Conserved determinants for membrane association of nonstructural protein 5A from hepatitis C virus and related viruses

Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.     

A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis

We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.     

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNA^Asn and Gln-tRNA^Gln are formed by amidation of aspartate and glutamate mischarged onto tRNA^Asn and tRNA^Gln, respectively. Coexistence in the organism of mischarged Asp-tRNA^Asn and Glu-tRNA^Gln and the homologous Asn-tRNA^Asn and Gln-tRNA^Gln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA^Asn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA^Asn from Asn-tRNA^Asn and Asp-tRNA^Asp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.     

Carbon isotope fractionation in species of the torrenticolous families Podostemaceae and Hydrostachyaceae

Carbon isotope ratios of herbarium material from members of the fresh-water families Podostemaceae and Hydrostachyaceae (Rosidae) were analyzed. The levels of ¹³C were highly variable (Podostemaceae −12.8‰ to −38.55‰; Hydrostachyaceae −10.78‰ to −30.42‰), across as well as within species and across a wide geographic range.

We suggest that the high variance observed is due neither to a constant attribute of the species like the photosynthetic CO₂-carboxylase (in water plants with very high discrimination of the ¹³CO₂ probably Rubisco) nor to the constant structural peculiarities of these species. Rather, it is likely due to the ‘diffusional resistance’ for the CO₂-flux from the turbulent and/or fast flowing water, causing a very variable boundary layer on the plant surface.     

Nitric oxide decreases ammonium release in tadpoles of the clawed frog, <Emphasis Type="Italic">Xenopus laevis</Emphasis>, Daudin

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or l-arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nω-methyl-l-arginine (l-NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.     

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida)

The pest potential of stored product mites depends on the reproduction rate that is affected by the environmental conditions. In this study we investigated the effect of temperature, ranging from 5 to 35°C, on the population growth of three important mite species, Acarus siro, Tyrophagus putrescentiae and Auleroglyphus ovatus at 85% r.h. Starting with 10 individuals the population increase of mites was observed after 3 weeks of cultivation, or after 6 weeks for those kept at low temperatures (5, 10, 12.5, and 15°C). The rate of increase was calculated for each temperature and species. The obtained data were fitted with polynomial models. The mite population growth rates increased with increasing moderate temperatures until 25°C, when r _m -values were 0.179, 0.177 and 0.190 for A. siro, A. ovatus and T. putrescentiae, respectively. The lower development threshold was 10.2°C in all three species. Estimated upper temperature threshold was higher in T. putrescentiae (49°C) than in A. siro and A. ovatus (38°C). Simulation of the rate of population increase under ideal conditions, using real temperature records obtained from Czech grain stores, showed that the pest mite populations increase only during 3.5 months within a typical 9-month storage season in Central Europe. These results indicate that control of mites, be it chemical, physical or biological, is recommended during the months when allergens and pests are produced, i.e. from September to mid November and in May.     

Membrane perturbation by an external electric field: a mechanism to permit molecular uptake

Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

26RFa, a novel orexigenic neuropeptide, inhibits insulin secretion in the rat pancreas

26RFa is a novel orexigenic neuropeptide identified as the endogenous ligand of the orphan G protein-coupled receptor GPR103. GPR103 shares sequence identity with the receptors for neuropeptide-Y and galanin, two peptides known to inhibit insulin secretion. We have investigated the effect of 26RFa on insulin and glucagon secretion in the perfused rat pancreas. 26RFa dose-dependently reduced glucose-induced insulin release, inhibited the insulin responses to both arginine and exendin-4 and did not affect glucagon output. The inhibitory effect of 26RFa on exendin-4-induced insulin secretion was not observed in pancreata from pertussis toxin-treated rats, thus suggesting that 26RFa may inhibit insulin secretion, at least in part, via a pertussis toxin-sensitive G(i) protein coupled to the adenylyl cyclase system.     

Glycan chains modulate prion protein binding to immobilized metal ions

PrP(c) is the normal isoform of the prion protein which can be converted into PrP(Sc), the pathology-associated conformer in prion diseases. It contains two N-linked glycan chains attached to the C-proximal globular domain. While the biological functions of PrP(c) are still unknown, its ability to bind Cu(2+) is well documented. The main Cu(2+)-binding sites are located in the N-proximal, unstructured region of the molecule. Here we report that PrP(c) glycans influence the capacity of PrP(c) from sheep brain or cultured Rov cells to bind IMAC columns loaded with Cu(2+) or Co(2+). Using different anti-PrP antibodies and PrP(c) glycosylation mutants, we show that the full length non-glycosylated form of PrP(c) has a higher binding efficiency for column-bound Cu(2+) and Co(2+) than the corresponding glycosylated form. Our findings raise the possibility that the accessibility of the PrP(c) metal ion-binding sites might be controlled by the glycan chains.