The antibiotic polymyxin B modulates P2X7 receptor function

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Investigation of de novo totally random biosequences, Part III: RNA Foster: A novel assay to investigate RNA folding structural properties

Fold is essential to RNA properties, and, in particular, its thermodynamic stability can be used to monitor RNA-protein or RNA-ligand interactions, and to engineer RNA with novel or improved properties. While clearly valuable, experimental determination of RNA folding stability by traditional biophysical techniques requires substantial amounts of pure sample and rather expensive equipment. In this paper, we report a new, simple approach to the determination of RNA folding stability by coupling enzymatic digestion and temperature denaturation. The assay, named RNA folding stability Test (RNA Foster), is designed to probe the fraction of folded RNA (f(fold)) in an equilibrium mixture of folded and unfolded ones as a function of temperature. The simplicity of RNA Foster suggests that it can easily be scaled up for high-throughput studies of RNA folding stability both in basic and applied research.     

Investigation of de novo totally random biosequences, Part IV: Folding Properties of de novo, totally random RNAs

This work lies within the framework of a broader project aimed at exploring the realm of all possible folded polypeptides; the main question addressed here is whether the corresponding RNAs also assume a folded conformation. We present an investigation on the structural properties of de novo, totally random RNAs by means of the 'RNA Foster' assay. Experimental results show that all RNAs studied are folded at 37 degrees , so that fold seems to be a common feature of RNAs. Random RNAs' fold shows a surprising thermal stability with an average T(m) value at ca. 50 degrees which prompts the idea that thermo-stable structures might not be as rare as they are commonly thought to be. The results are discussed within the general framework of random RNA properties such as those that might have been produced in a prebiotic scenario.     

Hydrodynamic interaction of two unsteady model microorganisms

The study of pair-wise interactions between swimming microorganisms is fundamental to the understanding of the rheological and transport properties of semi-dilute suspensions. In this paper, the hydrodynamic interaction of two ciliated microorganisms is investigated numerically using a boundary-element method, and the microorganisms are modeled as spherical squirmers that swim by time-dependent surface deformations. The results show that the inclusion of the unsteady terms in the ciliary propulsion model has a large impact on the trajectories of the interacting cells, and causes a significant change in scattering angles with potential important consequences on the diffusion properties of semi-dilute suspensions. Furthermore, the analysis of the shear stress acting on the surface of the microorganisms revealed that the duration and the intensity of the near-field interaction are significantly modified by the presence of unsteadiness. This observation may account for the hydrodynamic nature of randomness in some biological reactions, and supersedes the distinction between intrinsic randomness and hydrodynamic interactions, adding a further element to the understanding and modeling of interacting microorganisms.     

Assessing coral bleaching and recovery with a colour reference card in Watamu Marine Park,Kenya

With this study we estimated the changes in colour, bleaching and mortality of coral colonies from February to December 2007, using the colour reference card method. The study was developed in the Watamu Marine Park lagoon (Kenya), bridging the local summer when seawater temperatures were highest and coral bleaching risk was at its maximum. Seven coral genera were selected, and their colour recorded using a colour reference card (Coral Watch card). Seven different scenarios of bleaching and mortality were observed, varying among the coral genera and between two species in the genus Pocillopora. Twenty percent of the colonies bleached, of which 50% died. Only 15% of the coral that did not bleach died. Branching genera had a higher bleaching incidence than massive and sub-massive genera. Pocillopora showed the highest bleaching susceptibility, followed by Acropora, and the highest level of mortality. Of the two species of Pocillopora considered in this study, P. eydouxi showed higher bleaching and mortality levels, while P. verrucosa bleached less and experienced only partial mortality. Our results evidenced different patterns of coral bleaching and mortality which were easily and clearly detected with the colour card method during both bleaching and a post-bleaching events.     

Development and limitations of lentivirus vectors as tools for tracking differentiation in prostate epithelial cells

To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133⁺ cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133⁺ cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation.     

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25^+/− mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.     

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.