Isopeptide and Ester Bond Ubiquitination Regulate Degradation of the Human Dopamine Receptor 4

The human dopamine receptor 4 (hD4R) is a seven-transmembrane helical G protein-coupled receptor (GPCR) found in neural synaptic membranes. The neurotransmitter dopamine binds to and activates the hD4R, which is involved in central nervous system pathways that modulate cognition and circadian rhythms. The hD4R is the primary dopaminergic receptor for the atypical anti-psychotic drug clozapine, which is used to treat schizophrenia and other cognitive disorders. The hD4R gene is unique among the superfamily of GPCR-encoding genes because within the human population, it contains a variable number of tandem repeat (VNTR) exon polymorphism. Because of the VNTRs, the length of the primary structure of one of the intracellular loops of the hD4R can vary dramatically among individuals. Attempts have been made to correlate different VNTR structures with different behavioral traits – for example, a specific variant of hD4R is robustly correlated with attention deficit hyperactivity disorder. Like other GPCRs, hD4R functions at the plasma membrane by binding an extracellular ligand, in this case dopamine, to regulate an intracellular signaling cascade. The density of hD4R at the plasma membrane and its distribution within the neuron/synapse dictate the cellular response to dopamine. Despite the importance of hD4R in neuronal signaling, the molecular mechanisms regulating its cellular expression and degradation are unclear. Isopeptide ubiquitination of lysine residues on the cytoplasmic surface of various GPCRs regulates receptor abundance at the membrane by promoting protein degradation. I have studied the role of the ubiquitin-proteasome system in the cellular degradation of hD4R, and show here that hD4R protein levels are regulated through both a canonical and a non-canonical ubiquitination pathway. Site-directed mutagenesis of lysine residues, as well as mutagenesis of the atypical ubiquitin acceptors serine and threonine, led to an additive increase in mutant hD4R protein abundance in a cellular expression model. Chemical inhibition of the proteasome increased levels of the wild-type hD4R, but not the lysine, serine, and threonine null mutant. Both isopeptide ubiquitination of lysine and ester bond ubiquitination of serine and threonine were detected on hD4R in a model protein expression system using immunoprecipitation techniques. A proximity ligation assay was used to quantify isopeptide and ester bond ubiquitination in this protein expression system and to detect ubiquitination of hD4R in mouse primary cortical neurons. Together, these data support the hypothesis that hD4R is proteasomally degraded after isopeptide ubiquitination of lysine residues and ester ubiquitination of serine and threonine residues. The ubiquitination and subsequent degradation of hD4R represents a mechanism for cellular control over hD4R protein levels. While the low abundance of hD4R protein produced in heterologous expression systems has previously been limiting for biochemical and structural biology techniques, the degradation-resistant hD4R mutants presented here overcomes this limitation and may facilitate future research, including the identification of dopamine receptor interacting proteins (DRIPs). hD4R joins a small number of proteins that are known to be modified by ubiquitination via ester bonds. This work also describes novel techniques to confirm and quantify ester-bond ubiquitination for a given membrane protein within a cell

CO diffusion and desorption kinetics in CO2_2 ices

Diffusion of species in icy dust grain mantles is a fundamental process that shapes the chemistry of interstellar regions; yet measurements of diffusion in interstellar ice analogs are scarce. Here we present measurements of CO diffusion into CO$_2$ ice at low temperatures (T=11--23~K) using CO$_2$ longitudinal optical (LO) phonon modes to monitor the level of mixing of initially layered ices. We model the diffusion kinetics using Fick's second law and find the temperature dependent diffusion coefficients are well fit by an Arrhenius equation giving a diffusion barrier of 300 $\pm$ 40 K. The low barrier along with the diffusion kinetics through isotopically labeled layers suggest that CO diffuses through CO$_2$ along pore surfaces rather than through bulk diffusion. In complementary experiments, we measure the desorption energy of CO from CO$_2$ ices deposited at 11-50 K by temperature-programmed desorption (TPD) and find that the desorption barrier ranges from 1240 $\pm$ 90 K to 1410 $\pm$ 70 K depending on the CO$_2$ deposition temperature and resultant ice porosity. The measured CO-CO$_2$ desorption barriers demonstrate that CO binds equally well to CO$_2$ and H$_2$O ices when both are compact. The CO-CO$_2$ diffusion-desorption barrier ratio ranges from 0.21-0.24 dependent on the binding environment during diffusion. The diffusion-desorption ratio is consistent with the above hypothesis that the observed diffusion is a surface process and adds to previous experimental evidence on diffusion in water ice that suggests surface diffusion is important to the mobility of molecules within interstellar ices

A review assessing the current treatment strategies for postnatal psychological morbidity with a focus on post-traumatic stress disorder

Objective: to conduct a systematic review of randomised controlled trials investigating the efficacy of treatments used to manage postnatal psychological morbidity. Design: a systematic review was conducted of studies in English published from 1995 to 2011. Studies were included in the review if they were randomised controlled trials and had extractable data on symptoms of psychological morbidity after an intervention designed to manage the disorders in postnatal women. Eight studies met the criteria and were included in the review. Findings: the number of participants ranged from 58 to 1745. The interventions included group and individual counselling, debriefing and expressive writing. Authors of only three studies reported fewer symptoms of PTSD after the intervention. Those that appeared to be helpful were counselling and expressive writing. However most authors did not assess pre-existing PTSD. Key conclusions and implications for practice: the review revealed that there was no standardised scale used for diagnosis of post-traumatic stress disorder across the studies and no single efficacious treatment. A universal instrument for diagnosis of postnatal post-traumatic stress disorder is required. The intrapartum relationship with midwives appears to be an important contributor to prevention of PTSD and this requires further investigation. © 2012 Elsevier Ltd

European Non-native Species in Aquaculture Risk Analysis Scheme - a summary of assessment protocols and decision support tools for use of alien species in aquaculture

The European Non-native Species in Aquaculture Risk Analysis Scheme (ENSARS) was developed in response to European 'Council Regulation No. 708/2007 of 11 June 2007 concerning use of alien and locally absent species in aquaculture' to provide protocols for identifying and evaluating the potential risks of using non-native species in aquaculture. ENSARS is modular in structure and adapted from non-native species risk assessment schemes developed by the European and Mediterranean Plant Protection Organisation and for the UK. Seven of the eight ENSARS modules contain protocols for evaluating the risks of escape, introduction to and establishment in open waters, of any non-native aquatic organism being used (or associated with those used) in aquaculture, that is, transport pathways, rearing facilities, infectious agents, and the potential organism, ecosystem and socio-economic impacts. A concluding module is designed to summarise the risks and consider management options. During the assessments, each question requires the assessor to provide a response and confidence ranking for that response based on expert opinion. Each module can also be used individually, and each requires a specific form of expertise. Therefore, a multidisciplinary assessment team is recommended for its completion

EM-21 Higher Waste Loading Glasses for Enhanced DOE High-Level Waste Melter Throughput Studies -10194

ABSTRACT Supplemental validation data have been generated that will be used to determine the applicability of the current Defense Waste Processing Facility (DWPF) liquidus temperature (T L ) model to expanded DWPF glass regions of interest based on higher waste loadings. For those study glasses which had very close compositional overlap with the model development and/or model validation ranges (except TiO 2 and MgO concentrations), there was very little difference in the predicted and measured T L values, even though the TiO 2 contents were above the 2 wt% upper concentration covered by the model. The results indicate that the current T L model is applicable in these compositional regions. As the compositional overlap between the model validation ranges diverged from the target glass compositions, the T L data suggest that the model underpredicted the measured values. These discrepancies imply that the influence of individual oxides or their combinations at concentrations outside of the model development and/or previous validation regions may not be adequately assessed by the current model. These oxides include B 2 O 3 , SiO 2 , MnO, TiO 2 and/or their combinations. More data are required to fill in these anticipated DWPF compositional regions so that the model coefficients could be refit to account for these influences. INTRODUCTION High-level waste (HLW) throughput (i.e., the amount of waste processed per unit time) is a function of several parameters, two of which are extremely critical: waste loading (WL) and melt rate. For the Defense Waste Processing Facility (DWPF) at the Savannah River Site (SRS), increasing HLW throughput would significantly reduce the overall mission life cycle costs for the Department of Energy (DOE)

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

Experiments performed on the heat resistant organism CK 4-6 are described. Its response to dry heat at two temperatures (125 C and 135 C) at eight humidity levels (0.001 percent to 100 percent RH) in a closed can system is studied

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

Dry heat sterilization of spacecraft was investigated by studying the production of spore crops, and thermal inactivation of the spores, and bacillus subtillus. Spore assays were made by conventional plate count methods, and survival curves for the spores are presented. The results indicate that the inherent resistance of spores from a parent cell can be maintained