Human serum aryl acylamidase associated with serum cholinesterase was purified to homogeneity. Evidence for the identity of the two enzymes was based on co-elution profiles, co-purification in the different steps including affinity chromatography with constant ratios of specific activity and percentage recoveries, co-migration on gel electrophoresis, parallel inhibition by typical cholinesterase inhibitors and co-precipitation by antibody raised against the purified enzyme. Human liver aryl acylamidase was partially purified. Based on the elution profiles, purification data, inhibitory characteristics and gel electrophoresis it was concluded that aryl acrylamidase of liver was not associated with liver cholinesterase. More conclusive evidence for the non-association of the liver aryl acylamidase and cholinesterase came from their clear-cut separation on procainamide-Sepharose affinity chromatography. Both the serum and liver aryl acylamidase were compared with the purified erythrocyte aryl acylamidase associated with acetylcholinesterase. While the erythrocyte and serum aryl acylamidases showed some similarities in their sensitivities to amines like serotonin or tryptamine and choline derivatives, the liver enzyme was unaffected by any of these compounds. A notable observation was the activation by tyramine of the serum aryl acylamidase but not the erythrocyte and liver aryl acylamidases. The liver aryl acylamidase also differed from the other two in its relative insensitivity to inhibition by eserine, neostygmine and other cholinesterase inhibitors. Immunodiffusion and immunoprecipitation studies showed that the aryl acylamidases from the liver and erythrocytes were immunologically non-identical with the serum enzyme.
The delivery of oligonucleotides to appropriate intracellular compartments is crucial to their development as tools in gene function studies and as therapeutics. Here, we report the characterization of meso-substituted cationic porphyrins as a large class of water-soluble reagents for oligonucleotide delivery. These porphyrins form non-covalent complexes with single-stranded oligonucleotides and deliver these molecules into the nuclei of cell lines in culture. The porphyrins protect oligonucleotides from nuclease degradation, and delivery is unaffected by the presence of serum. Delivery capacity is dependent on the charge ratio and concentration of the oligonucleotide and porphyrin used to form the complex, on the chemical substituents of the oligonucleotide and on the identity of the cationic porphyrin. This class of molecules provides a versatile set of water-soluble delivery reagents that could contribute to the development of oligonucleotide drugs.
The beta1-adrenergic receptor (beta1-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat beta1-AR gene was assessed by transiently transfecting chimeric genes containing the beta1-AR promoter, driving the luciferase reporter gene into various cell lines. beta1-AR/luciferase vectors containing 3 kb of the 5'-flanking region and extending to -126 relative to the start site of translation were expressed at high levels in ventricular myocytes, SK-N-MC cells, and HepG2 cells. The addition of 26 nucleotides from -125 to -100 to the -3311 beta1-AR/luciferase chimeric gene reduced expression in myocytes and SK-N-MC cells while eliminating expression in HepG2 cells. This element is located 125 base-pairs 3' to the transcriptional start site. The mutation of four nucleotides between -121 and -118 diminished the inhibitory effect of this element. The inhibitory activity of the -125 to -100 sequence was completely dependent on promoter context and positioning. In addition to this 3' element, sequences between -3311 and -2740 in the 5'-flanking region of the beta1-AR gene were required for the full transcriptional suppression. Using DNase I footprinting and gel mobility assays, it was determined that within the 26-bp region, rat heart nuclear proteins bound to two sites between nucleotides -123 and -112 and -106 and -100. Therefore, appropriate basal expression of the beta1-AR gene involves widely separated sequences 3' and 5' to the transcriptional start site.
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