Inhibition of secreted phospholipase A2 by neuron survival and anti-inflammatory peptide CHEC-9

BACKGROUND: The nonapeptide CHEC-9 (CHEASAAQC), a putative inhibitor of secreted phospholipase A2 (sPLA2), has been shown previously to inhibit neuron death and aspects of the inflammatory response following systemic treatment of rats with cerebral cortex lesions. In this study, the properties of CHEC-9 inhibition of sPLA2 enzymes were investigated, using a venom-derived sPLA2 group I and the plasma of rats and humans as the sources of enzyme activity. The results highlight the advantages of inhibitors with uncompetitive properties for inflammatory disorders including those resulting in degeneration of neurons. METHODS: Samples of enzyme and plasma were reacted with 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine, a sPLA2 substrate that forms phospholipid vesicles in aqueous solutions. Some of the plasma samples were collected from restrained peptide-treated rats in order to confirm the validity of the in vitro assays for extrapolation to in vivo effects of the peptide. The enzyme reactions were analyzed in terms of well-studied relationships between the degree of inhibition and the concentrations of different reactants. We also examined interactions between different components of the reaction mixture on native polyacrylamide gels. RESULTS: In all cases, the peptide showed the properties of an uncompetitive (or anti-competitive) enzyme inhibitor with Ki values less than 100 nanomolar. The electrophoresis experiments suggested CHEC-9 modifies the binding properties of the enzyme only in the presence of substrate, consistent with its classification as an uncompetitive inhibitor. Both the in vitro observations and the analysis of plasma samples from restrained rats injected with peptide suggest the efficacy of the peptide increases under conditions of high enzyme activity. CONCLUSION: Modeling studies by others have shown that uncompetitive inhibitors may be optimal for enzyme inhibition therapy because, unlike competitive inhibitors, they are not rendered ineffective by the accumulation of unmodified substrate. Such conditions likely apply to several instances of neuroinflammation where there are cascading increases in sPLA2s and their substrates, both systemically and in the CNS. Thus, the present results may explain the efficacy of CHEC-9 in vivo

Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties

<p>Abstract</p> <p>Background</p> <p>Lysophosphatidylcholines (lysoPCs) are products of phospholipase A2 (PLA2) enzyme activity, and like the enzyme, have a direct role in toxic inflammatory responses in variety of organ systems. Paradoxically, reduced plasma lysoPC levels have been noted in sepsis patients and systemic treatment with lysoPCs is therapeutic in rodent models of sepsis and ischemia. These observations suggest that elevation of plasma levels of these lipids can actually help to relieve serious inflammatory conditions. We demonstrate that specific lysoPCs act as uncompetitive product inhibitors of plasma secreted PLA2 enzymes (sPLA2s), especially under conditions of elevated enzyme activity, thus providing a feedback mechanism for the observed anti-inflammatory effects of these compounds.</p> <p>Methods</p> <p>Thin layer chromatography and mass spectroscopy were used to estimate total lysoPC concentration and the relative contributions of different lysoPC species in rat plasma samples. Kinetic studies of sPLA2 enzyme activity were conducted on these samples <it>ex vivo </it>and on purified group IA sPLA2 <it>in vitro </it>after addition of specific lysoPC species to the reaction mixture. Enzyme activity was also measured in plasma samples of rats injected with these same lysoPCs.</p> <p>Results</p> <p>Palmitoyl (16:0), stearoyl (18:0) are the most abundant lysoPCs in rat plasma consistent with other reports. Kinetic studies demonstrated that both were uncompetitive inhibitors of plasma sPLA2 enzyme activity. <it>In vitro </it>experiments with group IA sPLA2 confirmed the inhibition and the kinetic properties of these lysoPC species. Decanoyl lysoPC (10:0), which was not detected in plasma, did not inhibit enzyme activity in vitro. LysoPC injections into normal rats resulted in "buffering" of plasma sPLA2 activity in a narrow low range, consistent with the activity-dependent inhibition suggested by the <it>ex vivo </it>and <it>in vitro </it>experiments.</p> <p>Conclusion</p> <p>The results may explain the efficacy of lysoPC therapy during periods of elevated inflammatory activity and further highlight the utility uncompetitive enzyme inhibitors. In this case, the inhibitor is a product of the enzyme reaction, and therefore represents an example of activity-driven feedback inhibition.</p

Substrate Induced Structural and Dynamics Changes in Human Phosphomevalonate Iinase and Implications for Mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P Kd\u27s were 6–60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (τc = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S2 ∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker-A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ-phosphate binding interactions. Indeed, the role of γ-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β-γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley-Liss, Inc

Time-Domain Methods for Diffusive Transport in Soft Matter

Passive microrheology [12] utilizes measurements of noisy, entropic fluctuations (i.e., diffusive properties) of micron-scale spheres in soft matter to infer bulk frequency-dependent loss and storage moduli. Here, we are concerned exclusively with diffusion of Brownian particles in viscoelastic media, for which the Mason-Weitz theoretical-experimental protocol is ideal, and the more challenging inference of bulk viscoelastic moduli is decoupled. The diffusive theory begins with a generalized Langevin equation (GLE) with a memory drag law specified by a kernel [7, 16, 22, 23]. We start with a discrete formulation of the GLE as an autoregressive stochastic process governing microbead paths measured by particle tracking. For the inverse problem (recovery of the memory kernel from experimental data) we apply time series analysis (maximum likelihood estimators via the Kalman filter) directly to bead position data, an alternative to formulas based on mean-squared displacement statistics in frequency space. For direct modeling, we present statistically exact GLE algorithms for individual particle paths as well as statistical correlations for displacement and velocity. Our time-domain methods rest upon a generalization of well-known results for a single-mode exponential kernel [1, 7, 22, 23] to an arbitrary M-mode exponential series, for which the GLE is transformed to a vector Ornstein-Uhlenbeck process

Founder Characteristics and Legitimacy-Seeking Behaviors

The entrepreneur’s experience, personality, and values affect the entrepreneur’s behaviors and decisions (Chrisman, Bauerschmidt, and Hofer 1998). Past research results show that (1) more experienced new venture founders have a greater likelihood of leading their ventures to early success than less experienced founders (Delmar and Shane 2006) and (2) founders who engage in legitimacy-seeking behaviors have a greater likelihood of leading their ventures to early success than founders who do not do so (Tornikoski and Newbert 2007). We propose that more experienced founders understand the importance of obtaining legitimacy for their ventures and therefore will engage in more legitimacy-seeking behaviors. In addition, we propose that entrepreneurs’ growth aspirations and internal locus of control are also associated with engagement in legitimacy-seeking behaviors. We test and find support for these propositions in a sample of new ventures and their founders

Preferred EAC initiation sites in 7xxx aluminum

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Mie resonance engineering in meta-shell supraparticles for nanoscale nonlinear optics

Supraparticles are coordinated assemblies of discrete nanoscale building blocks into complex and hierarchical colloidal superstructures. Holistic optical responses in such assemblies are not observed in an individual building block or in their bulk counterparts. Furthermore, subwavelength dimensions of the unit building blocks enable engraving optical metamaterials within the supraparticle, which thus far has been beyond the current pool of colloidal engineering. This can lead to effective optical features in a colloidal platform with unprecedented ability to tune the electromagnetic responses of these particles. Here, we introduce and demonstrate the nanophotonics of meta-shell supraparticle (MSP), an all dielectric colloidal superstructure having an optical nonlinear metamaterial shell conformed onto a spherical core. We show that the metamaterial shell facilitates engineering the Mie resonances in the MSP that enable significant enhancement of the second harmonic generation (SHG). We show several orders of magnitude enhancement of second-harmonic generation in an MSP compared to its building blocks. Furthermore, we show an absolute conversion efficiency as high as 10^-7 far from the damage threshold, setting a new benchmark for SHG with low-index colloids. The MSP provides pragmatic solutions for instantaneous wavelength conversions with colloidal platforms that are suitable for chemical and biological applications. Their engineerability and scalability promise a fertile ground for nonlinear nanophotonics in the colloidal platforms with structural and material diversity

Mie Resonance Engineering in Meta-Shell Supraparticles for Nanoscale Nonlinear Optics

Supraparticles are coordinated assemblies of discrete nanoscale building blocks into complex and hierarchical colloidal superstructures. Holistic optical responses in such assemblies are not observed in an individual building block or in their bulk counterparts. Furthermore, subwavelength dimensions of the unit building blocks enable engraving optical metamaterials within the supraparticle, which thus far has been beyond the current pool of colloidal engineering. This can lead to effective optical features in a colloidal platform with ability to tune the electromagnetic responses of these particles. Here, we introduce and demonstrate the nanophotonics of meta-shell supraparticle (MSP), an all dielectric colloidal superstructure having an optical nonlinear metamaterial shell conformed onto a spherical core. We show that the metamaterial shell facilitates engineering the Mie resonances in the MSP that enable significant enhancement of the second harmonic generation (SHG). We show several orders of magnitude enhancement of second-harmonic generation in an MSP compared to its building blocks. Furthermore, we show an absolute conversion efficiency as high as 10⁻⁷ far from the damage threshold, setting a benchmark for SHG with low-index colloids. The MSP provides pragmatic solutions for instantaneous wavelength conversions with colloidal platforms that are suitable for chemical and biological applications. Their engineerability and scalability promise a fertile ground for nonlinear nanophotonics in the colloidal platforms with structural and material diversity