Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

The impact of the Scheldt input on the trace metal distribution in the Belgian coastal area (results of 1981–1983 and 1995–1996)

Offshore fluxes of Cu, Zn, Cd, Pb and Hg were calculated based onresidual flow patterns and salinity gradients along the Belgian coast. Theresidual flow lines along the Belgian coast are more or less parallel to thecoast except in the area where the north-easterly flowing watermass comingfrom the Channel encounters the south-westerly-oriented Scheldt outflow,forming a residual hydrodynamical front. From the steady-state salinitypattern, diffusion coefficients perpendicular to the residual flow werededuced; they ranged from 21 to 108 m² s^-1. Offshore fluxes of dissolved and particulate trace metals based on diffusiveand mixing processes are calculated. The steady state profiles of dissolvedmetals show a dilution effect in the coastal waters, reaching an almostconstant concentration in the marine watermass in the 1981–1983dataset. The ratios of the Scheldt input of trace metals to the totaldissolved offshore flux vary from 38 to 55% (1981–1983),depending on the kind of metal, and from 55 to 91% (1995–1996).The ratio of the Scheldt input to the dissolved metal flow parallel to thecoast, is in both periods (1981–1983 and 1995–1996), smallerthan 1%. The steady-state concentration profiles of particular metalsversus salinity are fairly constant in the coastal-estuarine and marinewatermasses, but decrease very abruptly from the first to the secondwatermass. Assuming a conservative behaviour of the particular metals,offshore fluxes and the resulting concentration increases agree fairly wellwith the observed values. The ratios of the Scheldt input to the particulatetrace metal offshore flux vary between 30 to 46% (1981–1983)and 13 to 37% (1995–1996). The contribution of the Scheldtestuary to the flows parallel to the coast ranges from 1.6 to 2.9%(1981–1983) and from 0.6 to 1.6% (1995). This revised version was published online in July 2006 with corrections to the Cover Date.     

Sugar beet guard cell protoplasts demonstrate a remarkable capacity for cell division enabling applications in stomatal physiology and molecular breeding

A highly-efficient protocol for the large-scale isolation ofguard cell protoplasts from sugar beet (Beta vulgaris L.) hasbeen developed. Optimization of conditions for culturing theseprotoplasts resulted in extensive cell division and colony formation,at frequencies exceeding 50%. Plants can subsequently be regeneratedfrom these guard cell-derived colonies. This provides definitiveconfirmation that, in sugar beet leaf protoplast populations,only guard cells are the source of totipotent protoplasts. Thesefindings are the outcome of a directed, non-empirical approachto overcoming plant cell recalcitrance which was initiated byexploiting computer-assisted microscopy to couple in vitro responseto cell origin. The results reaffirm the conclusion that, inplants, extreme degrees of cytodifferentiation need not entailterminal specialization. The responsive nature of this systemcan be ascribed to the unique use of cultures essentially comprisinga single in vivo cell type. A uniform model system has thusbeen created with potential for widespread application. Theirdistinct morphological (and mechanical) features make guardcells a valuable choice for studying various fundamental aspects,not only of stomatal physiology, but also of plant cell (de)differentiation,differential gene expression etc. Furthermore, an applied valuefor such a system can also be envisaged. Results indicate thatthese cells are highly amenable to genetic manipulation techniques.The importance of these observations to our understanding ofplant cell function and behaviour is discussed. Key words: Beta, guard cells, stomatal physiology, totipotency, transformation     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.