RESUMO
We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.
Assuntos
Canabinoides/metabolismo , Receptores de Droga/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Canabinoides , Receptores de Droga/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.
Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , Sequência de Bases , Southern Blotting , Células Cultivadas , Ciclosporina/farmacologia , DNA de Cadeia Simples , Humanos , Interferon gama/genética , Interleucina-4/genética , Cinética , Dados de Sequência Molecular , Plasmídeos , Receptores de Interleucina-2/genética , Titulometria , Fator de Necrose Tumoral alfa/genética , Microglobulina beta-2/genéticaRESUMO
DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.
