Effect of pH on Na induced Ca deficiency

Although it is well known that high Na concentrations induce Ca deficiency in acidic conditions, the effect of high pH on this competitive mechanism is not so well understood. The effect of Ca activity ratio (CAR) and pH on the Ca uptake of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald) and Rhodes grass (Chloris gayana cv. Pioneer) in Na dominated solution cultures and in soil was investigated. Changes in pH in the alkaline range were shown not to affect the critical CAR of 0.024 (corresponding to 90 % relative root length) for mungbeans grown in solution culture. Results from soil grown mungbeans confirmed those from solution culture, with a critical CAR of 0.025. A critical CAR of 0.034 was also established for soil grown Rhodes grass. The similarity of critical values established for mungbeans and Rhodes grass in solution culture and soil justifies the use of both solution culture and soil solution measurement as techniques for studying plant growth and limitations across plant species.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Net uptake of dissolved free amino acids by the giant clam, Tridacna maxima: alternative sources of energy and nitrogen?

The role of dissolved free amino acids (DFAA) in nitrogen and energy budgets was investigated for the giant clam, Tridacna maxima, growing under field conditions at One Tree Island, at the southern end of the Great Barrier Reef, Australia. Giant clams (121.5-143.7 mm in shell length) took up neutral, acidic and basic amino acids. The rates of net uptake of DFAA did not differ between light and dark, nor for clams growing under normal or slightly enriched ammonium concentrations. Calculations based on the net uptake concentrations typical of the maximum concentrations of DFAA found in coral reef waters (similar to 0.1 mu M)revealed that DFAA could only contribute 0.1% and 1% of the energy and nitrogen demands of giant clams, respectively. These results suggest that DFAA does not supply significant amounts of energy or nitrogen for giant clams or their symbionts.

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Carbon structure and porosity of carbonaceous adsorbents in relation to their adsorption properties

A number of carbonaceous adsorbents were prepared by carbonisation at 600 degrees C following acidic oxidation under various conditions. Effects of the chemical nature of the precursor, such as the ratio of aromatic to aliphatic carbons and oxygen content, on the chemical and structural characteristics of the resultant chars were investigated using C-13 NMR and Raman spectroscopy, respectively. The C-13 NMR spectral parameters of the coal samples show that as the severity of oxidation conditions increased, the ratio of aromatic to aliphatic carbons increased. Furthermore, it was also found that the amount of disorganised carbon affects both the pore structure and the adsorption properties of carbonaceous adsorbents. It is demonstrated that higher amount of the disorganised carbon indicates smaller micropore size. (C) 1999 Elsevier Science Ltd. All rights reserved.

Friedmanniella spumicola sp nov and Friedmanniella capsulata sp nov from activated sludge foam: Gram-positive cocci that grow in aggregates of repeating groups of cocci

Two Gram-positive, non-motile, non-spore-forming, strictly aerobic, pigmented cocci, strains Ben 107(T) and Ben 108(T), growing in aggregates were isolated from activated sludge samples by micromanipulation. Both possessed the rare type A3 gamma' peptidoglycan. Major menaquinones of strain Ben 107(T) were MK-9(H-4) and MK-7(H-2), and the main cellular fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). In strain Ben 108(T), MK-9(H-4), MK-9(H-2) and MK-7(H-4) were the menaquinones and again the main fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). Polar lipids in both strains consisted of phosphatidyl inositol, phosphatidyl glycerol and diphosphatidyl glycerol with two other unidentified glycolipids and phospholipids also present in both. These data, together with the 16S rDNA sequence data, suggest that strain Ben 107(T) belongs to the genus Friedmanniella which presently includes a single recently described species, Friedmanniella antarctica. Although the taxonomic status of strain Ben 108(T) is far less certain, on the basis of its 16S rRNA sequence it is also adjudged to be best placed in the genus Friedmanniella, The chemotaxonomic characteristics and DNA-DNA hybridization data support the view that Ben 107(T) and Ben 108(T) are novel species of the genus Friedmanniella. Hence, it is proposed that strain Ben 107(T) (=ACM 5121(T)) is named as Friedmanniella spumicola sp. nov. and strain Ben 108(T) (=ACM 5120(T)) as Friedmanniella capsulata sp. nov.

The role of carbon surface chemistry in N2O conversion to N2 over Ni catalyst supported on activated carbon

The effect of acidic treatments on N2O reduction over Ni catalysts supported on activated carbon was systematically studied. The catalysts were characterized by N-2 adsorption, mass titration, temperature-programmed desorption (TPD), and X-ray photoelectron spectrometry (XPS). It is found that surface chemistry plays an important role in N2O-carbon reaction catalyzed by Ni catalyst. HNO3 treatment produces more active acidic surface groups such as carboxyl and lactone, resulting in a more uniform catalyst dispersion and higher catalytic activity. However, HCl treatment decreases active acidic groups and increases the inactive groups, playing an opposite role in the catalyst dispersion and catalytic activity. A thorough discussion of the mechanism of the N2O catalytic reduction is made based upon results from isothermal reactions, temperature-programmed reactions (TPR) and characterization of catalysts. The effect of acidic treatment on pore structure is also discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

Chemical synthesis, characterization and activity of RK-1, a novel alpha-defensin-related peptide

The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial alpha-defensin family, was synthesized by the continuous now Fmoc-solid phase method. The crude, cleaved and S-reduced Linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by H-1-NMR spectroscopy and confirmed to be a Cys(1)-Cys(6), Cys(2)-Cys(4), Cys(3)-Cys(5) arrangement similar to other mammalian alpha-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418, Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.

Prediction of rock alteration patterns: a potential tool in mineral exploration

Hydrothermal alteration of a quartz-K-feldspar rock is simulated numerically by coupling fluid flow and chemical reactions. Introduction of CO2 gas generates an acidic fluid and produces secondary quartz, muscovite and/or pyrophyllite at constant temperature and pressure of 300 degrees C and 200 MPa. The precipitation and/or dissolution of the secondary minerals is controlled by either mass-action relations or rate laws. In our simulations the mass of the primary elements are conserved and the mass-balance equations are solved sequentially using an implicit scheme in a finite-element code. The pore-fluid velocity is assumed to be constant. The change of rock volume due to the dissolution or precipitation of the minerals, which is directly related to their molar volume, is taken into account. Feedback into the rock porosity and the reaction rates is included in the model. The model produces zones of pyrophyllite quartz and muscovite due to the dissolution of K-feldspar. Our model simulates, in a simplified way, the acid-induced alteration assemblages observed in various guises in many significant mineral deposits. The particular aluminosilicate minerals produced in these experiments are associated with the gold deposits of the Witwatersrand Basin.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Interaction of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes, and its open chain analogue with membrane mimics

In contrast to other mammalian defensins, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue, oRTD-1, although both peptides adopt very similar structures in water. It was suggested that the additional charges at the termini of oRTD-1 are the cause for its lower antimicrobial activity. Therefore, we studied the interaction of both peptides with membrane mimics composed of zwitterionic (PC) and negatively charged (PG) phospholipids, major lipid components of erythrocyte and bacterial cell membranes, respectively. Microcalorimetry showed that RTD-1 and oRTD-1 did not affect the phase behavior of PC liposomes, while in PG liposomes both peptides induced new phase transitions above the chain melting transition of the lipid. The shape and fraction differed between both peptides, depending also on their concentration, which will be discussed in terms of their antimicrobial activity.

Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

Antibacterial activity of bovine lactoferrin-derived peptides

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified. one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf. one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond, These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ton-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC), They were characterized by. N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination, Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques, This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 mu M or less, Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC, Subfragment 1 (residues I to 10) was active against most of the test microorganisms at concentrations of 10 to 50 mu M. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 mu M. These antibacterial studies indicate that the activity of lactoferricin Is mainly, but not wholly, due to its N-terminal region.

Gold(III) template synthesis of a pendant-arm macrocycle

Gold(III)-directed condensation of ethane-1,2-diamine with nitroethane and formaldehyde yielded the gold-coloured macrocyclic complex (cis-6,13-dimethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecan-1-ido)gold(III) and the orange acyclic complex (1,9-diamino-5-methyl-5-nitro-3,7-diazanoran-3-ido)gold(III) in good yields. Dissolution in strongly acidic solution gave the colourless fully protonated complexes. The pendant nitro groups are disposed on the same side of the macrocycle in a cis geometry, as confirmed by crystal structure analysis. In both complexes the gold ion lies in a square-planar environment of four nitrogen donors, and the co-ordinate bond to the deprotonated amine is shorter than the remaining Au-N distances.

Crystal structure of Escherichia coli xanthine phosphoribosyltransferase

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coil is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl l-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 Angstrom by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel B-sheet surrounded by three or-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.