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1.
We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins. 相似文献
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Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle. 相似文献
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An Extreme Value Analysis of Pollutant Concentrations in Surface Soils Due to Atmospheric Deposition
Risk assessments often rely on deterministic models using long-term averages or “steady-state” values of input variables. Such models do not provide the information needed to estimate acute exposures. This study uses extreme value theory to examine the frequency and magnitude of daily pollutant concentrations in surface soils predicted at six U.S. locations. Concentrations are predicted using a deposition-leaching model and 50 years of historical precipitation data. A stochastic model also is used to generate 1000 years of precipitation data as modeling inputs for each location. The annual maximum concentrations at each site are fitted to a Gumbel type I distribution to estimate occurrence probability. For soluble pollutants, the predicted concentration varied substantially with precipitation, and the maximum daily concentrations exceeded annual averages by 4 to 8 times. Observed and synthetic precipitation data produced similar results at most study locations, though the synthetic data provided a slightly better fit to the Gumbel type I distribution. The precipitation model allows the generation of representative precipitation data that extend limited historical records. The extreme value analysis facilitates the evaluation of maximum pollutant concentrations, return periods, and other statistics that are important in evaluating acute exposures. 相似文献
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This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated. 相似文献
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Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin
Birgitta Nordenman Ingemar Björk 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,672(3):227-238
Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups. 相似文献
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《Cell calcium》2019
Alterations in the (bio)chemical and physical microenvironment of cells accompany and often promote disease formation and progression. This is particularly well established for solid cancers, which are typically stiffer than the healthy tissue in which they arise, and often display profound acidification of their interstitial fluid. Cell surface receptors can sense changes in the mechanical and (bio)chemical properties of the surrounding extracellular matrix and fluid, and signalling through these receptors is thought to play a key role in disease development and advancement. This review will look at ion channels and G protein coupled receptors that are activated by mechanical cues and extracellular acidosis, and stimulation of which results in increases in intracellular Ca2+ concentrations. Cellular Ca2+ levels are dysregulated in cancer as well as cancer-associated cells, and mechano- and proton-sensing proteins likely contribute to these aberrant intracellular Ca2+ signals, making them attractive targets for therapeutic intervention. 相似文献
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Abstract The growth, biomass δ13C values, and ability to accumulate titratable acidity at night were compared in eight environmental treatments for Cremnophila linguifolia, Sedum greggii, and their F1 hybrid. In the phytotron, differences in treatment daylength, day/night temperature and water availability were all found to have effects on total plant dry weight, nocturnal accumulation of titratable acidity and biomass δ13C value of at least some of the genotypes. However, there were differences between the genotypes both in the magnitude and direction of response of the phenotypic properties to the treatment variables. The phytotron δ13C values ranged from -12.9 to -19.2‰ for C. linguifolia, from -22.2 to -33.4‰ for S. greggii, and from -19.2 to -24.9‰ for the hybrid. After with-holding water for 76 h both C. linguifolia and the hybrid had midday Ψleaf values of -0.23 MPa; however, S. greggii had a value of -1.05 MPa. In contrast to past observations of other species, the daily watered plants of C. linguifolia had less negative δ13C values than did the plants watered only weekly. 相似文献
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