Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

The effect of electric field on callus induction with rape hypocotyls

The influence of electric field treatment on dedifferentiation and calli formation on rape hypocotyls was investigated. Segments, 10 mm long, of the upper part of rape (Brassica napus L., cv. Góczański) hypocotyls were stimulated by different combinations of voltage/time (1.5 V/120 h, 3 V/3 h, 10 V/15 min and 30 V/30 s) under in vitro conditions. With all electric field treatments, segments oriented with their apical part towards the cathode produced more calli as compared to control (non-treated with electric field). Under opposite orientation slight inhibition of callus growth was observed. As the strongest effect on callus growth was observed after treatment with 30 V/30 s, this electric field treatment was selected for following analyses: the incorporation of [14C]-2,4-D (2,4-dichlorophenoxyacetic acid) and [14C]-BAP (benzylaminopurine) from the culture medium, changes in ACC (1-aminocyclopropane-1-carboxylic acid) level and the redox activity in apical and bottom parts of hypocotyls during 18 d of culture. In contrast to changes in fresh weight, electric field treatment (30 V/30 s) stimulated a higher accumulation of 2,4-D and BAP in basal parts of hypocotyls than in apical ones. Moreover, orienting the apical part towards the cathode resulted in lower uptake of hormones as compared with the opposite orientation. The ACC concentration increased, especially in the basal parts of hypocotyls, independently on electric field application. However, the highest level was observed after electric field treatment with orientation of the apical part towards the anode. The distribution of oxidative substances (measured as the amount of ferric ions) between the apical and bottom part of hypocotyls was not changed when the apical parts were oriented towards the cathode. Under these conditions a decrease in apical and an increase in basal parts was observed during culture. Opposite orientation influenced the redistribution of oxidative substances from the first day of electric field treatment. Based on these results we suggest that electric field action can be connected with its influence on specific concentration of oxidative substances and hormone distribution in cells.     

Bacterial dynamics in spring water of alpine karst aquifers indicates the presence of stable autochthonous microbial endokarst communities

Spring water of two alpine karst aquifers differing in hydrogeology but of nearby catchments were investigated for their bacterial population dynamics. Dolomite karst aquifer spring 1 (DKAS 1) represents a dolomitic-limestone karst aquifer spring showing high average water residence time and relative constant flow. Limestone karst aquifer spring 2 (LKAS 2) constitutes a typical limestone karst aquifer spring with a dynamic hydrological regime and discharge. Dolomite karst aquifer spring 1 yielded constantly lower cell counts and biomasses (median of 15 x 10(6) cells l(-1) and 0.22 microg C l(-1)) as the LKAS 2 (median of 63 x 10(6) cells l(-1) and 1.1 microg C l(-1)) and distribution of morphotypes and mean cell volumes was also different between the considered systems, indicating the influence of hydrogeology on microbial spring water quality. Molecular bacterial V3 16S-rDNA profiles revealed remarkable constancy within each spring water throughout the investigation period. Time course analysis of a flood event in LKAS 2 further supported the trend of the temporal constancy of the microbial community. Except for one case, retrieval of partial and full length 16S rDNA gene sequences from the relative constant DKAS 1 revealed similarities to presently known sequences between 80% to 96%, supporting the discreteness of the microbial populations. The gathered results provide first evidence for the presence of autochthonous microbial endokarst communities (AMEC). Recovery of AMEC may be considered of relevance for the understanding of alpine karst aquifer biogeochemistry and ecology, which is of interest as many alpine and mountainous karst springs are important water resources throughout the world.     

IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.     

Mechanism of gamma-secretase cleavage activation: is gamma-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.     

G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.     

Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein

The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK(2) cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication.