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Gunther Ott Lubos Arnold Jiri Smrt Michal Sobkowski Stefan Limmer Hans-Peter Hofmann 《Nucleosides, nucleotides & nucleic acids》2013,32(5):1069-1085
Abstract Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations. 相似文献
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Vogtherr M Kirchner R Zuleeg T Limmer S 《Journal of biomolecular structure & dynamics》1999,17(2):355-365
A simple system derived from the acceptor stem of tRNA(Ala) is presented which undergoes a pH-dependent dimerization. This is brought about by formation of C+-G-C base triples of the pyrimidine motif type between protonated cytidines of the 3' single-stranded end and regularly paired G-C pairs in the double-helical stem. In addition, an unusual interaction between a protonated adenine and a regular G-C pair is suggested. The equilibrium between monomer and dimer forms can be monitored via NMR spectroscopy and UV melting curve analysis. A dimerization enthalpy of 159 kJ mol(-1) was found at pH 5.0. The system could serve as a model for inter- and intra-molecular association, respectively, of single-stranded and double-helical regions to enable optimal packing of large RNA molecules. 相似文献
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Linam Franklin Limmer Matt A. Tappero Ryan Seyfferth Angelia L. 《Plant and Soil》2022,477(1-2):135-152
Plant and Soil - Rice is a staple crop worldwide and a silicon (Si) hyperaccumulator with Si levels reaching 5–10% of its mass; this can result in desilication and Si-deficiency if plant... 相似文献
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Basner-Tschakarjan E Gaffal E O'Keeffe M Tormo D Limmer A Wagner H Hochrein H Tüting T 《The journal of gene medicine》2006,8(11):1300-1306
BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches. 相似文献
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A. G. C. Buguet S. D. Livingstone L. D. Reed R. E. Limmer 《International journal of biometeorology》1976,20(1):61-69
Two Caucasian males, aged 19 and 22, slept at night in sleeping bags (9.0 clo) in an unheated tent at ambient temperatures between –25 and –35°C in the Arctic. Electroencephalographic (EEG) sleep studies were conducted for two baseline nights (19–21°C), 10 cold exposure nights and 2 recovery nights (19–21°C). Rectal and skin temperatures, and heart rates were also recorded. The subjects suffered disturbances in sleep patterns involving an insomnia composed of an increased wakefulness, a decrease in slow wave sleep and a deprivation in rapid eye movement (REM) sleep. Dissimilarities appeared between the subjects which may be related to differences in thermoregulatory responses. 相似文献
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A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.
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The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition. 相似文献
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Kornek M Lukacs-Kornek V Limmer A Raskopf E Becker U Klöckner M Sauerbruch T Schmitz V 《Molecular medicine (Cambridge, Mass.)》2008,14(7-8):365-373
Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 microg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased beta-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC. 相似文献
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Schwandt T Schumak B Gielen GH Jüngerkes F Schmidbauer P Klocke K Staratschek-Jox A van Rooijen N Kraal G Ludwig-Portugall I Franken L Wehner S Kalff JC Weber O Kirschning C Coch C Kalinke U Wenzel J Kurts C Zawatzky R Holzmann B Layland L Schultze JL Burgdorf S den Haan JM Knolle PA Limmer A 《The EMBO journal》2012,31(1):201-213
Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis. 相似文献