Cloning, expression, and functional analysis of human dopamine D1 receptors

Aim : To construct an HEK293 cell line stably expressing human dopamine D 1 receptor (D 1 R). Methods : cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D 1 R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D 1 R in HEK293 cells was monitored by the [ 3 H]SCH23390 binding assay. The function of D 1 R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. Results : An HEK293 cell line stably expressing human D 1 R was obtained. A saturation radioligand binding experiment with [ 3 H]SCH23390 demonstrated that the K d and B max values were 1.5±0.2 nmol/L and 2.94±0.15 nmol/g of protein, respectively. In the [ 3 H]SCH23390 competition assay, D 1 R agonist SKF38393 displaced [ 3 H]SCH23390 with an IC 50 value of 2.0 (1.5–2.8) Μmol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D 1 R expressed in HEK293 cells in a concentration-dependent manner with an EC 50 value of 0.25 (0.12–0.53) Μmol/L and 0.39 (0.27–0.57) Μmol/L at 6 h/0.59 (0.22–1.58) Μmol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC 50 value of 27 (8.6–70) nmol/L. Conclusion : An HEK293 cell line stably expressing human D 1 R was obtained successfuly. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75187/1/j.1745-7254.2005.00017.x.pd

Quinpramine Ameliorates Rat Experimental Autoimmune Neuritis and Redistributes MHC Class II Molecules

Activation of inflammatory cells is central to the pathogenesis of autoimmune demyelinating diseases of the peripheral nervous system. The novel chimeric compound quinpramine—generated from imipramine and quinacrine—redistributes cholesterol rich membrane domains to intracellular compartments. We studied the immunological and clinical effects of quinpramine in myelin homogenate induced Lewis rat experimental autoimmune neuritis (EAN), a model system for acute human inflammatory neuropathies, such as the Guillain-Barré syndrome. EAN animals develop paresis of all limbs due to autoimmune inflammation of peripheral nerves. Quinpramine treatment ameliorated clinical disease severity of EAN and infiltration of macrophages into peripheral nerves. It reduced expression of MHC class II molecules on antigen presenting cells and antigen specific T cell proliferation both in vitro and in vivo. Quinpramine exerted its anti-proliferatory effect on antigen presenting cells, but not on responder T cells. Our data suggest that quinpramine represents a candidate pharmaceutical for inflammatory neuropathies

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Cannulated screw and hexapodal fixator reconstruction for compound upper tibial fractures

OBJECTIVES: The aim of the treatment of tibial plateau fractures is to obtain a pain-free and fully functional knee with closed reduction, percutaneous cannulated screw fixation and hexapodal external fixator reconstruction for high energy compound upper tibial fractures. METHODS: Patients with comminuted tibial plateau fractures underwent closed reduction, percutaneous fixation with cannulated screws, and reconstruction with hexapodal external fixator. The follow-up period was 24 months. RESULTS: The clinical and radiological results were good or excellent. The average knee flexion was 125°. CONCLUSION: Our results are successful in the initial stage, however, it should be pointed out that during the long term follow-up osteoarthritis may develop leading to worsening of the condition. Level of Evidence IV, Case Series

Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

Metallic, magnetic and molecular nanocontacts

Scanning tunnelling microscopy and break-junction experiments realize metallic and molecular nanocontacts that act as ideal one-dimensional channels between macroscopic electrodes. Emergent nanoscale phenomena typical of these systems encompass structural, mechanical, electronic, transport, and magnetic properties. This Review focuses on the theoretical explanation of some of these properties obtained with the help of first-principles methods. By tracing parallel theoretical and experimental developments from the discovery of nanowire formation and conductance quantization in gold nanowires to recent observations of emergent magnetism and Kondo correlations, we exemplify the main concepts and ingredients needed to bring together ab initio calculations and physical observations. It can be anticipated that diode, sensor, spin-valve and spin-filter functionalities relevant for spintronics and molecular electronics applications will benefit from the physical understanding thus obtained