Substitution of a mutant α2a-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and α2 adrenergic receptor (α2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each α2AR subtype (α2A, α2B, and α2C) plays in these central effects. Here we demonstrate that α2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated α2AAR, D79N α2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N α2AAR to inhibit voltage-gated Ca2+ currents and spontaneous neuronal firing, a measure of K+ current activation. These results provide definitive evidence that the α2AAR subtype is the primary mediator of clinically important central actions of α2AR agonists and suggest that the D79N α2AAR mouse may serve as a model for exploring other possible α2AAR functions in vivo.

Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit α4

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the α4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged α4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of α4. Localization of Mid1 and α4 was influenced by one another in transiently transfected cells. Mid1 could recruit α4 onto microtubules, and high levels of α4 could displace Mid1 into the cytosol. Metabolic 32P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length α4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein–Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by α4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Core derived downhole logs for holes CRP-2 and CRP-2A

In the northern McMurdo Sound (Ross Sea, Antarctica), the CRP-2/2A drillhole targeted the western margin of the Victoria Land Basin to investigate Neogene to Palaeogene climatic and tectonic history by obtaining continuous core and downhole logs. Well logging of CRP-2/2A has provided a complete and comprehensive dataset of in situ geophysical measurements. This paper describes the evaluation and interpretation of the downhole logging data using multivariate statistical methods. Two major types of multivariate statistical methods were each yielding a different perspective: (1) Factor analysis was used as an objective tool for classification of the drilled sequence based on physical and chemical properties. The factor logs are mirroring the basic geological controls (i.e., grain size, porosity, clay mineralogy) behind the measured geophysical properties, thereby making them easier to interpret geologically. (2) Cluster analysis of the logs groups similar downhole geophysical properties into one cluster, delineating individual logging or sedimentological units. These objectively and independently defined units, or statistical electrofacies, are helpful in differentiating lithological and sedimentological characterisations (e.g. grain size, provenance). The multivariate statistical methods of factor and cluster analysis proved to be powerful tools for fast, reliable, and objective characterisation of downhole geophysical properties at CRP-2/2A, resulting in interpretations which are consistent with sedimentological findings.

Clay and smectite analysis of sediment core CRP-2A

Sediments of the CRP-2/2A drill core from the continental shelf of McMurdo Sound in Ross Sea, Pacific sector of the Southern Ocean, have been investigated for their clay mineral assemblages, especially for the smectite contents and smectite crystallinities. Highest smectite amounts and best crystallinities occur in three intervals below 485 mbsf in CRP-2/2A. They indicate deposition of sediments during a time when chemical weathering was active on large ice-free areas on the nearby Antarctic continent. In he upper part of the core, smectite contents are much lower and crystallinities are worse. This clay mineral composition indicates deposition of sediments during a time when physical weathering prevailed on an ice-covered continent. At deep-sea sites around Antarctica the shift from smectite-dominated to smectite-poor and illite-rich assemblages is well dated as earliest Oligocene, 33.9-33.1 Ma, and documents the onset of continental glaciation in East Antarctica. At CIROS-1 a corresponding shift in the clay mineralogy was observed at a depth of 425-445 mbsf.

Direct support, general support and depot maintenance manual, including repair parts and special tools lists for engine, with container, turbosupercharged, diesel, fuel injection, 90-degree "V" type, air cooled, 12-cylinder, assembly; models AVDS-1790-2M (2815-856-4996), AVDS-1790-2A and AVDS-1790-2AM (2815-856-9005).

Includes indexes.