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Selected AbstractsMyosin-II negatively regulates minor process extension and the temporal development of neuronal polarityDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2009K.M. Kollins Abstract The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Surfactant-coated single-walled carbon nanotubes as a novel pseudostationary phase in capillary EKCELECTROPHORESIS, Issue 11 2007Beatriz Suárez Abstract The analytical potential of the use of surfactant-coated single-walled carbon nanotubes (SC-SWNTs) as pseudostationary phase in CE is described. The pseudostationary phase shows an efficient alternative in enhancing electrochromatographic resolution of compounds which are capable of interacting with a nanotube surface, such as aromatic compounds. In general, the resolution is enhanced by increasing nanotube concentration in the buffer but the maximum amount of SWNTs that can be added to background electrolyte was found limited by compatibility with the UV/visible detection. As an alternative, a low-extension partial filling was used, consisting of the introduction into the capillary of concentrated SC-SWNT, just before the sample, with a plug length similar to the sample one. This has been showed as a reliable procedure in increasing resolution and sensitivity by sweeping phenomena. Finally, the potential of SC-SWNTs to perform chiral separations is discussed. [source] Search for direct empirical spatial correlation signatures of the critical triggering earthquake modelGEOPHYSICAL JOURNAL INTERNATIONAL, Issue 3 2004G. Ouillon SUMMARY We propose a new test of the critical earthquake model based on the hypothesis that precursory earthquakes are ,actors' that create fluctuations in the stress field which exhibit an increasing correlation length as the critical large event becomes imminent. Our approach constitutes an attempt to build a more physically based time-dependent indicator (cumulative scalar stress function), in the spirit of, but improving on, the cumulative Benioff strain used in previous works documenting the phenomenon of accelerating seismicity. Using a simplified scalar space and time-dependent viscoelastic Green's function in a two-layer model of the Earth's lithosphere, we compute spatiotemporal pseudo-stress fluctuations induced by a series of events before four of the largest recent shocks in southern California. Through an appropriate spatial wavelet transform, we then estimate the contribution of each event in the series to the correlation properties of the simplified pseudo-stress field around the location of the mainshock at different scales. This allows us to define a cumulative scalar pseudo-stress function which reveals neither an acceleration of stress storage at the epicentre of the mainshock nor an increase of the spatial stress,stress correlation length similar to those observed previously for the cumulative Benioff strain. The earthquakes we studied are thus either simple ,witnesses' of a large-scale tectonic organization, or are simply unrelated, and/or the Green's function describing interactions between earthquakes has a significantly longer range than predicted for standard viscoelastic media used here. [source] Specificity and reactive loop length requirements for crmA inhibition of serine proteasesPROTEIN SCIENCE, Issue 2 2005Lisa D. Tesch Abstract The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (,102,104 M,1sec,1), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (kdiss ,10,7 sec,1), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (kcat/KM of ,106 M,1 sec,1). N-terminal sequencing confirmed that the P1 Arg,P1,Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1,Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ,10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (105,106 M,1sec,1), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1,Cys,P2,Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites. [source] Rational shape engineering of the filamentous protein , prefoldin through incremental gene truncationBIOPOLYMERS, Issue 6 2009Timothy A. Whitehead Abstract An enticing possibility in nanotechnology is to use proteins as templates for the positioning of molecules in regular patterns with nanometer precision over large surface areas. However, the ability to redesign protein quaternary structure to construct new shapes remains underdeveloped. In the present work, we have engineered the dimensions of a filamentous protein, the , prefoldin (, PFD) from the hyperthermophile Methanocaldococcus jannaschii, and have achieved controllable attachment of filaments in a specific orientation on a carbon surface. Four different constructs of , PFD were generated in which the coiled coils extending from the association domain are progressively truncated. Three of the truncation constructs form well-defined filaments with predictable dimensions according to transmission electron microscopy. Two of these constructs had 2D persistence lengths similar to that of , PFD at 300,740 nm. In contrast, the 2D persistence length of the shortest truncation mutant was 3500 nm, indicating that the filament adsorbs along a different axis than the other constructs with its two rows of coiled coils facing out from the surface. The elastic moduli of the filaments range from 0.7,2.1 GPa, similar to rigid plastics and within the lower limit for proteins whose primary intermolecular interaction is hydrogen bonding. These results demonstrate a versatile approach for controlling the overall dimensions and surface orientation of protein filaments, and expand the toolbox by which to tune two overall dimensions in protein space for the creation of templated materials over a wide variety of conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 496,503, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | |||||||||||||