Effect of lin-benzoadenosine and lin-benzoadenosine 3':5'-monophosphate on cyclic AMP-dependent protein kinase activity in vitro.

We have shown that the fluorescent "stretched-out" analog of cyclic AMP, namely linear-benzo-cyclic AMP, maximally activates brain protein kinase and protein kinase from skeletal muscle. The corresponding linear-benzoadenosine inhibits kinase activity slightly less than does adenosine. Thus, the 2.4A-wider versions of cyclic AMP and of adenosine interact with protein kinase in a manner similar to that of the natural compounds.     

Stimulation of polypeptide synthesis by cyclic 3'-5'-guanosine monophosphate

3′-5′-Cyclic GMP was found to stimulate polyphenylalanine synthesis in a cell-free system derived from rat liver. The effect of cyclic GMP is detected either in the presence or absence of GTP and appears quite specific since it is not produced by other cyclic nucleotides.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

A mini-library of TBA analogues containing 3'-3' and 5'-5' inversion of polarity sites

Several researches have been devoted to structure-activity relationship and to post-SELEX modifications of the thrombin binding aptamer (TBA), one of the first aptamers discovered by the SELEX methodology. However, no studies on TBA dealing with the effects of introduction of inversion of polarity sites have been reported yet. In this frame, we have undertaken the synthesis and the study of a mini-library composed of several TBA analogues containing a 3'-3' or a 5'-5' inversion of polarity site at different positions into the sequence. Particularly, in this article, we present preliminary results about their structural and biological properties.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Stimulation of rat liver cyclic 3':5'-nucleotide phosphodiesterase by cyclic GMP is dependent on enzyme concentration

A high-speed supernatant of rat liver extract displayed multiple forms of cyclic nucleotide phosphodiesterase (EC 3.1.4.17). One of the forms catalyzed the hydrolysis of cyclic AMP and cyclic GMP, with approximately comparable facility. One salient feature of the enzyme is that at micromolar concentrations, cyclic GMP stimulated the hydrolysis of cyclic AMP, but not vice versa. Another is that the activity of phosphodiesterase varied as a function of enzyme concentration in the assayed system: the enzyme activity was higher at low than at high enzyme concentrations. A concentrated enzyme was not stimulated by cyclic GMP but was stimulated by cyclic GMP upon dilution of the enzyme. Conversely, stimulation of the enzyme by cyclic GMP could be reversed by increasing the enzyme concentration. The cyclic GMP-stimulated cyclic AMP phosphodiesterase was partially purified by a continuous sucrose density gradient. The apparent change of phosphodiesterase activity as a function of enzyme concentration was also observed after partial purification by the sucrose density gradient. High enzyme concentrations favored the aggregated form of phosphodiesterase, whereas low concentrations favored the dissociated form. Dilution of the enzyme shifted the equilibrium toward the dissociated form, which presumably exposed the cyclic GMP regulatory site on the enzyme molecule.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).