Structural features of the C-terminus from the human neurokinin-1 receptor

The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and β-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% β-sheets and β-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions.     

FTIR spectroscopy of secondary-structure reorientation of melibiose permease modulated by substrate binding

Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from Escherichia coli shows that the oriented structures correspond mainly to tilted transmembrane α-helices, forming an average angle of ∼26° with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H₂O and D₂O makes apparent two populations of α-helices differing by their tilt angle (helix types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na⁺ binding, whereas it decreases upon subsequent melibiose binding in the presence of Na⁺. In contrast, melibiose binding in the presence of H⁺ causes virtually no change in the average tilt angle. The data also suggest that the two helix populations change their tilting and H/D exchange level in different ways depending on the bound substrate(s). Notably, cation binding essentially influences type I helices, whereas melibiose binding modifies the tilting of both helix populations.     

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 μM blebbistatin, disordering was at least 10 times slower. In the M·ADP state, myosin heads are also disordered. When blebbistatin was added to M·ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.     

Synthesis, in vitro cytotoxicity and in vivo anti-inflammatory activity of long chain 3-amino-1,2-diols

The synthesis of long chain 3-amino-1,2-diols was carried out based on Sharpless asymmetric epoxidation of long chain allylic alcohols and regioselective nucleophilic ring opening by azido group. The in vitro cytotoxicity of the compounds prepared was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10, UMSCC-22B). Free 3-amino-1,2-diols exhibited IC50 values between 1.45 microM and 32 microM. These compounds also presented interesting inhibition of carrageenin-induced paw edema in rats (85.3% - 79.6% at a concentration of 0.15 mmol/kg).     

Vertical migration of phototrophic bacterial populations in a hypersaline microbial mat from Salins-de-Giraud (Camargue, France)

The spatio-temporal distribution of phototrophic communities of the hypersaline photosynthetic Camarguc microbial mat (Salins-de-Giraud, France) was investigated over a diel cycle by combining microscopic and molecular approaches. Microcoleus chthonoplastes and Halomicronema excentricum, the dominant cyanobacteria of this oxyphotrophic community, were observed with confocal laser scanning microscopy to determine their biomass profiles. Both bacteria have similar vertical distributions, varying from a homogenous distribution through the mat during the night, to a specific localization in the upper oxic zone of 1.5 mm during the day. Terminal restriction fragment length polymorphism of PCR-amplified pufM gene fragments revealed three groups of anoxyphototrophic populations, which varied according to the two opposite periods of the diel cycle under study. They were either specifically detected in only one period, or homogenously distributed through the mat in all periods, or located in specific zones of the mat depending on the period considered. Oxygen concentrations, pH and biomass of the major filamentous cyanobacteria were the determinative factors in the distribution of these anoxyphototrophs across the mat. Thus, vertical migration, cell-cell aggregate formation and metabolic switches were the most evident defence of the photosynthetic populations against the adverse effects of sulfide and oxygen fluxes during a diel cycle.     

In silico study of the human rhodopsin and meta rhodopsin II/S-arrestin complexes: impact of single point mutations related to retina degenerative diseases

We propose two models of the human S-arrestin/rhodopsin complex in the inactive dark adapted rhodopsin and meta rhodopsin II form, obtained by homology modeling and knowledge based docking. First, a homology model for the human S-arrestin was built and validated by molecular dynamics, showing an average root mean square deviation difference from the pattern behavior of 0.76 A. Then, combining the human S-arrestin model and the modeled structure of the two human rhodopsin forms, we propose two models of interaction for the human S-arrestin/rhodopsin complex. The models involve two S-arrestin regions related to the N domain (residues 68-78; 170-182) and a third constituent of the C domain (248-253), with the rhodopsin C terminus (330-348). Of the 22 single point mutations related to retinitis pigmentosa and congenital night blindness located in the cytoplasmatic portion of rhodopsin or in S-arrestin, our models locate 16 in the interaction region and relate two others to possible dimer formation. Our calculations also predict that the light activated complex is more stable than the dark adapted rhodopsin and, therefore, of higher affinity to S-arrestin.     

Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areas

Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed ≥ 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed ≥ 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time.     

Selective isolation and identification of N-terminal blocked peptides from tryptic protein digests.

A method for the easy isolation and direct sequencing of N-terminally blocked peptide in proteins refractory to N-terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the Nalpha-blocked peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N-terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.     

Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy

Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested to be involved in epileptogenesis, via modulation of intracellular calcium levels, excitotoxicity, activation of inflammatory cascades, and cell death, among other mechanisms. These processes have been described to be involved in pilocarpine-induced status epilepticus (SE) and contribute to hyperexcitability, resulting in spontaneous and recurrent seizures. Here, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using RNA interference (RNAi) to inhibit the expression of this receptor. Small interfering RNA (siRNA) targeting P2X7R mRNA was injected into the lateral ventricles (icv) 6 h after SE. Four groups were studied: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle, and Pilo-siRNA. P2X7R was quantified by western blotting and neuronal death assessed by Fluoro-Jade B histochemistry. The hippocampal volume (edema) was determined 48 h following RNAi. Behavioral parameters as latency to the appearance of spontaneous seizures and the number of seizures were determined until 60 days after the SE onset. The Saline-siRNA and Pilo-siRNA groups showed a 43 and 37% reduction, respectively, in P2X7R protein levels compared to respective vehicle groups. Neuroprotection was observed in CA1 and CA3 of the Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the edema detected in the hilus, suprapyramidal dentate gyrus, CA1, and CA3; reduced mortality rate following SE; increased the time to onset of spontaneous seizure; and reduced the number of seizures, when compared to the Pilo-Vehicle group. Therefore, our data highlights the potential of P2X7R as a therapeutic target for the adjunct treatment of epilepsy.     

O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels

Abstract The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs]) and outer membrane proteins [OMPs] was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB⁺ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB⁻ strains antigenically diverse that either exhibited a heterogenous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB⁺ strains, whereas major OMPs detected in PAB⁻ strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.