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排序方式: 共有302条查询结果,搜索用时 31 毫秒
31.
Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism. 相似文献
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In order to purify milligram quantities of turkey beta-adrenergic receptor (betaAR) for structural analysis, we have expressed mutant betaARs using the baculovirus system. The initial betaAR construct was truncated at both N- and C-termini thus removing an N-glycosylation site. Cys 116 was mutated to leucine and a histidine tag was added at the C-terminus resulting in the betaAR construct 20-424/His6. Expression of this construct in Sf9 cells produced 0.5 mg of unpurified receptor per liter of culture which necessitated the use of a fermenter for large-scale production. The yield was improved more than 2-fold to 1.2 mg/l culture by using Tni cells which facilitated the production of receptor on a 4 litre scale in shake cultures. The receptor was purified to homogeneity with 35% recovery giving a yield of 2 mg receptor. A further deletion at the N-terminus (betaAR 34-424/His6) eliminated proteolysis which had been observed with the original construct and also increased expression more than 5-fold to 360 pmol/mg solubilized membrane protein. This expression level is one of the highest reported for a G protein-coupled receptor (GPCR) and has enabled us to purify 10 mg betaAR for large-scale crystallization experiments. 相似文献
33.
The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant. 相似文献
34.
Transformation of Acinetobacter sp. Strain BD413 by Transgenic Sugar Beet DNA 总被引:3,自引:0,他引:3 下载免费PDF全文
The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate transgenic plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA. 相似文献
35.
Gille C Spring B Bernhard W Gebhard C Basile D Lauber K Poets CF Orlikowsky TW 《Journal of lipid research》2007,48(2):307-317
Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and alphaCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf(R)), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissaminetrade mark-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited alphaCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions. 相似文献
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Taylor J Jensen Sung K Kim Zhanyang Zhu Christine Chin Claudia Gebhard Tim Lu Cosmin Deciu Dirk van den Boom Mathias Ehrich 《Genome biology》2015,16(1)
BackgroundCirculating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.ResultsWe perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures.ConclusionsWe generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0645-x) contains supplementary material, which is available to authorized users. 相似文献38.
Palmfeldt J Vang S Stenbroen V Pavlou E Baycheva M Buchal G Monavari AA Augoustides-Savvopoulou P Mandel H Gregersen N 《Journal of proteome research》2011,10(5):2389-2396
Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide. 相似文献
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Josefsson EC Gebhard HH Stossel TP Hartwig JH Hoffmeister KM 《The Journal of biological chemistry》2005,280(18):18025-18032
alpha(M)beta(2) integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands. Ligand binding occurs in the alpha(M) chain, which is composed of an I domain and a lectin domain. The alpha(M) I domain binds iC3b, fibrinogen, intercellular adhesion molecule-1, and other ligands and mediates the adhesion of neutrophils to platelet glycoprotein Ibalpha (GPIbalpha). alpha(M)beta(2) also recognizes beta-GlcNAc residues on GPIbalpha that are clustered on platelets after cooling. The phagocytosis of chilled platelets could be reconstituted when Chinese hamster ovary cells were transfected with alpha(M)beta(2). Replacement of the I domain or the lectin domain of the alpha(M) chain with the corresponding domain from the alpha(X) chain (p150) revealed that the activity of the alpha(M)beta(2) integrin toward chilled platelets resides within the lectin domain and does not require the I domain. Additional evidences for this conclusion are: 1) Sf9 cells expressing solely the alpha(M) lectin domain bound chilled platelets, and 2) soluble recombinant alpha(M) lectin domain inhibited the phagocytosis of chilled platelets by alpha(M)beta(2)-expressing THP-1 cells, whereas I domain substrates showed no inhibitory effect. Therefore chilled platelets are removed from blood by an interaction between beta-GlcNAc residues on clustered GPIbalpha and the lectin domain of alpha(M) chain of the alpha(M)beta(2) integrin, distinguishing this interaction from those mediated by the alpha(M) I domain. 相似文献