The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.     

Determination of dihedral Psi angles in large proteins by combining NH(N)/C(alpha)H(alpha) dipole/dipole cross-correlation and chemical shifts

We propose a strategy based on the combination of experimental NH(N)/C(alpha)H(alpha) dipole/dipole cross-correlated relaxation rates and chemical shift analysis for the determination of Psi torsion angles in proteins. The method allows the determination of a dihedral angle that is not easily accessible by nuclear magnetic resonance (NMR). The measurement of dihedral angle restraints can be used for structure calculation, which is known to improve the quality of NMR structures. The method is of particular interest in the case of large proteins, for which spectral assignment of the nuclear Overhauser effect spectra, and therefore straightforward structural determination, is out of reach. One advantage of the method is that it is reasonably simple to implement, and could be used in association with other methods aiming at obtaining structural information on complex systems, such as residual dipolar coupling measurements. An illustrative example is analyzed in the case of the 30-kDa protein 6-phosphogluconolactonase.     

Size distribution dependence of prion aggregates infectivity

We consider a model for the polymerization (fragmentation) process involved in infectious prion self-replication and study both its dynamics and non-zero steady state. We address several issues. Firstly, we extend a previous study of the nucleated polymerization model [M.L. Greer, L. Pujo-Menjouet, G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006) 598; H. Engler, J. Pruss, G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006) 98] to take into account size dependent replicative properties of prion aggregates. This is achieved by a choice of coefficients in the model that are not constant. Secondly, we show stability results for this steady state for general coefficients where reduction to a system of differential equations is not possible. We use a duality method based on recent ideas developed for population models. These results confirm the potential influence of the amyloid precursor production rate in promoting amyloidogenic diseases. Finally, we investigate how the converting factor may depend upon the aggregate size. Besides the confirmation that size-independent parameters are unlikely to occur, the present study suggests that the PrPsc aggregate size repartition is amongst the most relevant experimental data in order to investigate this dependence. In terms of prion strain, our results indicate that the PrPsc aggregate repartition could be a constraint during the adaptation mechanism of the species barrier overcoming, that opens experimental perspectives for prion amyloid polymerization and prion strain investigation.     

Genetic patchiness of the shore crab Pachygrapsus marmoratus along the Portuguese coast

The population genetic structure of the shore crab Pachygrapsus marmoratus was studied along the Portuguese coast based on six variable microsatellite loci. Genetic differentiation among populations according to a geographic gradient was not detected. This lack of genetic structure reflects the continuous distribution of the species along the Portuguese coast and suggests that gene flow occurs within the studied distribution range. Gene flow is probably maintained by the planktonic larvae of P. marmoratus that can last up to 31 days in the plankton. Tests for population differentiation demonstrated that the Praia das Avencas population is genetically more separated from all other populations, and Bayesian methodologies tend to form 4 groups that clustered together populations that are several hundred kilometres apart. This grouping pattern could be due to coastal hydrological events that are apparently influencing larval flux. Other hypotheses to explain the significant genetic heterogeneity among populations on a local scale and the absence of geographic variation are pre- and post-settlement natural selection events. Results suggest that the forces causing genetic differentiation may be acting on a local scale and that the larval pool is possibly not always mixed homogeneously.     

Toxoplasma gondii transmembrane microneme proteins and their modular design

Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM‐MICs). Toxoplasma gondii possesses functionally non‐redundant MIC complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM‐MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase. During invasion these adhesive complexes are shed from the surface notably via intramembrane cleavage of the TM‐MICs by a rhomboid protease. Some TM‐MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM‐MICs lack targeting information within their C‐terminal domains, indicating that trafficking depends on yet unidentified proteins interacting with their ectodomains. Most TM‐MICs serve as substrates for a rhomboid protease and some of them are able to bind to aldolase. We also show that the residues responsible for binding to aldolase are essential for TgAMA1 but dispensable for TgMIC6 function during invasion.     

Old and new inhibitors of quinone reductase 2

Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC₅₀ in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin < resveratrol < S29434. These molecular tools might permit to explore and better understand the relationship existing between QR2 catalytic activity and the various pathological situations in which QR2 has a key role.     

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

Background  

Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.