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121.
Summary Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences. It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histonie H4, regardless whether induced or uninduced cells were analysed. The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin. 相似文献
122.
Sajad Sofi Julia C. Fitzgerald Désirée Jähn Bernhard Dumoulin Sabine Stehling Hartmut Kuhn Christoph Ufer 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(4):866-876
The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling. 相似文献
123.
Martini M Gnann A Scheikl D Holzmann B Janssen KP 《The international journal of biochemistry & cell biology》2011,43(11):1630-1640
SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell–matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells. 相似文献
124.
The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific
binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions −578 to −501 relative to the translational start
of the first open reading frame of the operon. This region includes stretches of homology to E. coliσ
70 promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide
gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact
with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and
RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28° C, relative to expression at 37° C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora.
Received: 26 February 1997 / Accepted: 23 May 1997 相似文献
125.
l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy
sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases,
including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded
by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological
important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization
of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose. 相似文献
126.
Bernhard Benkel Sonja Kollers Ruedi Fries Alexei Sazanov Erin Yoshida Edith Valle Jon Davoren Donal Hickey 《Mammalian genome》2005,16(3):194-200
AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits. 相似文献
127.
128.
Large-scale candidate gene analysis of HDL particle features 总被引:1,自引:0,他引:1
Kaess BM Tomaszewski M Braund PS Stark K Rafelt S Fischer M Hardwick R Nelson CP Debiec R Huber F Kremer W Kalbitzer HR Rose LM Chasman DI Hopewell J Clarke R Burton PR Tobin MD Hengstenberg C Samani NJ 《PloS one》2011,6(1):e14529
Background
HDL cholesterol (HDL-C) is an established marker of cardiovascular risk with significant genetic determination. However, HDL particles are not homogenous, and refined HDL phenotyping may improve insight into regulation of HDL metabolism. We therefore assessed HDL particles by NMR spectroscopy and conducted a large-scale candidate gene association analysis.Methodology/Principal Findings
We measured plasma HDL-C and determined mean HDL particle size and particle number by NMR spectroscopy in 2024 individuals from 512 British Caucasian families. Genotypes were 49,094 SNPs in >2,100 cardiometabolic candidate genes/loci as represented on the HumanCVD BeadChip version 2. False discovery rates (FDR) were calculated to account for multiple testing. Analyses on classical HDL-C revealed significant associations (FDR<0.05) only for CETP (cholesteryl ester transfer protein; lead SNP rs3764261: p = 5.6*10−15) and SGCD (sarcoglycan delta; rs6877118: p = 8.6*10−6). In contrast, analysis with HDL mean particle size yielded additional associations in LIPC (hepatic lipase; rs261332: p = 6.1*10−9), PLTP (phospholipid transfer protein, rs4810479: p = 1.7*10−8) and FBLN5 (fibulin-5; rs2246416: p = 6.2*10−6). The associations of SGCD and Fibulin-5 with HDL particle size could not be replicated in PROCARDIS (n = 3,078) and/or the Women''s Genome Health Study (n = 23,170).Conclusions
We show that refined HDL phenotyping by NMR spectroscopy can detect known genes of HDL metabolism better than analyses on HDL-C. 相似文献129.
Loll B Rückert C Hee CS Saenger W Uchanska-Ziegler B Ziegler A 《Protein science : a publication of the Protein Society》2011,20(2):278-290
The human major histocompatibility complex class I antigen HLA‐B*2705 binds several sequence‐related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross‐reactivity of cytotoxic T cells (CTL) against these HLA‐B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging‐in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross‐reactivity of these CTL with a peptide derived from voltage‐dependent calcium channel α1 subunit (pCAC, SRRWRRWNR), in which the C‐terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA‐B*2705:pCAC complex by X‐ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence‐related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F‐pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA‐B*2705 heavy chain near the N‐terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg‐Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR‐directed, HLA‐B*2705‐restricted CTL to cross‐react with HLA‐B*2705:pCAC complexes. 相似文献
130.
Rath P Demange P Saurel O Tropis M Daffé M Dötsch V Ghazi A Bernhard F Milon A 《The Journal of biological chemistry》2011,286(37):32525-32532
PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. (15)N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties. 相似文献