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Pigott CR Mikolajek H Moore CE Finn SJ Phippen CW Werner JM Proud CG 《The Biochemical journal》2012,442(1):105-118
eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K. 相似文献
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Blaney Davidson EN Scharstuhl A Vitters EL van der Kraan PM van den Berg WB 《Arthritis research & therapy》2005,7(6):R1338-R1347
Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance
in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and
transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we
observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and
its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional
consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to
IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1,
-2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined
in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days
Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (35S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression
decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered,
the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked
the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis
and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished
level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic
absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection.
In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in
old mice might be at the root of OA development. 相似文献
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W. B. Phippen S. Kresovich F. G. Candelas J. R. McFerson 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(2):227-234
To better characterize and conserve crop genetic resources, the assessment of genetic identity, relatedness, and structure
among entries and collections becomes a priority. In the present study, a random amplified polymorphic DNA (RAPD) assay was
applied as a quick, cost-effective, and preliminary screen to quantify and partition the molecular variation among accessions.
Fourteen phenotypically uniform accessions of Brassica oleracea var. capitata L. (cabbage) similarly designated as `Golden Acre' were tested with nine decamer oligonucleotide primers. These amplifications
generated 110 fragments, of which 80 were polymorphic ranging in size from 370 to 1720 bp. The 80 polymorphic fragments were
sufficient to distinguish between all 14 accessions. Data based on the partitioning of variation among accessions indicated
that `Golden Acre' entries could be reduced to as few as four groups, with the potential loss of variation being only 4.6%
of the absolute current genetic variation in those holdings as estimated from RAPD analysis. This proposed grouping would
concurrently save approximately 70% [$750–1000 (US) per accession] for each cycle of regeneration (approximately 20–25 years
at most) which alternatively could then be used for other priorities in B. oleracea conservation and use. This case represents but one example where targeted use of a molecular-marker assay linked with rigorous
statistical analysis will be useful for plant genebank management, particularly for questions at the intraspecific level.
Molecular markers will provide genebank curators with additional sources of information to better plan and organize collection
holdings and use finite financial support in a more effective manner.
Received: 10 June 1996 / Accepted: 23 August 1996 相似文献
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R Simões WB Feitosa CM Mendes AC Nicacio FRO de Barros 《Biotechnic & histochemistry》2013,88(3):79-83
Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A3 (CMA3) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA3 staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA3-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA3-positive sperm cells compared to the others. CMA3 is a simple and useful tool for detecting sperm protamine deficiency in bulls. 相似文献
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