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.     

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.     

Genetic markers for identification of Patagonian toothfish and Antarctic toothfish

Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.     

Photoproduction of hydrogen, nitrogenase and hydrogenase activities of free and immobilized whole cells of Rhodobacter sphaeroides O.U. 001

Abstract Photoproduction of hydrogen, nitrogenase activity (acetylene reduction) and hydrogenase activity (methylene blue dye reduction) were studied in free and alginate immobilized whole cells of a purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides O.U. 001. Four-fold increase in hydrogen production, two-fold increase in nitrogenase activity and 1.2-fold increase in the hydrogenase activity were observed in immobilized cells compared to free cells. Effect of various inhibitors (CO and C₂H₂) and electron donor (H₂) on the above three functions by free and immobilized cells has also been studied.     

Marine incursions,cryptic species and ecological diversification in Amazonia: the biogeographic history of the croaker genus Plagioscion (Sciaenidae)

Aim We propose a phylogenetic hypothesis for the marine‐derived sciaenid genus Plagioscion in the context of geomorphology and adaptation to freshwaters of South America, and assess the extent to which contemporary freshwater hydrochemical gradients influence diversification within a widely distributed Plagioscion species, Plagioscion squamosissimus. Location Amazon Basin and South America. Methods Using nuclear and mitochondrial DNA sequence data, phylogenetic analyses were conducted on the five nominal Plagioscion species, together with representatives from Pachyurus and Pachypops, using character and model‐based methods. Genealogical relationships and population genetic structure of 152 P. squamosissimus specimens sampled from the five major rivers and three hydrochemical settings/‘colours’ (i.e. white, black and clear water) of the Amazon Basin were assessed. Results Phylogenetic analyses support the monophyly of Plagioscion in South America and identify two putative cryptic species of Plagioscion. Divergence estimates suggest that the Plagioscion ancestor invaded South America via a northern route during the late Oligocene to early Miocene. Within P. squamosissimus a strong association of haplotype and water colour was observed, together with significant population structure detected between water colours. Main conclusions Our analyses of Plagioscion are consistent with a biogeographic scenario of early Miocene marine incursions into South America. Based on our phylogenetic results, the fossil record, geomorphological history and distributional data of extant Plagioscion species, we propose that marine incursions into western Venezuela between the late Oligocene and early Miocene were responsible for the adaptation to freshwaters in Plagioscion species. Following the termination of the marine incursions during the late Miocene and the establishment of the modern Amazon River, Plagioscion experienced a rapid diversification. Plagioscion squamosissimus arose during that time. The formation of the Amazon River probably facilitated population and range expansions for this species. Further, the large‐scale hydrochemical gradients within the Amazon Basin appear to be acting as ecological barriers maintaining population discontinuities in P. squamosissimus even in the face of gene flow. Our results highlight the importance of divergent natural selection through time in the generation and maintenance of sciaenid diversity in Amazonia.