N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

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.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

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.     

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.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

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.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

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.     

Mechano- and pH-sensing convergence on Ca2+-mobilising proteins – A recipe for cancer?

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.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

格网尺度下典型旅游城市生态服务价值估算和时空分异特征——以三亚为例

以典型旅游城市三亚市为案例地，利用2006-2018年4期Landsat遥感影像数据，借助ENVI、ArcGIS平台定量识别土地利用演变特征，在1km×1km格网尺度下估算旅游地生态系统服务价值，并结合空间探索性数据分析揭示生态系统服务价值时空分异特征及其与旅游地发展的时空耦合关系。结果表明：（1）2006-2018年间，三亚市生态系统服务价值总量呈逐年下降趋势，由6.73×10⁹元降至5.76×10⁹元，累计减少9.78×10⁸元；（2）空间格局上，三亚市呈"南低北高"空间分异格局，2006-2018年增值区域连片分布于崖州区、天涯区、吉阳区南部区域，且呈逐年减少趋势，减值区域集聚于天涯区东北部、海棠区；（3）空间集聚上，生态系统服务价值截面各年份均呈显著空间正相关且相关性先降后增。高高集聚区位于天涯区北部区域，低低集聚区分布于沿海、海湾地区；（4）旅游发展与生态系统服务价值时空演化特征关联性较强。三亚市天涯区北部林地生态环境良好，生态系统服务价值略有下降但绝对数值稳定，是生态系统服务价值主要来源；旅游发展较为迅速的三亚湾、崖州湾以及海棠湾，相对增值区域较多，但绝对生态系统服务价值损失显著，严重滞后于其他区域。