Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P(2) as monitored by the PLCdelta1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca(2+) signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P(2) was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P(2) is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P(2) depletion.     

Binding of N-acetyl-N '-beta-D-glucopyranosyl urea and N-benzoyl-N '-beta-D-glucopyranosyl urea to glycogen phosphorylase b: kinetic and crystallographic studies.

Two substituted ureas of beta-D-glucose, N-acetyl-N'-beta-D-glucopyranosyl urea (Acurea) and N-benzoyl-N'-beta-D-glucopyranosyl urea (Bzurea), have been identified as inhibitors of glycogen phosphorylase, a potential target for therapeutic intervention in type 2 diabetes. To elucidate the structural basis of inhibition, we determined the structure of muscle glycogen phosphorylase b (GPb) complexed with the two compounds at 2.0 A and 1.8 A resolution, respectively. The structure of the GPb-Acurea complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change in the tertiary structure. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the GPb-glucose complex, while the acetyl urea moiety is in a favourable electrostatic environment and makes additional polar contacts with the protein. The structure of the GPb-Bzurea complex shows that Bzurea binds tightly at the catalytic site and induces substantial conformational changes in the vicinity of the catalytic site. In particular, the loop of the polypeptide chain containing residues 282-287 shifts 1.3-3.7 A (Calpha atoms) to accommodate Bzurea. Bzurea can also occupy the new allosteric site, some 33 A from the catalytic site, which is currently the target for the design of antidiabetic drugs.     

Kinetic and crystallographic studies of glucopyranosylidene spirothiohydantoin binding to glycogen phosphorylase B.

Glucopyranosylidene spirothiohydantoin (TH) has been identified as a potential inhibitor of both muscle and liver glycogen phosphorylase b (GPb) and a (GPa) and shown to diminish liver GPa activity in vitro. Kinetic experiments reported here show that TH inhibits muscle GPb competitively with respect to both substrates phosphate (K(i)=2.3 microM) and glycogen (K(i)=2.8 microM). The structure of the GPb-TH complex has been determined at a resolution of 2.26 A and refined to a crystallographic R value of 0.193 (R(free)=0.211). The structure of GPb-TH complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provides a basis of understanding potency and specificity of the inhibitor. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the glucose complex, while the rigid thiohydantoin group is in a favourable electrostatic environment and makes additional polar contacts to the protein.     

Comparison of Fish Assemblage Diversity in Natural and Artificial Rip‐Rap Habitats in the Littoral Zone of a Large River (River Danube,Hungary)

We studied day‐night patterns in fish diversity in natural, gravel‐sand stretches and boulder covered rip‐rap habitats in the littoral zone of the River Danube. Sample‐based rarefaction indicated marked differences in species richness between day and night, and smaller differences between habitats for both day and night. Whereas, individual‐based rarefaction indicated no such substantial differences in species richness. However, distinct fish assemblages were found based on relative abundance data, and species of great conservation concern tended to link to natural habitats. The diversity of biological traits/attributes were generally higher in rip‐rap habitats. The differences in fish assemblage characteristics between habitats revealed the importance of gravel‐sand habitats in maintaining natural assemblages and that rip‐rap sections increase compositional beta diversity at the mesohabitat scale. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Development of a PCR-based DNA marker for <Emphasis Type="Italic">Glu-1By</Emphasis> alleles in the old Hungarian Bánkúti wheat

Based on sequence alignment, phylogenetic, and dotplot analyses, primers were designed in order to distinguish the wheat high-molecular-weight glutenin subunit alleles By8 and By9. The primers were tested on 26 lines of Bánkúti wheat, an old Hungarian variety, and a number of other varieties. Consistency was observed between their known By protein subunit and the obtained DNA marker. Comparison of the B subunit content and the By alleles of the Bánkúti lines was also in agreement with the previous prediction that the By8 and By9 subunits are linked to the Bx7 and Bx7? subunits, which are responsible for dough quality in Bánkúti wheat, respectively. Thus, the developed molecular marker would be appropriate for marker assisted selection of the dough quality trait in the introgressive breeding of Bánkúti lines into modern cultivars.     

Enrichment of dissimilatory Fe(III)-reducing bacteria from groundwater of the Siklós BTEX-contaminated site (Hungary)

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.     

Edge responses are different in edges under natural versus anthropogenic influence: a meta‐analysis using ground beetles

Most edges are anthropogenic in origin, but are distinguishable by their maintaining processes (natural vs. continued anthropogenic interventions: forestry, agriculture, urbanization). We hypothesized that the dissimilar edge histories will be reflected in the diversity and assemblage composition of inhabitants. Testing this “history‐based edge effect” hypothesis, we evaluated published information on a common insect group, ground beetles (Coleoptera: Carabidae) in forest edges. A meta‐analysis showed that the diversity‐enhancing properties of edges significantly differed according to their history. Forest edges maintained by natural processes had significantly higher species richness than their interiors, while edges with continued anthropogenic influence did not. The filter function of edges was also essentially different depending on their history. For forest specialist species, edges maintained by natural processes were penetrable, allowing these species to move right through the edges, while edges still under anthropogenic interventions were impenetrable, preventing the dispersal of forest specialists out of the forest. For species inhabiting the surrounding matrix (open‐habitat and generalist species), edges created by forestry activities were penetrable, and such species also invaded the forest interior. However, natural forest edges constituted a barrier and prevented the invasion of matrix species into the forest interior. Preserving and protecting all edges maintained by natural processes, and preventing anthropogenic changes to their structure, composition, and characteristics are key factors to sustain biodiversity in forests. Moreover, the increasing presence of anthropogenic edges in a landscape is to be avoided, as they contribute to the loss of biodiversity. Simultaneously, edges under continued anthropogenic disturbance should be restored by increasing habitat heterogeneity.     

Amino acid-amino acid interactions studied by charge-transfer chromatography

The interaction between amino acids was studied by charge-transfer reversed-phase thin-layer chromatography. The dependence of the lipophilicity of Trp on the concentration of other amino acids in the eluent was considered to be linearly related to the strength of interaction. Arg, Asn, Glu, Met, Phe and Thr interacted with Trp; Ala, Gly and Ser showed no interaction. Stepwise regression analysis indicated that the pK value of the amino acid side-chain and the lipophilicity of the amino acid had the greatest impact on the interaction, suggesting the simultaneous presence of weak hydrophilic and hydrophobic bonding forces between amino acids. Sodium acetate in the eluent increased the interactive strength between Phe and Trp; acetic acid and sodium chloride did not influence the interaction significantly. No significant difference was found between the effects of l- and d-Asn.     

Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella.

The lipopolysaccharide (LPS) is up to now the only identified major virulence determinant of Brucella. This bacterium is responsible for brucellosis in animals and for Malta fever in humans. Several monoclonal antibodies (mAbs) directed against various LPS epitopes have been characterized. Two mAbs, named A15-6B3 and B66-2C8, directed against distinct LPS epitopes have been used to select peptides from 11 phage display libraries. The sequences of the selected peptides contain an overrepresentation of either proline or tryptophan residues when selected with either A15-6B3 or B66-2C8 mAbs, respectively. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed. These data suggest that a subset of the selected peptides are mimotopes of the LPS epitopes.