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31.
Dr. Jan Hansmann Florian Groeber Alexander Kahlig Claudia Kleinhans Heike Walles 《Biotechnology journal》2013,8(3):298-307
Bioreactor technology is vital for tissue engineering. Usually, bioreactors are used to provide a tissue-specific physiological in vitro environment during tissue maturation. In addition to this most obvious application, bioreactors have the potential to improve the efficiency of the overall tissue-engineering concept. To date, a variety of bioreactor systems for tissue-specific applications have been developed. Of these, some systems are already commercially available. With bioreactor technology, various functional tissues of different types were generated and cultured in vitro. Nevertheless, these efforts and achievements alone have not yet led to many clinically successful tissue-engineered implants. We review possible applications for bioreactor systems within a tissue-engineering process and present basic principles and requirements for bioreactor development. Moreover, the use of bioreactor systems for the expansion of clinically relevant cell types is addressed. In contrast to cell expansion, for the generation of functional three-dimensional tissue equivalents, additional physical cues must be provided. Therefore, bioreactors for musculoskeletal tissue engineering are discussed. Finally, bioreactor technology is reviewed in the context of commercial constraints. 相似文献
32.
Marion Haensler Heike Schuerer Ulrich Hahn Hans-Dieter Jakubke 《Nucleosides, nucleotides & nucleic acids》2013,32(7):1267-1274
Abstract Synthesis of guanylyl(3′→5′)cytidine catalysed by RNase T1 variants (Tyr42Trp, Tyr24Trp and GluSSAla) was studied in frozen aqueous systems at-10°C and in solution at 0°C. Freezing the reaction mixture resulted in significantly enhanced dinucleoside monophosphate yields independently of the effect of mutation on substrate binding and catalytic mechanism. We assume that the protonation state of the catalytic residues is influenced by freezing, possibly due to conformational changes of the enzyme proteins. 相似文献
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The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. 相似文献
35.
Liewen H Meinhold-Heerlein I Oliveira V Schwarzenbacher R Luo G Wadle A Jung M Pfreundschuh M Stenner-Liewen F 《Experimental cell research》2005,306(1):24-34
The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells. 相似文献
36.
Blad H Reiter NJ Abildgaard F Markley JL Butcher SE 《Journal of molecular biology》2005,353(3):540-555
The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 micros. The U80 nucleotide displays low internal mobility on the picosecond time-scale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K(d) values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13C nuclei. Addition of Mg2+ shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK(a) of A79. 相似文献
37.
The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein 下载免费PDF全文
U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome. 相似文献
38.
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
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Carrai M Steinke V Vodicka P Pardini B Rahner N Holinski-Feder E Morak M Schackert HK Görgens H Stemmler S Betz B Kloor M Engel C Büttner R Naccarati A Vodickova L Novotny J Stein A Hemminki K Propping P Försti A Canzian F Barale R Campa D 《PloS one》2011,6(6):e20464
Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; Pvalue = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; Pvalue = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; Pvalue = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin. 相似文献