplification of the 279 bp fragment of the pCS20 region detected E. ruminantium DNA in 142 (28.4 %) of the 500 ticks with a higher infection rate (47/115; 40.9 %) observed in ticks from SDR and 24.7 % (95/385) of ticks collected from cattle at UFR. Twenty five randomly selected PCR products were sequenced and results indicated that some of the isolates shared homology with one another and to sequences of E. ruminantium in the GenBank. This report represents the first molecular evidence of E. ruminantium infection in A. variegatum ticks in Cameroon and suggests possible exposure of cattle to this pathogen in our environment. 相似文献
64.
65.
Kate J. Howell Lucy A. Weinert Shi-Lu Luan Sarah E. Peters Roy R. Chaudhuri David Harris ?ystein Angen Virginia Aragon Julian Parkhill Paul R. Langford Andrew N. Rycroft Brendan W. Wren Alexander W. Tucker Duncan J. Maskell 《Journal of bacteriology》2013,195(18):4264-4273
Haemophilus parasuis is the causative agent of Glässer''s disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer''s disease. 相似文献
66.
David Fowler Mhairi Coyle Ute Skiba Mark A. Sutton J. Neil Cape Stefan Reis Lucy J. Sheppard Alan Jenkins Bruna Grizzetti James N. Galloway Peter Vitousek Allison Leach Alexander F. Bouwman Klaus Butterbach-Bahl Frank Dentener David Stevenson Marcus Amann Maren Voss 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2013,368(1621)
Global nitrogen fixation contributes 413 Tg of reactive nitrogen (Nr) to terrestrial and marine ecosystems annually of which anthropogenic activities are responsible for half, 210 Tg N. The majority of the transformations of anthropogenic Nr are on land (240 Tg N yr−1) within soils and vegetation where reduced Nr contributes most of the input through the use of fertilizer nitrogen in agriculture. Leakages from the use of fertilizer Nr contribute to nitrate (NO3−) in drainage waters from agricultural land and emissions of trace Nr compounds to the atmosphere. Emissions, mainly of ammonia (NH3) from land together with combustion related emissions of nitrogen oxides (NOx), contribute 100 Tg N yr−1 to the atmosphere, which are transported between countries and processed within the atmosphere, generating secondary pollutants, including ozone and other photochemical oxidants and aerosols, especially ammonium nitrate (NH4NO3) and ammonium sulfate (NH4)2SO4. Leaching and riverine transport of NO3 contribute 40–70 Tg N yr−1 to coastal waters and the open ocean, which together with the 30 Tg input to oceans from atmospheric deposition combine with marine biological nitrogen fixation (140 Tg N yr−1) to double the ocean processing of Nr. Some of the marine Nr is buried in sediments, the remainder being denitrified back to the atmosphere as N2 or N2O. The marine processing is of a similar magnitude to that in terrestrial soils and vegetation, but has a larger fraction of natural origin. The lifetime of Nr in the atmosphere, with the exception of N2O, is only a few weeks, while in terrestrial ecosystems, with the exception of peatlands (where it can be 102–103 years), the lifetime is a few decades. In the ocean, the lifetime of Nr is less well known but seems to be longer than in terrestrial ecosystems and may represent an important long-term source of N2O that will respond very slowly to control measures on the sources of Nr from which it is produced. 相似文献
67.
68.
Terry A. Gray Euan Murray Matthew W. Nowicki Lucy Remnant Alexander Scherl Petr Muller Borek Vojtesek Ted R. Hupp 《Protein science : a publication of the Protein Society》2013,22(9):1266-1278
Many regulatory proteins are homo‐oligomeric and designing assays that measure self‐assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer‐associated oncoprotein AGR2. A two site‐sandwich microtiter assay (2SMTA) was designed using a DyLight800‐labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self‐peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N‐terminal region of AGR2 increase in trans oligomer stability as defined using the 2SMTA assay. A DSS‐crosslinking assay that traps the AGR2 dimer through K95‐K95 adducts confirmed that Δ45‐AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt‐AGR2, Δ45‐AGR2 (more stable dimer), and monomeric AGR2E60A revealed that Δ45‐AGR2 was more active in binding to Reptin than either wt‐AGR2 or the AGR2E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential. 相似文献
69.
Susan M. Gribble Frances K. Wiseman Stephen Clayton Elena Prigmore Elizabeth Langley Fengtang Yang Sean Maguire Beiyuan Fu Diana Rajan Olivia Sheppard Carol Scott Heidi Hauser Philip J. Stephens Lucy A. Stebbings Bee Ling Ng Tomas Fitzgerald Michael A. Quail Ruby Banerjee Kai Rothkamm Victor L. J. Tybulewicz Elizabeth M. C. Fisher Nigel P. Carter 《PloS one》2013,8(4)
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439. 相似文献
70.
Xuehui Yang Yan Gong Yuefeng Tang Hongfang Li Qing He Lindsey Gower Lucy Liaw Robert E. Friesel 《PloS one》2013,8(3)