Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]triphosphate

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O

Evolutionary Dynamics in a Simple Model of Self-Assembly

We investigate the evolutionary dynamics of an idealised model for the robust self-assembly of two-dimensional structures called polyominoes. The model includes rules that encode interactions between sets of square tiles that drive the self-assembly process. The relationship between the model's rule set and its resulting self-assembled structure can be viewed as a genotype-phenotype map and incorporated into a genetic algorithm. The rule sets evolve under selection for specified target structures. The corresponding, complex fitness landscape generates rich evolutionary dynamics as a function of parameters such as the population size, search space size, mutation rate, and method of recombination. Furthermore, these systems are simple enough that in some cases the associated model genome space can be completely characterised, shedding light on how the evolutionary dynamics depends on the detailed structure of the fitness landscape. Finally, we apply the model to study the emergence of the preference for dihedral over cyclic symmetry observed for homomeric protein tetramers

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+

General implementation of all possible positive-operator-value measurements of single photon polarization states

Positive Operator Value Measures (POVMs) are the most general class of quantum measurements. We propose a setup in which all possible POVMs of a single photon polarization state (corresponding to all possible sets of two-dimensional Kraus operators) can be implemented easily using linear optics elements. This method makes it possible to experimentally realize any projective orthogonal, projective non-orthogonal or non-projective sets of any number of POVM operators. Furthermore our implementation only requires vacuum ancillas, and is deterministic rather than probabilistic. Thus it realizes every POVM with the correct set of output states. We give the settings required to implement two different well-known non-orthogonal projective POVMs.Comment: 5 pages, newer version with minor addition

Nondeterministic self-assembly with asymmetric interactions.

We investigate general properties of nondeterministic self-assembly with asymmetric interactions, using a computational model and DNA tile assembly experiments. By contrasting symmetric and asymmetric interactions we show that the latter can lead to self-limiting cluster growth. Furthermore, by adjusting the relative abundance of self-assembly particles in a two-particle mixture, we are able to tune the final sizes of these clusters. We show that this is a fundamental property of asymmetric interactions, which has potential applications in bioengineering, and provides insights into the study of diseases caused by protein aggregation.Winton Programme for the Physics of Sustainability, Gates Cambridge, Oppenheimer PhD studentship, NanoDTC Cambridge (Grant ID: EP/L015978/1), Engineering and Physical Sciences Research Council (Grant ID: EP/L504920/1), Royal SocietyThis is the author accepted manuscript. The final version is available from the American Physical Society via http://dx.doi.org/10.1103/PhysRevE.94.02240

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis

Numerical and experimental assessment of the modal curvature method for damage detection in plate structures

This paper is concerned with the use of numerically obtained modal curvatures for damage detection in both isotropic and composite laminated plates. Numerical simulations are carried out by using COMSOL Multiphysics as FEM solver of the governing equations, in which a Mindlin-Reissner plate model is assumed and defects are introduced as localized smoothed variations of the baseline (healthy) configuration. Experiments are also performed on steel and aluminum plates using scanning laser vibrometry. This study confirms that the central difference method greatly amplifies the measurement errors and its application leads to ineffective predictions for damage detection, even after denoising. As a consequence, different numerical techniques should be explored to allow the use of numerically obtained modal curvatures for structural health monitoring. Herein, the Savitzky-Golay filter (or least-square smoothing filter) is considered for the numerical differentiation of noisy data

Self-assembly, modularity and physical complexity

We present a quantitative measure of physical complexity, based on the amount of information required to build a given physical structure through self-assembly. Our procedure can be adapted to any given geometry, and thus to any given type of physical system. We illustrate our approach using self-assembling polyominoes, and demonstrate the breadth of its potential applications by quantifying the physical complexity of molecules and protein complexes. This measure is particularly well suited for the detection of symmetry and modularity in the underlying structure, and allows for a quantitative definition of structural modularity. Furthermore we use our approach to show that symmetric and modular structures are favoured in biological self-assembly, for example of protein complexes. Lastly, we also introduce the notions of joint, mutual and conditional complexity, which provide a useful distance measure between physical structures.Comment: 9 pages, submitted for publicatio

Numerical and experimental assessment of the modal curvature method for damage detection in plate structures

Use of modal curvatures obtained from modal displacement data for damage detection in isotropic and composite laminated plates is addressed through numerical examples and experimental tests. Numerical simulations are carried out employing COMSOL Multiphysics as finite element solver of the equations governing the Mindlin-Reissner plate model. Damages are introduced as localized non-smooth variations of the bending stiffness of the baseline (healthy) configuration. Experiments are also performed on steel and aluminum plates using scanning laser vibrometry. The obtained results confirm that use of the central difference method to compute modal curvatures greatly amplifies the measurement errors and its application leads to unreliable predictions for damage detection, even after denoising. Therefore, specialized ad hoc numerical techniques must be suitably implemented to enable structural health monitoring via modal curvature changes. In this study, the Savitzky-Golay filter (also referred to as least-square smoothing filter) is considered for the numerical differentiation of noisy data. Numerical and experimental results show that this filter is effective for the reliable computation of modal curvature changes in plate structures due to defects and/or damages