Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase

The specific chemical modification by sodium cyanate of highly reactive cysteine residues at pH 7.5 in pig kidney fructose 1,6-bisphosphatase results in the reversible loss of activation of the enzyme by monovalent cations. No loss of activation by potassium ions occurs when modification is carried out in the presence of fructose 2,6-bisphosphate. The effect of Mg2+ on native and cyanate-modified enzyme activities implicates the above cysteine residue as being directly linked to the inhibition by both the divalent cation and fructose 2,6-bisphosphate. Incorporation of [14C]cyanate to the enzyme shows that the blockage of two reactive residues per tetramer is sufficient to eliminate the activation of the enzyme by K+.     

Coping with Pleistocene climatic fluctuations: Demographic responses in remote endemic reef fishes

Elucidating demographic history during the settlement of ecological communities is crucial for properly inferring the mechanisms that shape patterns of species diversity and their persistence through time. Here, we used genomic data and coalescent‐based approaches to elucidate for the first time the demographic dynamics associated with the settlement by endemic reef fish fauna of one of the most remote peripheral islands of the Pacific Ocean, Rapa Nui (Easter Island). We compared the demographic history of nine endemic species in order to explore their demographic responses to Pleistocene climatic fluctuations. We found that species endemic to Rapa Nui share a common demographic history, as signatures of population expansions were retrieved for almost all of the species studied here, and synchronous demographic expansions initiated during the last glacial period were recovered for more than half of the studied species. These results suggest that eustatic fluctuations associated with Milankovitch cycles have played a central role in species demographic histories and in the final stage of the community assembly of many Rapa Nui reef fishes. Specifically, sea level lowstands resulted in the maximum reef habitat extension for Rapa Nui endemic species; we discuss the potential role of seamounts in allowing endemic species to cope with Pleistocene climatic fluctuations, and we highlight the importance of local historical processes over regional ones. Overall, our results shed light on the mechanisms by which endemism arises and is maintained in peripheral reef fish fauna.     

Gene expression patterns in Atlantic salmon Salmo salar: gene expression during osmoregulation in intestine tissue

Ireland has the world's largest stocks of wild Atlantic salmon. A better understanding of gene expression will benefit conservation of wild stock as well as salmon aquaculture. We describe the PRTLI project designed to advance the fundamental understanding of the genome of Atlantic Salmon Salmo salar . The major objective is to create the first comprehensive database of gene expression and functional information using cDNA libraries and Microarray technology. One key area of interest to salmon biology is osmoregulation, which is critical to the ability of salmon to adapt in seawater. Tissues implicated in this process are the gills, intestine and skin. To initiate studies, SSH (suppression subtractive hybridization) libraries were constructed from intestine RNA extracted from smolts sampled in January and May. A number of potentially interesting clones have been identified, among those a heat shock protein, hsp90 in the reverse library. Others SSH libraries from various tissues (pituitary, hypothalamus, brain, gill, intestine, head kidney and spleen) have also been constructed and will be used to construct a 5000 clone microarray slide. This slide will then be used to elucidate gene expression profiles in various tissues. Further sample collection has been carried out to answer questions regarding biologicaldifferences between one‐ year and two‐year old parr and wild and hatchery smolt.     

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.     

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.