Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.     

Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes

A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4'-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.     

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.     

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution‐dependent curves in plasma and concentration‐dependent curves of full‐length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity‐free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.     

Changes in soil mineralogy and exchangeable cation pools in stands of Norway spruce planted on former pasture land

Chemical and mineralogical properties of the soils in 35- and 70-year-old stands of Norway spruce (Picea abies (L.) Karst), planted on former pasture and were studied at Asa Experimental Forest, southern Sweden. Remnant deciduous forests bordering the spruce stands were used as controls to assess possible tree-species-related effects on soil development. All soils are acid with little difference in soil pH between the spruce and deciduous stands. However, the saturation of the exchange complex with Mg is lower beneath spruce and the total exchangeable Mg pool in the upper meter of these soils is one third of the Mg store beneath the deciduous stands. Amphibole, biotite and chlorite are the major sources of Mg in the parent soil. The clay fraction of the topsoil beneath spruce has been depleted of all these easily weatherable ferromagnesian minerals. Apart from weathering-resistant primary silicates, the clay fraction consists almost exclusively of expandable, smectitic mixed-layer minerals, which are believed to be the products of advanced stages of biotite weathering. In contrast, vermiculite is the dominant secondary mineral in the A-horizon in the deciduous stands, and some chlorite has survived. Moreover, a greater depth of in situ weathering is indicated for the soil of the old spruce stands where biotite/vermiculite mixed-layers have formed in the C-horizon as products of early stages of biotite weathering. Thus, differences between the paired sites in soil solution chemistry are supported by the qualitative differences in soil mineralogy, and are believed to reflect divergent biotic and/or abiotic processes in the different stand types. This revised version was published online in August 2006 with corrections to the Cover Date.     

A mouse model utilising human transferrin to study protection against Neisseria meningitidis serogroup B induced by outer membrane vesicle vaccination

We have previously developed a mouse model based on transient bacteraemia in normal B10.M mice to evaluate the protective efficacy of outer membrane vesicle vaccines against serogroup B meningococci. To obtain a course of infection similar to that observed in man, we have in this work modified the mouse model by administration of human holo-transferrin upon bacterial challenge. Co-challenge with holo-transferrin induced increasing bacteraemia and subsequent death in normal non-immune mice, but not in vaccinated animals. The model system is dependent on challenge with meningococci expressing the transferrin receptor which is obtained by culturing the bacteria under iron restriction. The modified model system for protection against meningococcal infection presented here makes it possible to measure outer membrane vesicle vaccine induced protection by using bacteraemia as well as survival as parameters.     

The Relationship between the Growth Rate of Young Plants and their Total-N Concentration is Unique and Simple: a Comment

A series of experiments with cabbage and lettuce, where nitrogensupply is interrupted at a given and the future developmentof plants mass and of nitrogen concentration are observed, isre-analysed. Using the nitrogen productivity theory, it is shownthat plant properties determined from growth under unlimitednitrogen supply also explain the behaviour under interruptednitrogen supply. This is in contrast to the suggestion of theoriginal author Burns 1994, Annals of Botany 74: 143-157, whoquestions the existence of a unique relationship between plantrelative growth rate (RGR) and nitrogen concentration. Someserious problems in the models suggested by the author are alsoindicated.Copyright 1995, 1999 Academic Press Cabbage, lettuce, model, nitrogen limitation, nitrogen productivity, relative growth rate     

Intercalated discs: multiple proteins perform multiple functions in non-failing and failing human hearts

The intercalated disc (ICD) occupies a central position in the transmission of force, electrical continuity and chemical communication between cardiomyocytes. Changes in its structure and composition are strongly implicated in heart failure. ICD functions include: maintenance of electrical continuity across the ICD; physical links between membranes and the cytoskeleton; intercellular adhesion; maintenance of ICD structure and function; and growth. About 200 known proteins are associated with ICDs, 40% of which change in disease. We systemically reviewed cardiac immunohistochemical data on the Human Protein Atlas (HPA) web site, ExPASy protein binding data and published papers on ICDs. We identified 43 proteins not previously reported, and confirmed 37 proteins that have previously been described. In addition, 102 proteins not present on the HPA web site but were described in ICDs in the literature. We group these into clusters that demonstrate functionally interactive groups of proteins demonstrating that ICDs play a key role in cardiomyocyte function.     

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.