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  • 1
    Publication Date: 2019
    Description: The tRNAHis guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3′ to 5′ synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3′ to 5′ chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed.
    Electronic ISSN: 2073-4425
    Topics: Biology
    Published by MDPI
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  • 2
    Publication Date: 2019
    Description: Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.
    Electronic ISSN: 2073-4425
    Topics: Biology
    Published by MDPI
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  • 3
    Publication Date: 2003-07-26
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wozny, Christian -- Kivi, Anatol -- Lehmann, Thomas-Nicolas -- Dehnicke, Christoph -- Heinemann, Uwe -- Behr, Joachim -- New York, N.Y. -- Science. 2003 Jul 25;301(5632):463; author reply 463.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Neuroscience Research Center, at the Charite, Humboldt University of Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/12881553" target="_blank"〉PubMed〈/a〉
    Keywords: Action Potentials ; Electroencephalography ; Epilepsy, Temporal Lobe/pathology/*physiopathology ; Excitatory Amino Acid Antagonists ; Hippocampus/pathology/*physiopathology ; Humans ; In Vitro Techniques ; Neurons/physiology ; Neurons, Afferent/physiology ; Receptors, GABA-A/metabolism ; Receptors, Glutamate/metabolism ; Synaptic Transmission ; Temporal Lobe/physiopathology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 1987-10-23
    Description: The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Prive, G G -- Heinemann, U -- Chandrasegaran, S -- Kan, L S -- Kopka, M L -- Dickerson, R E -- New York, N.Y. -- Science. 1987 Oct 23;238(4826):498-504.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Molecular Biology Institute, University of California, Los Angeles 90024.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/3310237" target="_blank"〉PubMed〈/a〉
    Keywords: Base Composition ; Base Sequence ; Crystallization ; *Dna ; *Nucleic Acid Conformation ; *Oligodeoxyribonucleotides ; Phosphates ; Water
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2018-06-20
    Description: Genes, Vol. 9, Pages 305: MiRAR—miRNA Activity Reporter for Living Cells Genes doi: 10.3390/genes9060305 Authors: Matthew A. Turk Christina Z. Chung Emad Manni Stephanie A. Zukowski Anish Engineer Yasaman Badakhshi Yumin Bi Ilka U. Heinemann microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.
    Electronic ISSN: 2073-4425
    Topics: Biology
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 158 (1997), S. 159 -166 
    ISSN: 1432-1424
    Keywords: Key words: Nuclear ion channels — Cation channel — Cell nucleus — Patch clamp — Ion selectivity – Nuclear envelope
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K+ is 166 pS. The channels are selective for cations with P K/P Na= 2. They are neither permeable to, nor gated by Ca2+. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-1424
    Keywords: Microglia ; Granulocyte/macrophage colony-stimulating factor ; Whole-cell recording ; Outward K+ currents ; Frequency-independent K+ current ; Peptide toxins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Voltage-gated whole-cell currents were recorded from cultured microglial cells which had been developed in the presence of the macrophage/microglial growth factor granulocyte/macrophage colony-stimulating factor. Outward K+ currents (I K) were most prominent in these cells. I Kcould be activated at potentials more positive than −40 mV. Half-maximal activation of I Kwas achieved at −13.8 mV and half-maximal inactivation of I Kwas determined at −33.8 mV. The recovery of I Kfrom inactivation was described by a time constant of 7.9 sec. For a tenfold change in extracellular K+ concentration the reversal potential of I Kshifted by 54 mV. Extracellularly applied 10 mm tetraethylammonium chloride reduced I K by about 50%, while 5 mm 4-aminopyridine almost completely abolished I K. Several divalent cations (Ba2+, Cd2+, Co2+, Zn2+) reduced current amplitudes and shifted the activation curve of I Kto more positive values. Charybdotoxin (IC50 = 1.14 nm) and noxiustoxin (IC50=0.89 nm) blocked I Kin a concentration-dependent manner, whereas dendrotoxin and mast cell degranulating peptide had no effect on the current amplitudes. The outward K+ currents showed a frequency dependence when depolarizing pulses were applied at a frequency of 1 Hz. A frequency-independent outward current (I K′) characterized by the same activation behavior as I Kwas detected. I K′was blocked completely by 10 nm charybdotoxin or by 10 nm noxiustoxin. In contrast to its effect on I K, 10 mm tetraethylammonium chloride did not reduce I K′.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 16 (1988), S. 287-297 
    ISSN: 1432-1017
    Keywords: Ribonuclease T1 ; protein dynamics ; molecular dynamics ; protein-nucleic acid interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2′GMP-RNase T1 complex. The presence of water led to the maintenance of the 2′-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2′GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of activesite residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2′GMP, which did show an increase in mobility. Analysis of the 2′GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2′GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.
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  • 9
    Electronic Resource
    Electronic Resource
    Copenhagen : International Union of Crystallography (IUCr)
    Acta crystallographica 49 (1993), S. 468-477 
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: In an earlier study [Heinemann & Hahn (1992). J. Biol. Chem. 267, 7332–7341], the crystal structure of the double-stranded B-DNA decamer d(CCAGGCm5CTGG) was refined with NUCLSQ to R = 17.4% against 3799 2σ structure amplitudes in the resolution range 8–1.7 Å. This structure has now been re-refined against the same diffraction data using either TNT or X-PLOR in order to determine to what extent the resulting DNA conformations would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. The R value from the NUCLSQ refinement could not be reached with either TNT or X-PLOR, although both programs yield reasonably refined DNA models showing root-mean-square deviations against the NUCLSQ model of the decamer duplex of 0.25 and 0.32 Å, respectively. Some derived local structure parameters differ depending on the refinement procedure used. This holds true for several exocyclic torsion angles of the sugar-phosphate backbone, whereas sugar puckers as well as helical and base-pair stacking parameters are only weakly influenced. A subset of 15 solvent sites with low temperature factors is conserved in all three models.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Copenhagen : International Union of Crystallography (IUCr)
    Acta crystallographica 50 (1994), S. 75-84 
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Chloromuconate cycloisomerase (E.C. 5.5.1.7) is an enzyme involved in the 2,4-dichlorophenoxyacetate degradation pathway of Alcaligenes eutrophus JMP134 (pJP4). The crystal structure of this protein was determined at 3 Å resolution by molecular-replacement techniques using atomic coordinates from the reported crystal structure of the homologous muconate cycloisomerase (E.C. 5.5.1.1) from Pseudomonas putida as the search model (42% identical positions in the sequences). Structure refinement by simulated-annealing and restrained least-squares techniques converged at R = 0.195. In the crystals studied, space group I4, the protein is present as two octamers per unit cell with two subunits per asymmetric unit. Each subunit consists of two globular domains, one of which forms an α/β-barrel. Comparison of this structure with that of muconate cycloisomerase reveals the reasons for the altered substrate specificity of chloromuconate cycloisomerase. Marked differences are observed in polarity, accessibility and hydrogen-bonding potential in the channel leading into the active site as well as in the active center itself.
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