The G protein-coupling specificity of D2-like dopamine receptors

The G-protein coupling specificity of D2-like dopamine receptors was investigated using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion. A range of ligands displayed agonism at the long isoform of the human dopamine D2 receptor. However, varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [35S] GTPγS onto each of Gi1, Gi2, Gi3 and Go1. By contrast, S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine (S-(-)-3PPP) was a partial agonist when Go1 was the target G protein but an antagonist/inverse agonist at Gi1, Gi2, Gi3. In ligand binding assays dopamine identified both high and low affinity states at each of the dopamine D2 receptor-G protein fusion proteins and the high affinity state was eliminated by guanine nucleotide. S-(-)-3PPP bound to an apparent single state of the constructs where the D2 receptor was fused to Gi1, Gi2 or Gi3. However, it bound to distinct high and low affinity states of the D2 receptor-Go1 fusion with the high affinity state being eliminated by guanine nucleotide. Similarly, although dopamine identified guanine nucleotide-sensitive high affinity states of the D2 receptor when expression of pertussis toxin-resistant forms of either Gi2 or Go1 was induced, S-(-)-3PPP identified a high affinity site only in the presence of Go1. These results demonstrate S-(-)-3PPP to be a protean agonist at the D2l receptor and may explain in vivo actions of this ligand. Furthermore, in agreement with previous studies, the ability of the dopamine D2l receptor to couple promiscuously to Gαi1-3, and Gαo1 was demonstrated. However, despite high homology between dopamine D2l and D3 receptors, the G protein-coupling specificity of the D3 receptor has not been well characterised. Again using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion, we confirmed the selective coupling of the D3 receptor to Gαo1. A range of ligands displayed agonism at the D2l receptor and the D3 receptor when coupled to Gαo1. As a general trend, agonists, including dopamine, displayed a higher potency at the D3 receptor. This perhaps reflects the role of D3 as an autoreceptor. Of particular interest was the demonstration that S-(-)-3PPP has both a higher efficacy and potency at the D3 receptor when coupled to Gαo1. The investigations into dopamine receptor-G protein coupling highlighted the utility of the [35S]GTPγS binding assay as a method of directly measuring receptor catalysed nucleotide exchange on the α subunit of G proteins. However, the expense associated with the use of radiolabels makes this assay less attractive, particularly for high-throughput screening programmes. In an attempt to develop a non-radioactive assay equivalent to the [35S] GTPγS assay an immunisation programme was initiated to generate antibodies selective against the active (GTP bound) conformation of G proteins. 4 way primary screening of 1632 hybridomas generated from mice immunized with GTPS-loaded Gi1 and isolated using an automated robotic colony picker, identified 3 antibodies that interacted with the constitutively active Q204L but neither the constitutively inactive G203A nor wild type form of Gi1. This profile extended to other closely related Gi-family G proteins but not to the less closely related Gs and Gq/G11 families. Each of these antibodies was, however, also able to identify wild type, GDP-bound Gi- family G proteins in the presence of AlF4- which mimics the presence of the terminal phosphate of GTP and hence generates an active conformation of the G protein. Stimulation of cells co-expressing a wild type Gisubunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformation selective antibodies to bind the G protein. Such reagents allow the development of label- free assays for G protein-coupled receptor-mediated activation of Gi- family G proteins

Oralism: a sign of the times? The contest for deaf communication in education provision in late nineteenth-century Scotland

Disability history is a diverse field. In focussing upon children within deaf education in late nineteenth-century Scotland, this essay reflects some of that diversity. In 1880, the International Congress on the Education of the Deaf in Milan stipulated that speech should have ‘preference’ over signs in the education of deaf children. The mode of achieving this, however, effectively banned sign language. Endeavours to teach deaf children to articulate were not new, but this decision placed pressures on deaf institutions to favour the oral system of deaf communication over other methods. In Scotland efforts were made to adopt oralism, and yet educators were faced with the reality that this was not good educational practice for most pupils. This article will consider responses of Scottish educators of deaf children from the 1870s until the beginning of the twentieth century

Reconstructive Plastic Surgery An Atlas of Essential Procedures

A reader-friendly, how-to guide on reconstructive plastic surgery from international experts Reconstructive Plastic Surgery: An Atlas of Essential Procedures edited by esteemed authors, educators, and surgeons Robert X. Murphy Jr. and Charles K. Herman is a comprehensive resource detailing head-to-toe surgical procedures for a broad range of conditions. The senior editors have more than 50 years of collective surgical experience and expertise training hundreds of medical students and plastic surgery residents. A distinguished and diverse group of contributors from more than 15 countries and five continents share clinical pearls throughout the book. Sixty-seven chapters organized in five sections start with head and neck chapters detailing cleft palate defects and repair, followed by functional rhinoplasty, neoplasms, and trauma. Section two encompasses breast reduction/reconstruction techniques and other breast deformities; and management of trunk ulcers, deep wounds, and defects. The hand and upper extremity section details reconstructive techniques for infections, trauma, and Dupuytren's contracture. The final two sections cover a wide spectrum of nerve-related conditions and syndromes, followed by burns, melanoma, and vascular anomalies. Key Features High-quality illustrations and intraoperative photographs enhance understanding of step-by-step operative procedures More than 30 procedural videos provide hands-on guidance on how to perform specific steps in reconstructive plastic surgery A broad range of reconstructive techniques cover trauma, tumor resection, burns, congenital deformities, and degenerative conditions Consistent chapter formatting includes a clear and concise introduction, discussion of pertinent anatomy, surgical indications, operative techniques, complications, and long-term results This highly accessible yet comprehensive procedural guide is must-have reading for medical students, plastic surgery residents, and early-career plastic surgeons. It will also benefit veteran reconstructive plastic surgeons looking for a robust refresher with an international perspective

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used

Search for gravitational waves from Scorpius X-1 with a hidden Markov model in O3 LIGO data

Results are presented for a semicoherent search for continuous gravitational waves from the low-mass x-ray binary Scorpius X-1, using a hidden Markov model (HMM) to allow for spin wandering. This search improves on previous HMM-based searches of Laser Interferometer Gravitational-Wave Observatory data by including the orbital period in the search template grid, and by analyzing data from the latest (third) observing run. In the frequency range searched, from 60 to 500 Hz, we find no evidence of gravitational radiation. This is the most sensitive search for Scorpius X-1 using a HMM to date. For the most sensitive subband, starting at 256.06 Hz, we report an upper limit on gravitational wave strain (at 95% confidence) of h 95 % 0 = 6.16 × 10 − 26 , assuming the orbital inclination angle takes its electromagnetically restricted value ι = 4 4 ° . The upper limits on gravitational wave strain reported here are on average a factor of ∼ 3 lower than in the second observing run HMM search. This is the first Scorpius X-1 HMM search with upper limits that reach below the indirect torque-balance limit for certain subbands, assuming ι = 4 4 °