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141.
Multiple GTPases participate in the assembly of the large ribosomal subunit in Bacillus subtilis 下载免费PDF全文
Schaefer L Uicker WC Wicker-Planquart C Foucher AE Jault JM Britton RA 《Journal of bacteriology》2006,188(23):8252-8258
GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases. 相似文献
142.
143.
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
144.
Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling 总被引:8,自引:0,他引:8
Melendez J Welch S Schaefer E Moravec CS Avraham S Avraham H Sussman MA 《The Journal of biological chemistry》2002,277(47):45203-45210
Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility. 相似文献
145.
Diego Santiago‐Alarcon Raeann Mettler Gernot Segelbacher H. Martin Schaefer 《Journal of avian biology》2013,44(6):521-530
Parasites can exert strong selection on hosts. Throughout the year migrants are exposed to different sets of parasites, which may affect life history traits such as migratory schedules. Here, we studied the relationship between parasite infection and arrival date of blackcaps Sylvia atricapilla to their breeding grounds in Germany throughout a period of six years (2007–2012). We used two data sets, one that included all blackcaps and one that included only recaptured birds. We assesed whether parasites influence spring arrival to breeding grounds, and for the recaptured data set, we analysed temporal variation in parasitism (i.e. infection status and parasitaemia) throughout the breeding season. We used both microscopy and PCR (a fragment of ? 479 bp of the mtDNA cyt b) to determine haemosporidian infection. Blackcaps were mostly infected with Haemoproteus parabelopolskyi (lineages SYAT01 and SYAT02). Infection status, but not parasitaemia, was constant through time for individual birds; meaning that once a bird is infected, it most likely will retain the infection for life. We found that infection by haemosporidian parasites has no relationship to arrival date in this blackcap population; however, infection by H. parabelopolskyi has a marginally significant effect on arrival date of recaptured blackcaps, somewhat delaying their arrival to breeding grounds. Birds captured later in the season were more likely to be infected than those from early spring, and parasitaemia was frequently lower in birds captured earlier in the season compared to those captured later (summer). 相似文献
146.
The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP3) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4−/− mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle. 相似文献
147.
Efficient gene targeting in the moss Physcomitrella patens 总被引:16,自引:2,他引:16
Didier G. Schaefer Jean-Pierre Zrÿd 《The Plant journal : for cell and molecular biology》1997,11(6):1195-1206
The moss Physcomitrella patens is used as a genetic model system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. Transformation experiments designed to target three single-copy genomic loci were performed to determine the efficiency of gene targeting in this plant. Mean transformation rates were 10-fold higher with the targeting vectors and molecular evidence for the integration of exogenous DNA into each targeted locus by homologous recombination is provided. The efficiency of gene targeting determined in these experiments is above 90%, which is in the range of that observed in yeast and several orders of magnitude higher than previous reports of gene targeting in plants. Thus, gene knock-out and allele replacement approaches are directly accessible to study plant development in the moss Physcomitrella patens . Moreover, efficient gene targeting has so far only been observed in lower eukaryotes such as protozoa, yeasts and filamentous fungi, and, as shown here the first example from the plant kingdom is a haplobiontic moss. This suggests a possible correlation between efficient gene targeting and haplo-phase in eukaryotes. 相似文献
148.
Juan F Masello Petra Quillfeldt Gopi K Munimanda Nadine Klauke Gernot Segelbacher H Martin Schaefer Mauricio Failla Maritza Cortés Yoshan Moodley 《Frontiers in zoology》2011,8(1):1-17
Background
While the gene flow in some organisms is strongly affected by physical barriers and geographical distance, other highly mobile species are able to overcome such constraints. In southern South America, the Andes (here up to 6,900 m) may constitute a formidable barrier to dispersal. In addition, this region was affected by cycles of intercalating arid/moist periods during the Upper/Late Pleistocene and Holocene. These factors may have been crucial in driving the phylogeographic structure of the vertebrate fauna of the region. Here we test these hypotheses in the burrowing parrot Cyanoliseus patagonus (Aves, Psittaciformes) across its wide distributional range in Chile and Argentina.Results
Our data show a Chilean origin for this species, with a single migration event across the Andes during the Upper/Late Pleistocene, which gave rise to all extant Argentinean mitochondrial lineages. Analyses suggest a complex population structure for burrowing parrots in Argentina, which includes a hybrid zone that has remained stable for several thousand years. Within this zone, introgression by expanding haplotypes has resulted in the evolution of an intermediate phenotype. Multivariate regressions show that present day climatic variables have a strong influence on the distribution of genetic heterogeneity, accounting for almost half of the variation in the data.Conclusions
Here we show how huge barriers like the Andes and the regional environmental conditions imposed constraints on the ability of a parrot species to colonise new habitats, affecting the way in which populations diverged and thus, genetic structure. When contact between divergent populations was re-established, a stable hybrid zone was formed, functioning as a channel for genetic exchange between populations. 相似文献149.
Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca2+‐sensitive protein kinase C α fusion protein are demonstrated. 相似文献
150.
Sastre AP Grossmann S Reusch HP Schaefer M 《The Journal of biological chemistry》2008,283(38):25871-25878
The expression of contractile proteins in vascular smooth muscle cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific alpha-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of ERK1/2. Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerization of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be critical intermediates. Analysis of the expression of protease-activated receptor 1, heparin-binding EGF (HB-EGF), and the EGF receptor revealed that pro-HB-EGF is significantly up-regulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Because inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The small interfering RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a yellow fluorescent protein-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signaling strength within the Ras/Raf/MEK/ERK cascade. 相似文献