Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low- K m GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the Μ-selective opioid agonist [ 3 H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([ 3 H]DAMGO) bound to two populations of sites with K D values of 3.9 and 160 n M , with <10% of the sites in the high-affinity state. Both sites were also detected at 4°C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [ 3 H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [ 3 H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [ 3 H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the B max for [ 3 H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP. Opioid receptor coupling to adenylate cyclase in intact and fragmented cells occurred with similar efficiency: DAMGO inhibited adenylate cyclase with K i , values of 11 n M in cells and 26 n M in lysates, with 30% maximal inhibition in both preparations. Receptor coupling to G protein in membranes occurred with similar parameters: DAMGO stimulated low- K m GTPase with a K s of 31 n M and an S max of 48%. Both effector responses were blocked by naloxone and were strongly impaired by rigorous cell homogenization. These results indicate that opioid signal transduction in intact SH-SY5Y cells and their appropriately isolated membranes functions with similar efficiencies involving a large reserve of uncoupled receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66450/1/j.1471-4159.1992.tb10032.x.pd

Provincial and Territorial Interests in Land Use Planning and Development

Report: iv., pp.156, appendix A-G.; ill., digital file.Land use planning in Canada is in the process of undergoing a radical shift as legislation, much of which dates from the 1980s or even the 1970s, is less and less able to cope with the increasing complexity of land use issues faced in every region of the country. Growing metropolitan regions, mechanization and intensification of farming practices and a growing concern over environmental issues have radically altered the context within which planning takes place. Concerns with water quality, particularly in light of the Walkerton tragedy, as well as the advent of Intensive Livestock Operations and rapidly expanding urban fringes are at the fore of land use issues facing planners in Canada today.The Intergovernmental Committee on Urban and Regional Researc

Autonomous space processor for orbital debris

This work continues to develop advanced designs toward the ultimate goal of a GETAWAY SPECIAL to demonstrate economical removal of orbital debris utilizing local resources in orbit. The fundamental technical feasibility was demonstrated last year through theoretical calculations, quantitative computer animation, a solar focal point cutter, a robotic arm design and a subscale model. During this reporting period, several improvements are made in the solar cutter, such as auto track capabilities, better quality reflectors and a more versatile framework. The major advance has been in the design, fabrication and working demonstration of a ROBOTIC ARM that has several degrees of freedom. The functions were specifically tailored for the orbital debris handling. These advances are discussed here. Also a small fraction of the resources were allocated towards research in flame augmentation in SCRAMJETS for the NASP. Here, the fundamental advance was the attainment of Mach numbers up to 0.6 in the flame zone and a vastly improved injection system; the current work is expected to achieve supersonic combustion in the laboratory and an advanced monitoring system

Dynein Modifiers in C. elegans: Light Chains Suppress Conditional Heavy Chain Mutants

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis

A Comparative Study of the ReCell® Device and Autologous Spit-Thickness Meshed Skin Graft in the Treatment of Acute Burn Injuries.

Early excision and autografting are standard care for deeper burns. However, donor sites are a source of significant morbidity. To address this, the ReCell® Autologous Cell Harvesting Device (ReCell) was designed for use at the point-of-care to prepare a noncultured, autologous skin cell suspension (ASCS) capable of epidermal regeneration using minimal donor skin. A prospective study was conducted to evaluate the clinical performance of ReCell vs meshed split-thickness skin grafts (STSG, Control) for the treatment of deep partial-thickness burns. Effectiveness measures were assessed to 1 year for both ASCS and Control treatment sites and donor sites, including the incidence of healing, scarring, and pain. At 4 weeks, 98% of the ASCS-treated sites were healed compared with 100% of the Controls. Pain and assessments of scarring at the treatment sites were reported to be similar between groups. Significant differences were observed between ReCell and Control donor sites. The mean ReCell donor area was approximately 40 times smaller than that of the Control (P &lt; .0001), and after 1 week, significantly more ReCell donor sites were healed than Controls (P = .04). Over the first 16 weeks, patients reported significantly less pain at the ReCell donor sites compared with Controls (P ≤ .05 at each time point). Long-term patients reported higher satisfaction with ReCell donor site outcomes compared with the Controls. This study provides evidence that the treatment of deep partial-thickness burns with ASCS results in comparable healing, with significantly reduced donor site size and pain and improved appearance relative to STSG

Host pathogen interactions in relation to management of light leaf spot disease (caused by Pyrenopeziza brassicae) on Brassica species

Light leaf spot, caused by Pyrenopeziza brassicae, is currently the most damaging disease problem in oilseed rape in the UK. According to recent survey data, the severity of epidemics has increased progressively across the UK, with current yield losses of up to £160M per annum in England and more severe epidemics in Scotland. Light leaf spot is a polycyclic disease with primary inoculum consisting of air-borne ascospores produced on diseased debris from the previous cropping season. Splash-dispersed conidia produced on diseased leaves are the main component of the secondary inoculum. P. brassicae is also able to infect and cause considerable yield losses on vegetable brassicas, especially Brussels sprouts. There may be spread of light leaf spot among different brassica species. Since they have a wide host range, Pyrenopeziza brassicae populations are likely to have considerable genetic diversity and there is evidence suggesting population variations between different regions, which need further study. Available disease-management tools are not sufficient to provide adequate control of the disease. There is a need to identify new sources of resistance, which can be integrated with fungicide applications to achieve sustainable management of light leaf spot. Several major resistance genes and quantitative trait loci have been identified in previous studies, but rapid improvements in the understanding of molecular mechanisms underpinning B. napus – P. brassicae interactions can be expected through exploitation of novel genetic and genomic information for brassicas and extracellular fungal pathogens.Peer reviewe

HIPed Tailored Ceramic Waste Forms for the Immobilization of Cs, Sr and Tc

The Advanced Fuel Cycle Initiative is developing advanced technologies to allow for the safe and economical disposal of waste from nuclear reactors. An important element of this initiative is the separation of key radionuclides . One of the systems being developed to separate key radionuclides is the UREX+1 process. The Tc and Cs/Sr solutions from UREX+1 process will require treatment and solidification for managed storage. This paper illustrates the benefits of HIPed tailored ceramic waste forms, to provide for the immobilization of separated Cs, Sr and Tc. Experimental data are presented for a Cs and Sr-bearing hollandite-rich tailored ceramic prepared with 12 wt% waste (on an oxide basis). Normalized MCC-1 type leach testing at 90oC for 28 days revealed extremely low Cs and Sr release rates of 0.003 and 0.004 g/m2/day respectively. Experimental data on the immobilization of Tc in titanate ceramics containing up to 40wt% TcO2 are also be presented

Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers

Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits

Determining the Electronic Confinement of a Subsurface Metallic State

Dopant profiles in semiconductors are important for understanding nanoscale electronics. Highly conductive and extremely confined phosphorus doping profiles in silicon, known as Si:P δ-layers, are of particular interest for quantum computer applications, yet a quantitative measure of their electronic profile has been lacking. Using resonantly enhanced photoemission spectroscopy, we reveal the real-space breadth of the Si:P δ-layer occupied states and gain a rare view into the nature of the confined orbitals. We find that the occupied valley-split states of the δ-layer, the so-called 1Γ and 2Γ, are exceptionally confined with an electronic profile of a mere 0.40 to 0.52 nm at full width at half-maximum, a result that is in excellent agreement with density functional theory calculations. Furthermore, the bulk-like Si 3pz orbital from which the occupied states are derived is sufficiently confined to lose most of its pz-like character, explaining the strikingly large valley splitting observed for the 1Γ and 2Γ states