Insensitivity to Abeta42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

Social runaway: Fisherian elaboration (or reduction) of socially selected traits via indirect genetic effects

Our understanding of the evolutionary stability of socially selected traits is dominated by sexual selection models originating with R. A. Fisher, in which genetic covariance arising through assortative mating can trigger exponential, runaway trait evolution. To examine whether nonreproductive, socially selected traits experience similar dynamics—social runaway—when assortative mating does not automatically generate a covariance, we modeled the evolution of socially selected badge and donation phenotypes incorporating indirect genetic effects (IGEs) arising from the social environment. We establish a social runaway criterion based on the interaction coefficient, ψ , which describes social effects on badge and donation traits. Our models make several predictions. (1) IGEs can drive the original evolution of altruistic interactions that depend on receiver badges. (2) Donation traits are more likely to be susceptible to IGEs than badge traits. (3) Runaway dynamics in nonsexual, social contexts can occur in the absence of a genetic covariance. (4) Traits elaborated by social runaway are more likely to involve reciprocal, but nonsymmetrical, social plasticity. Models incorporating plasticity to the social environment via IGEs illustrate conditions favoring social runaway, describe a mechanism underlying the origins of costly traits, such as altruism, and support a fundamental role for phenotypic plasticity in rapid social evolution.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.     

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

Anatomy and histochemistry of spread-wing posture in birds. 2. Gliding flight in the California Gull,Larus californicus: A paradox of fast fibers and posture

Gliding flight is a postural activity which requires the wings to be held in a horizontal position to support the weight of the body. Postural behaviors typically utilize isometric contractions in which no change in length takes place. Due to longer actin-myosin interactions, slow contracting muscle fibers represent an economical means for this type of contraction. In specialized soaring birds, such as vultures and pelicans, a deep layer of the pectoralis muscle, composed entirely of slow fibers, is believed to perform this function. Muscles involved in gliding posture were examined in California gulls (Larus californicus) and tested for the presence of slow fibers using myosin ATPase histochemistry and antibodies. Surprisingly small numbers of slow fibers were found in the M. extensor metacarpi radialis, M. coracobrachialis cranialis, and M. coracobrachialis caudalis, which function in wrist extension, wing protraction, and body support, respectively. The low number of slow fibers in these muscles and the absence of slow fibers in muscles associated with wing extension and primary body support suggest that gulls do not require slow fibers for their postural behaviors. Gulls also lack the deep belly to the pectoralis found in other gliding birds. Since bird muscle is highly oxidative, we hypothesize that fast muscle fibers may function to maintain wing position during gliding flight in California gulls. J. Morphol. 233:237–247, 1997. © 1997 Wiley-Liss, Inc.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.