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排序方式: 共有124条查询结果,搜索用时 31 毫秒
51.
Volker Patzel Ralf Kronenwett Karola Rittner Georg Sczakiel 《Nucleosides, nucleotides & nucleic acids》2013,32(5-6):711-715
Abstract Fast annealing of complementary RNA in vitro is related to effective antisense RNA-mediated regulation of gene expression and inhibition of viral replication in living cells. Pools of antisense RNA can be selected for species that anneal fast with a given target strand. In this study, we compare the technical and biological advantages and disadvantages of a single round selection assay with extensive multiple cycle selection for fast-annealing antisense RNA species. 相似文献
52.
Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus oriP Sequences and EBNA1: Implications of the Nuclear Import of Plasmids 总被引:6,自引:1,他引:5
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Franoise Lngle-Rouault Volker Patzel Annie Benavente Martine Taillez Nathalie Silvestre Albine Bompard Georg Sczakiel Eric Jacobs Karola Rittner 《Journal of virology》1998,72(7):6181-6185
A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect. 相似文献
53.
Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica
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The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen. 相似文献
54.
Phenylphosphate synthase: a new phosphotransferase catalyzing the first step in anaerobic phenol metabolism in Thauera aromatica
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Schmeling S Narmandakh A Schmitt O Gad'on N Schühle K Fuchs G 《Journal of bacteriology》2004,186(23):8044-8057
The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were investigated. Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [(14)C]phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase. Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H(2)O-->phenylphosphate + MgAMP + orthophosphate. The phosphoryl group in phenylphosphate is derived from the beta-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (K(m), 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-beta-synthase domains of unknown function but does not show significant overall similarity to known proteins. The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications. 相似文献
55.
K Vorauer-Uhl M Skias A Trkola W Sch?nhofer F Unterluggauer P Schulz A Jungbauer 《Bioseparation》1992,3(4):217-226
Flocculation using pellicular charged flocculants was investigated as an alternative process to conventional chromatography in purification of recombinant proteins using human recombinant superoxide dismutase expressed in E. coli as a model system. The removal of pyrogens, proteins, debris and the yield were determined. At laboratory scale, the starting conditions were optimized to yield a stable solution and the flocculation process fitted into a purification scheme. 100 L fermentation broth was the initial volume at pilot scale. The process parameters determined at the laboratory scale were tested and the results were compared to the pilot scale. The method was also compared to an ion-exchange chromatography. 相似文献
56.
Renate Kunert Willibald Steinfellner Friedrich Altmann Jakob Wallner Hermann Katinger Karola Vorauer-Uhl 《Applied microbiology and biotechnology》2009,84(4):693-699
Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially
available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology,
50(1–3):49–56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible
in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised
products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported
in the literature (Gu et al. in Biotech Bioeng 56 (4):353–341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO
cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease
activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134–150.
In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested
molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH
to be an ideal candidate for media and component screening in mammalian cell culture. 相似文献
57.
Christina Theisen Susanne Fuchs-Winkelmann Karola Knappstein Turgay Efe Jan Schmitt Juergen RJ Paletta Markus D Schofer 《Biomedical engineering online》2010,9(1):9
Background
Rotator cuff tears are a common and frequent lesion especially in older patients. The mechanisms of tendon repair are not fully understood. Common therapy options for tendon repair include mini-open or arthroscopic surgery. The use of growth factors in experimental studies is mentioned in the literature. Nanofiber scaffolds, which provide several criteria for the healing process, might be a suitable therapy option for operative treatment. The aim of this study was to explore the effects of nanofiber scaffolds on human tendon derived fibroblasts (TDF's), as well as the gene expression and matrix deposition of these fibroblasts. 相似文献58.
Stotz K 《History and philosophy of the life sciences》2006,28(4):533-548
59.
60.
Filip Mundt Gustav Nilsonne Serta? Arslan Karola Csür?s Gunnar Hillerdal Huseyin Yildirim Muzaffer Metintas Katalin Dobra Anders Hjerpe 《PloS one》2013,8(8)