N-acylhomoserine lactone (AHL) quorum-sensing molecules modulate the swimming behaviour of zoospores of the macroalga Ulva to facilitate the location of bacterial biofilms. Here we show that the intertidal surfaces colonized by Ulva are dominated by Alphaproteobacteria, particularly the Rhodobacteraceae family, and the Bacteroidetes family Flavobacteriaceae, and that this diverse assemblage both produces and degrades AHLs. N-acylhomoserine lactones could also be extracted from the surfaces of pebbles recovered from intertidal rock-pools. Bacteria representative of this assemblage were isolated and tested for the production and degradation of AHLs, and for their ability to modulate zoospore settlement at different biofilm densities. Of particular interest was a Shewanella sp. This strain produced three major AHLs (OC4, OC10 and OC12) in the late exponential phase, but the longer-chain AHLs were rapidly degraded in the stationary phase. Degradation occurred via both lactonase and amidase activity. A close relationship was found between AHL synthesis and Ulva zoospore settlement. The Shewanella isolate also interfered with AHL production by a Sulfitobacter isolate and its ability to enhance zoospore settlement in a polymicrobial biofilm. This influence on the attachment of Ulva zoospores suggests that AHL-degrading strains can affect bacterial community behaviour by interfering with quorum sensing between neighbouring bacteria. More importantly, these interactions may exert wider ecological effects across different kingdoms.
SF5Phe, para-pentafluorosulfanyl phenylalanine, is an unnatural amino acid with extreme physicochemical properties, which is stable in physiological conditions. Here we present newly developed aminoacyl-tRNA synthetases that enable genetic encoding of SF5Phe for site-specific incorporation into proteins in high yields. Owing to the SF5 moiety’s dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins. The potential of SF5Phe in protein research is illustrated by (i) increasing the binding affinity of a consensus pentapeptide motif toward the β subunit of Escherichia coli DNA polymerase III holoenzyme by mutation of a phenylalanine to a SF5Phe residue, (ii) site-specifically adhering β-cyclodextrin to the surface of ubiquitin, and (iii) selective detection of 19F–19F nuclear Overhauser effects in the Escherichia coli peptidyl-prolyl cis/trans-isomerase B following mutation of two phenylalanine residues in the core of the protein to SF5Phe. With increasing use of the SF5 moiety in pharmaceutical chemistry, this general method of functionalizing proteins with SF5 groups opens unique opportunities for structural biology and in vivo studies.
Remarkable differences in action potential properties between the Purkinje fibre and ventricular cells may lead to abnormalities in excitation conduction through the Purkinje-ventricular junction (PVJ) IntroductionExcitation conducted through the Purkinje fibre (PF) network to the ventricles determines their normal electrical activation and contraction sequence. However, remarkable differences in action potential (AP) properties between the PF and ventricular cells [1,2] may lead to abnormalities in excitation conduction through the Purkinje-ventricular junction (PVJ) and arrhythmogenic behaviour [3][4][5]. Primarily, as action potentials (APs) from PF cells are typically longer than ventricular APs, it has been suggested that dispersion of the action potential duration (APD) at the PVJ can result in unidirectional conduction block [3], triggered activity [4] and reentry [5] under both physiological and pathological conditions.Computer simulations have been used previously in order to reconstruct APs in a single PF cell [6] and AP conduction in the whole fibre [7], as well as characterize electrotonic modulation of the APD dispersion at the PVJ [8]. However, the DiFrancesco-Noble model for a single rabbit PF cell [6] used in the latter simulations has been based on limited experimental data, whereas APs in ventricular cells were simulated using the Luo-Rudy model for a guinea-pig ventricular myocyte [9]. Hence, there is a demand for up-to-date non-chimeric models of the PVJ with realistic APD distributions.The aim of this study is (i) to develop a family of electrophysiologically detailed computer models for rabbit epicardial (epi), midmyocardial (M) and endocardial (endo) ventricular myocytes, (ii) develop a detailed model for the rabbit PF cell, (iii) simulate a realistic APD dispersion due to intercellular electrotonic interactions during conduction through the PVJ under normal conditions, and (iv) explore changes of the APD dispersion under pathological conditions of the short QT syndrome (SQTS) associated with a gain-in-function of I Kr channel due to HERG N588K mutation [10,11]. MethodsDynamics of electrical variables in cardiac tissues can be described by the Hodgkin-Huxley-type nonlinear partial differential equation (PDE) [7,8]:Here V (mV) is the membrane potential, t is time (s), ∇ is a spatial gradient operator defined within the tissue geometry. D is the effective diffusion coefficient (mm 2 ms -1) that characterizes electrotonic spread of voltage via gap junctions. C m (pF) is the cell membrane capacitance, I ion is the total membrane ionic current (pA). Various biophysically detailed mathematical models have been developed to describe the voltage and time dependent current I ion , and hence, action potential (AP) propertiesprimarily in a rabbit ventricular cell [12].The equation (1) is solved for the 1D strand geometry using a finite-difference PDE solver that implements the explicit Euler's method with time and space steps ∆t = 0.005 ms and ∆x = 0.1 mm, respectively. Ventricular modelsT...
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