Diatom species–environment relationships and inference models from Isachsen, Ellef Ringnes Island, Canadian High Arctic

Surface sediment diatom assemblages were examined from 26 freshwater sites near Isachsen (78°47N, 103°32W), Ellef Ringnes Island, a region of diverse and atypical water chemistry for high arctic sites. One hundred and sixty eight diatom taxa were identified from these samples, over 50% of which had not previously been recorded in the Canadian High Arctic. Variations in diatom assemblages were related to changes in measured environmental variables using multivariate techniques. Canonical correspondence analysis (CCA) indicated that five variables contributed significantly to explaining patterns of diatom variation (i.e., COND, DIC, Mn, TPF, TPU). The first CCA axis (=0.44) was primarily controlled by conductivity-related variables, while CCA axis 2 (=0.21) was related to particulate concentrations. Diatom-based inference models were generated for the reconstruction of conductivity (RMSEP_jack=0.32, r²_jack=0.76) and pH (RMSEP_jack=0.40, r²_jack=0.69). The strengths of these models indicate that it will be possible to reliably infer past trends in conductivity and pH from diatom assemblages preserved in dated sediment cores from the Isachsen region.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

Structural studies on a twin-arginine signal sequence

Translocation of folded proteins across biological membranes can be mediated by the so-called ‘twin-arginine translocation’ (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.     

Conversion of the nitrogen content in liquid manure into biomass and polyglutamic acid by a newly isolated strain of Bacillus licheniformis

Extensive spreading of liquid manure onto agricultural fields causes eutrophication of ground and surface water and also pollution of the atmosphere due to the high ammonium nitrogen content. A poly(gamma-glutamic acid) (PGA)-producing strain of Bacillus licheniformis was isolated in this study and investigated for its ability to reduce the ammonium nitrogen by converting ammonium into biomass and PGA as depot forms of nitrogen. In batch cultivations swine manure and an optimized mineral salts medium were used for PGA production. For example the cultivation of B. licheniformis strain S2 in liquid manure, which was modified by adding of 18 g citrate and 80 g glycerol l(-1) and exhibited a carbon to nitrogen ratio of 15.5:1, led to severe reduction of the ammonium content from 2.83 to 0.1 g x l(-1) and to the production of 0.16 g PGA and 7.5 g cell dry mass l(-1) within 410 h. Approximately 28% (w/w) of the total nitrogen was converted into cellular biomass, whereas 0.1% (w/w) was used for the production of PGA. In addition, approximately 33% (w/v) of the original ammonium was lost by stripping.     

Chromatographic determination of L- and D-amino acids in plants

Quantities of free L- and D-amino acids (L- and D-AAs) in plants (leaves of coniferous and decidious trees, fleshy fruits, leaf blades of fodder grasses, and seeds and seedlings of edible legumes) were determined. Amino acid (AA) enantiomers were converted into diastereomers using pre-column derivatization with o-phthaldialdehyde together with N-isobutyryl-L(or D)-cysteine followed by separation of the resulting fluorescent isoindol derivatives on an octadecylsilyl stationary phase using high-performance liquid chromatography. Relative amounts of D-AAs were also determined by enantioselective gas chromatography-mass spectrometry on Chirasil-L-Val. Free D-AAs acids in the range of about 0.2% up to 8% relative to the corresponding L-AAs acids were found in plants. D-Asp, D-Asn, D-Glu, D-Gln, D-Ser and D-Ala could be detected in most of the plants, and D-Pro, D-Val, D-Leu and D-Lys in certain plants. As D-AAs were detected in gymnosperms as well as mono- and dicotyledonous angiosperms of major plant families it is concluded that free D-AAs in the low percentage range are principle constituents of plants.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: dodecanoic and 3-phenylpropionic acids system

Dimer structure and liquid structure of fatty acids in the binary liquid mixture of dodecanoic (LA) and 3-phenylpropionic acids (PPA) were studied through the measurements of DSC, self-diffusion coefficient (D), density, viscosity, 13C NMR spin-lattice relaxation time, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS). The phase diagram of LA/PPA mixture exhibited a typical eutectic pattern, which means that LA and PPA are completely immiscible in solid phase. In the liquid phase of the LA/PPA mixture, D of LA always differed from that of PPA irrespective of their compositions. This exhibited that, in the liquid phase of the binary mixture of fatty acids giving a complete eutectic in the solid phase, the fatty acid dimers are composed of the same fatty acid species irrespective of their compositions. The liquid structure of the LA/PPA mixture was clarified through the SAXS and also the SANS measurements.     

Purification and characterization of phytocystatins from kiwifruit cortex and seeds

Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (K(I)) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The K(I) (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESLNSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of approximately 11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrusxparadisi (52% identity). This is the first report of phytocystatins from the Ericales.     

Evaluation of the mass spectrometric fragmentation of codeine and morphine after 13C-isotope biosynthetic labeling

All major fragment ions of codeine and morphine were elucidated using LC-electrospray MS/MS and high resolution FT-ICR-MS combined with an IRMPD system. Nanogram quantities of labeled codeine were isolated and purified from Papaver somniferum seedlings, which were grown for up to 9 days in the presence of [ring-13C6]-l-tyrosine, [ring-13C6]-tyramine and [1,2-13C2], [6-O-methyl 13C]-(R,S)-coclaurine. The labeling degree of codeine up to 57% into morphinans was observed.     

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time (t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.     

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.