Aberration measurement using in-situ two-beam interferometry

A reticle with phase-only blazed gratings ofvaiying azimuthal orientations diffracts light into only two orders, 0 & +1, discretely illuminating a lens pupil. The image ofeach grating is a sinusoidal interference pattern and is recorded as a surface relief in a highly absorbing photoresist. The maximum image contrast occurs when focus is set such that the RMS wavefront error over the two beams is minimized. This maximum contrast vs focus is recorded by a CCD array mounted on a dark-field optical microscope and the aberrations are obtained from an analysis of this record. Repeatability of equivalent primary aberrations ofless than O.OO17RMS are achieved and used to monitor lens stability.published_or_final_versio

Activation and modulation of recombinant glycine and GABAA receptors by 4-halogenated analogues of propofol

BACKGROUND AND PURPOSE: Glycine receptors are important players in pain perception and movement disorders, and therefore an important therapeutic target. Glycine receptors can be modulated by the intravenous anesthetic propofol (2,6-diisopropylphenol); however, the drug is more potent, by at least one order of magnitude, on GABAA receptors. It has been proposed that halogenation of the propofol molecule generates compounds with selective enhancement of glycinergic modulatory properties. EXPERIMENTAL APPROACH: We synthesized 4-bromopropofol, 4-chloropropofol, and 4-fluoropropofol. The direct activating and modulatory effects of these drugs and propofol were compared on recombinant rat glycine and human GABAA receptors expressed in oocytes. Behavioral effects of the compounds were compared in the tadpole loss-of-righting assay. KEY RESULTS: The concentration-response curves for potentiation of homomeric α1, α2, and α3 glycine receptors were shifted to lower drug concentrations by 2-10-fold for the halogenated compounds. Direct activation by all compounds was minimal with all subtypes of the glycine receptor. The four compounds were essentially equally potent modulators of the α1β3γ2L GABAA receptor with EC50 s between 4 and 7 μM. The EC50 s for loss-of-righting in Xenopus tadpoles, a proxy for loss of consciousness and considered to be mediated by actions on GABAA receptors, ranged from 0.35 to 0.87 μM. Conclusions and Implications We confirm that halogenation of propofol more strongly affects modulation of homomeric glycine receptors than α1β3γ2L GABAA receptors. However, the effective concentrations of all tested halogenated compounds remained lower for GABAA receptors. We infer that 4-bromo-, 4-chloro, or 4-fluoropropofol are not selective homomeric glycine receptor modulators

Gating at the Mouth of the Acetylcholine Receptor Channel: Energetic Consequences of Mutations in the αM2-Cap

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C↔O gating motions generate a low↔high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the α subunit (‘αM2-cap’). This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. αM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three αM2-cap residues (αI260, αP265 and αS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the αM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance

Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs

Ligand Activation of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC

While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors

Biallelic loss of LDB3 leads to a lethal pediatric dilated cardiomyopathy.

Autosomal dominant variants in LDB3 (also known as ZASP), encoding the PDZ-LIM domain-binding factor, have been linked to a late onset phenotype of cardiomyopathy and myofibrillar myopathy in humans. However, despite knockout mice displaying a much more severe phenotype with premature death, bi-allelic variants in LDB3 have not yet been reported. Here we identify biallelic loss-of-function variants in five unrelated cardiomyopathy families by next-generation sequencing. In the first family, we identified compound heterozygous LOF variants in LDB3 in a fetus with bilateral talipes and mild left cardiac ventricular enlargement. Ultra-structural examination revealed highly irregular Z-disc formation, and RNA analysis demonstrated little/no expression of LDB3 protein with a functional C-terminal LIM domain in muscle tissue from the affected fetus. In a second family, a homozygous LDB3 nonsense variant was identified in a young girl with severe early-onset dilated cardiomyopathy with left ventricular non-compaction; the same homozygous nonsense variant was identified in a third unrelated female infant with dilated cardiomyopathy. We further identified homozygous LDB3 frameshift variants in two unrelated probands diagnosed with cardiomegaly and severely reduced left ventricular ejection fraction. Our findings demonstrate that recessive LDB3 variants can lead to an early-onset severe human phenotype of cardiomyopathy and myopathy, reminiscent of the knockout mouse phenotype, and supporting a loss of function mechanism

Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens

The signal sequence influences post-translational ER translocation at distinct stages

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.Nicholas Johnson, Sarah Haßdenteufel, Melanie Theis, Adrienne W. Paton, James C. Paton, Richard Zimmermann, Stephen Hig