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Granulated agarose gels suitable for gel exclusion chromatography of proteins of any molecular weight may now be prepared. This was made possible by the observation that agarose solutions of 16% polysaccharide may be prepared by displacing 8% agarose from solution with 8% polyethylene glycol Mr 6000. The displaced polysaccharide concentrates in a viscous mass occupying half the volume of the original carbohydrate solution. By diluting the displaced polysaccharide with hot watery solutions of electrolyte and allowing the solutions to congeal, gels of any desired concentration, ranging from low to the maximum of 16%, may be prepared. 相似文献
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Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen 总被引:7,自引:1,他引:7 下载免费PDF全文
Andrew G. Polson Herbert L. Ley III Brenda L. Bass John L. Casey 《Molecular and cellular biology》1998,18(4):1919-1926
RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication. 相似文献
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High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes 总被引:3,自引:0,他引:3
S A Robicsek J J Krzanowski A Szentivanyi J B Polson 《Biochemical and biophysical research communications》1989,163(1):554-560
The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes. 相似文献
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Wallace OB Smith DW Deshpande MS Polson C Felsenstein KM 《Bioorganic & medicinal chemistry letters》2003,13(6):1203-1206
Inhibitors of amyloid-beta (Abeta) protein production have been widely pursued as a potential treatment for Alzheimer's disease. Following the identification of a 5 microM screening hit, SAR was initiated using solid-phase synthetic techniques. Two series of alpha-hydroxy esters and ketones which are sub-micromolar inhibitors of Abeta production were identified. The most potent alpha-hydroxyketone identified is approximately 30-fold more potent than the initial lead. 相似文献
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Parker MF Barten DM Bergstrom CP Bronson JJ Corsa JA Deshpande MS Felsenstein KM Guss VL Hansel SB Johnson G Keavy DJ Lau WY Mock J Prasad CV Polson CT Sloan CP Smith DW Wallace OB Wang HH Williams A Zheng M 《Bioorganic & medicinal chemistry letters》2007,17(16):4432-4436
A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity. 相似文献
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A Polson 《Preparative biochemistry》1977,7(2):129-154
The displacement action of polyethylene glycol of different molecular weights may be linked to the ability of the polymers to form coiled particles in solution. From conclusions drawn from their sedimentating properties in centrifugal fields the polyethylene glycols of low molecular weights, as expected, are less randomly coiled than those of higher molecular weight. It is suggested that protein molecules have the ability to diffuse into the coils of the polyethylene glycol from which they are excluded when the random coiling increases with increasing polymer concentration. From considerations based on the interaction of the polymer filament with the displaced particle the distribution of the substance between the coils and the intermolecular spaces may be predicted semi-quantitatively. 相似文献
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Craig Polson Joseph L. Cantone Cong Wei Dieter M. Drexler Jere E. Meredith Jr. 《Analytical biochemistry》2014
Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [13C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [13C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease. 相似文献