In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

The role of glucosinolates and their hydrolysis products in oviposition and host-plant finding by cabbage webworm, Hellula undalis

The cabbage webworm, Hellula undalis (Fabricius) (Lepidoptera: Pyralidae), a tropical pest on crucifers (Brassicaceae), differentiated among host‐plant species for oviposition in laboratory and field tests. White mustard, Sinapis alba (L.) var. Selinda, was the preferred host‐plant, followed by Brassica juncea (L.) Czern. et. Coss var. Canadian brown mustard, and pak‐choi, Brassica campestris L. ssp. chinensis var. Joi Choi, Black Behi and Bai Tsai. Glucosinolates (GS), secondary plant compounds characteristic to the Cruciferae plant family, and their breakdown products were analyzed by using HPLC and GC‐MS‐techniques. Species differed in GS composition and concentration. Content of GS was highest in S. alba with progressively lower contents detected in B. juncea and B. chinensis. The aromatic GS, 4‐hydroxybenzyl‐GS and benzyl‐GS, were detected in S. alba. In B. juncea the alkenyl GS, allyl‐GS, dominated, whereas in varieties of B. chinensis indolyl and alkenyl GS predominated. Oviposition of H. undalis females on the non‐host‐plant Vigna unguiculata ssp. sesquipedalis (L.) Fruwirth was stimulated by application of GS extracts from the crucifer species; the extract from S. alba was preferred, followed by extracts from B. juncea and B. chinensis. Hydrolysis of GS in the plant extract from B. chinensis causes loss of the oviposition stimulatory effect of the extract. Application of the GS, allyl‐GS, and benzyl‐GS also stimulated oviposition by H. undalis. Significantly more eggs were laid on leaves treated with the aromatic GS, benzyl‐GS, than with the alkenyl GS, allyl‐GS. Host‐plant odor attracted H. undalis females but not males, in behavioral assays conducted in a Y‐tube olfactometer. Low concentrations of the GS hydrolysis product, allyl‐isothiocyanate, induced anemotaxis of females, but a high concentration of allyl‐isothiocyanate was repellent. Oviposition by H. undalis females was not stimulated by host‐plant volatiles. Females laid eggs on inserted traps and the walls of the Y‐tube regardless of presence or absence of host‐plant odor. The relevance of these results in the context of crucifer‐insect interactions is discussed.     

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.     

Altitudinal differences in herbivory on montane Compositae species

To test whether the range of montane Compositae species may be restricted to higher sites because of greater herbivory levels in the lowlands, we transplanted six species, combining them in three species pairs each consisting of a rare montane and a widespread species. Individuals of all species were planted at four sites of different altitude ranging from the lowlands to the subalpine Mt. Brocken in the Harz mountains, Germany. Food choice experiments with three mollusc species indicated that the montane plant was more palatable in the species pair Senecio hercynicus/S. ovatus, but the widespread plant was more palatable in the species pair Petasites albus/Tussilago farfara. In the third pair (Cicerbita alpina/Mycelis muralis), neither species was preferred. In the field, species-specific herbivory levels differed in their amount, in their interaction with plant phenology and in their effect on mortality. They only partially reflected the laboratory food choice results. We found clear differences between the lowest and the highest site for all species, but a continuous decrease in herbivory with altitude was only detected in three of the six species.     

Biogenesis of Respiratory Complex I

Proteins specifically involved in the biogenesis of respiratory complex I in eukaryotes have been characterized. The complex I intermediate associated proteins CIA30 and CIA84 are tightly bound to an assembly intermediate of the membrane arm. Like chaperones, they are involved in multiple rounds of membrane arm assembly without being part of the mature structure. Two biosynthetic subunits of eukaryotic complex I have been characterized. The acyl carrier subunit is needed for proper assembly of the peripheral arm as well as the membrane arm of complex I. It may interact with enzymes of a mitochondrial fatty acid synthetase. The 39/40-kDa subunit appears to be an isomerase with a tightly bound NADPH. It is related to a protein family of reductases/isomerases. Both subunits have been discussed to be involved in the synthesis of a postulated, novel, high-potential redox group.     

Molecular characterization of the brassinosteroid-deficient lkb mutant in pea

The brassinosteriod-deficient lkb mutant of garden pea (Pisum sativum L.) is characterized by an erectoides phenotype (reduced internode length, thickened stems, epinastic leaves), which is rescued by application of exogenous brassinolide. We show that the LKB gene is the Arabidopsis DIMINUTO/DWARF-1 (DIM/DWF1) homologue of pea. The DIM/DWF1 homologue from lkb plants contains a mutation that may result in reduced enzyme function, thus resulting in the previously shown accumulation of 24-methylenecholesterol and a deficiency of its hydrogenated product, campesterol. This ultimately leads to a deficiency of the biologically active brassionolide. The mutation in the lkb sequence cosegregates with the lkb phenotype. Northern analyis of the LKB gene revealed that the gene is ubiquitously expressed around the plant and that there is no evidence for negative feedback regulation of the gene.