Characterization and functional divergence of the alpha 1-adrenoceptor gene family: insights from rainbow trout (Oncorhynchus mykiss).

Presently, three alpha(1)-adrenoceptor (AR) types are recognized in vertebrates: alpha(1A)-, alpha(1B)-, and alpha(1D)-ARs. These alpha(1)-subtypes have distinct pharmacology and molecular profiles, play crucial roles in metabolic and vascular control, and are the targets for numerous pharmaceuticals, especially those affecting blood pressure and vascular resistance. To better understand the functional divergence within the alpha(1)-AR gene family, we sequenced these alpha(1)-AR paralogs in the rainbow trout and performed an extensive phylogenetic analysis. We show that these AR genes evolved by duplication events just before the origin of the jawed vertebrates. Our computational analyses suggest that the differences between the three alpha(1)-AR subtypes may affect their tissue specificity, ligand specificity, and possibly signal transduction processes and desensitization. We also show that, within each subtype, differences exist between fish and mammalian receptors, both at the transcriptional and at the physiological level. These differences, however, suggest that the role of alpha(1)-ARs in fish is more complex than previously thought. Our integrated analysis of the alpha(1)-AR gene family suggests that these receptors evolved these distinct features very early within vertebrates.

Trehalose preserves the integrity of lyophilized phycoerythrin-antihuman CD8 antibody conjugates and enhances their thermal stability in flow cytometric assays.

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption-emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8-RPE (CD8-RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose-CD8-RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8(+) lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the "cold chain" distribution, particularly suited for use in less-developed countries of the tropical areas.

Identification and properties of a Gs protein in catfish liver membranes.

The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5'-O-(gamma-thiotriphosphate) (GTPgammaS) (maximal activation of about eightfold at 10(-5) M; half-maximal activation at 1.31 x 10(-7) M), and reduced by the competitive inhibitor of GTP, GDPbetaS (70% maximal inhibition at 10(-4) M; half-maximal inhibition at 1.98 x 10(-7) M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 microM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [(35)S]GTPgammaS binding assay. At 100 microM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20 degrees, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPgammaS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 microM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a G(s)alpha-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-G(s)alpha antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein alpha(s) subunits in the liver of an ectothermal vertebrate.