The Batten Disease Palmitoyl Protein Thioesterase 1 Gene Regulates Neural Specification and Axon Connectivity during Drosophila Embryonic Development

Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL

Rotaatiodopplerilmiö kaksiatomisen molekyylin fotoelektronispektroskopiassa

Tiivistelmä. Tässä tutkielmassa käsittelen aluksi perusasioita molekyylin rakenteen kuvaamisesta molekyyliorbitaaliteorian avulla, sekä tutustun molekyylin tutkimiseen käytettyihin spektroskooppisiin menetelmiin erityisesti kaksiatomisten molekyylien ja fotoelektronispektroskopian kannalta. Tämän jälkeen esittelen kaksi tutkimusta. Ensimmäisessä raportoidaan kokeesta, jolla on havaittu ensimmäistä kertaa rotaatiodopplerleveneminen typpimolekyylin fotoelektronispektrissä. Toisessa tutkimuksessa kerrotaan uudesta menetelmästä, jolla elektronin emittoitumisen puoli on saatu selville analysoimalla vetykloridin spektrin rotaatiokaistoja. Lopuksi pohdin mahdollisuutta käyttää molekyylin rotaatioon liittyvää Doppler-ilmiötä hyödyksi molekyylin rakenteen tutkimisessa

Interaction of huntingtin fragments with brain membranes - clues to early dysfunction in Huntington's disease

Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis

The Pharmacokinetics and Pharmacodynamics of SM-1

Introduction: The most common form of sleep disturbance is transient insomnia, and in parallel the most common pattern of use for hypnotic medications is short-term use. We report here the results of a daytime sedation clinical trial with a staged-release combination drug product, SM-1, comprised of immediate release diphenhydramine 50 mg, delayed-release zolpidem 5 mg, and delayed-release lorazepam 0.5 mg. Methods: This was a double blind, placebo controlled, randomized, crossover study to assess the pharmacokinetics and pharmacodynamics of SM-1 in 24 healthy volunteers. Blood samples were collected prior to and at the following time points after the dose: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 10, 12, 16 and 24 hours to determine the concentrations of diphenhydramine, zolpidem and lorazepam in plasma. Sedative drug effects were assessed with visual analogue scales (VAS) and the digit symbol substitution test (DSST) prior to and at the following time points after dosing: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 10, 12 and 24 hours. Memory acquisition and immediate recall were assessed with a word list test at 1, 2, 4, 6, 8, 10 and 12 hours after the dose administration. Electroencephalographic (EEG) recording were carried out prior to and at the following time points after the dose: 1, 2, 4, 6, 8, 10 and 12 hours, for off-line analysis of the proportion of beta frequency amplitude in the EEG spectrum. Results: The drug combination was well tolerated, as evidenced by similar number and severity of adverse events following administration of SM-1 and placebo. The pharmacodynamic profile of SM-1 showed onset of activity, as determined by subjective, performance, and EEG surrogates for sedation, at 0.5-1 hour post-dose, lasting about 7-7.5 hours. Pharmacokinetic profiles showed that the shapes of the plasma concentration curves for the two delayed-release components were altered compared with published data with unmodified drugs, while for all three agents the exposure values obtained with the combination product are in good agreement with published or historical values of the drugs given individually. Conclusion:A single 15 mg dose of GBX reduces ORP and arousal index to the same extent as four nights of SR alone. Augmentation of sleep depth by GBX remains stable across four nights of SR. The correlation between changes in ORP and in arousal index further supports the use of ORP as a continuous measure of sleep depth

Mol Cell

Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis