A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate     

Histochemical method for localization of cyclic 3′, 5′-nucleotide phosphodiesterase

Summary A histochemical method has been described for demonstration of cyclic 3, 5-nucleotide phosphodiesterase in tissues. 5-AMP is formed due to splitting of substrate cyclic 3, 5-AMP by cyclic 3, 5-AMPase. The 5-AMP is split into adenosine and phosphate by the 5-nucleotidase from added snake venom. Endogenous tissue 5-nucleotidase would contribute to this activity. The phosphate was in turn visualized by conversion to the lead salt in the presence of lead acetate and finally into brownish-black lead sulphide by treatment with yellow ammonia sulphide. Control studies with and without substrate and snake venom, as well as inhibition by theophylline, indicate the test to be specific for cyclic 3, 5-AMPase.In the eye the conjunctiva, ciliary process, choroid and retina all showed strongly positive activity. In the kidney the proximal and distal tubules both ascending and descending and the loop of Henle show strongly positive activity — the rest of the elements being negative. The cardiac and skeletal muscle exhibited very little positive activity. The liver showed only mildly positive activity. The villi of the small intestine showed strongly positive activity at the apical part of the cells. Neurons showed very little positive activity in either the cerebral cortex or the cerebellum. On the other hand, the molecular layer in the cerebellum and the plexiform layer of the cerebral cortex showed strongly positive activity. The significance of these findings are briefly discussed. T. R. Shanthaveerappa — in previous publications.     

Studies of effects of cyclic adenosine 3′,5′-monophosphate in regulation of human hemopoiesis in vitro

Summary Prostaglandin E₁ (PGE₁), high concentrations of dibutyryl cyclic AMP (dbcAMP), and theophylline were strikingly inhibitory both to tritiated thymidine ([³H]TdR) incorporation into bone marrow deoxyribonucleic acid (DNA) in vitro and to granulocytic colony growth. Autoradiography revealed that lower concentrations of dbcAMP were stimulatory to red blood cell precursors. This study was supported in part by United States Public Health Service Grant AM15163, by Health Research Council of the City of New York Career Scientist Award I-683, and by a Veterans Administration Medical Investigatorship to V. H.     

Calcium,cyclic adenosine 3′, 5′-monophosphate,and the control of cell proliferation: A review

Conclusion By manipulating the rat's calcium balance, we have discovered that the calcium homeostatic system is a main regulator of cell proliferation in the bone marrow and thymus gland. Although the limits of the system's sphere of influence have yet to be completely defined, it is already known to include such diverse elements as chicken fibroblasts, liver parenchymal cells, and circulating small lymphocytes. Of even greater significance is the possibility that the ubiquitous cyclic AMP is calcium's partner and may even be the ion's intracellular agent for the control of cell proliferation. Thus, we now have a wide variety of possible explanations for diseases involving uncontrolled cell proliferation. Issued as NRCC No. 12996. Supported by a grant from the American Cancer Society.     

New Approach to the Synthesis of 2′,3′-dideoxyadenosine and 2′,3′-Dideoxyinosine

Abstract

A new approach to the synthesis of 2′,3′-dideoxyadenosine and 2′,3′-dideoxyinosine based on deoxygenation of 2′,3′-di-O-mesylnucleosides was developed.     

Stannylation Approach to the Synthesis of 2′- and 3′-Substituted Analogues of 2′,3′-Didehydro-2′,3′-dideoxynucleosides

Abstract

Three methods are described for the introduction of a tributylstannyl group to the sp²-carbon of 2′,3′-didehydro-2′,3′-dideoxy nucleosides (d44Ns). The resulting stannylated products serve as versatile intermediates for the synthesis of d4Ns having various types of carbon-substituent.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Diastereoselective Synthesis of 2′,3′-Dideoxy- β-C- Glucopyranosides as Intermediates for the Synthesis of 2′,3′-Dideoxy-β-D-Glucopyranosyl-C-Nucleosides

An extension of the Vorbrüggen method of nucleotide synthesis for the synthesis of C-glucopyranosides, as intermediates for C-nucleosides, is described. It could be shown that the diastereoselectivity of the reaction can be tuned by a simple change of protecting groups.     

Synthesis of 2′,3′-Didehydro-2′,3′-Dideoxy-3′-C-Methyl Substituted Nucleosides

Abstract

1-(2,3-Dideoxy-3-C-hydroxmethyl-β-D-threo-pentofuranosyl) -,1- (2,3-didehydro-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl) -and 1-(3-C-azidomethyl-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl)uracil, thymine and cytosine were synthesized and evaluated for anti-HIV activity. The synthetic strategy was based on an allylic alcohol transposition of the corresponding 3′-C-methylene-nucleoside analogues.     

Phosphonates Derivatives of 2′,3′-Dideoxy-2′,3′-didehydro-pentopyranosyl Nucleosides

Abstract

Adenine and thymine derivatives of 2′,3′-dideoxy-2′,3′-didehydropento-pyranosyl nucleosides carrying a phosphonomethyl moiety at their 4′-O-position and in a cis relationship with the heterocyclic base have been synthesized.     

Immunofluorescence studies on the effect of cyclic adenosine 3′,5′-monophosphate in tissue culture preparations of the cervicovaginal anlage from neonatal mice

Summary The authors have studied the production of antigenic material, specific for the cervicovaginal epithelium, in organ cultures of the cervicovaginal region from neonatal mice. In control cultures the antigenic material appeared at a time corresponding to the normal appearance under in vivo conditions. When cyclic adenosine 3, 5-monophosphate (dibutyryl derivate) was added to the medium, a strong increase of the fluorescent material was seen.This investigations was supported by grants from the Norwegian Research Council and the Norwegian Cancer Society (Landsforeningen mot Kreft).     

Effect of dibutyryl cyclic adenosine 3′5′-monophosphate on mitotic activity in the thyroid of hypophysectomized rats

Summary Effect of dibutyryl cyclic adenosine 35-monophosphate (dbcAMP) on mitotic activity in the thyroid of hypophysectomized rats has been examined. It has been demonstrated that dbcAMP stimulates the incidence of mitoses in the thyroid follicular cells. It is therefore suggested that cAMP may be a mediator of the proliferogenic effect of TSH on the thyroid in vivo. Cyclic AMP could also release some unidentified growth-promoting factors for the thyroid. A direct stimulating effect of dbcAMP on the proliferation of the thyroid follicular cells is assumed to be possible as well.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

The influence of adenosine 3′,5′-monophosphate upon the activity of the membrane-associated (Ca2++Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

The phosphohydrolase activity of the membrane-associated $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+Mg}{}^{\mathrm{2+}}\text{)-dependent}$ adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar or nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline.     

Effect of adenosine 3′,5′ monophosphate on the mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine

The effect of increased cellular concentrations of adenosine 3′,5′ monophosphate (cAMP) upon mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.     

Occurrence of adenosine 2′,5′-bisphosphate in rat liver

Adenosine 2′,5′-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 μM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5′-AMP than 2′-AMP was obtained as an intermediate of the reaction.     

The Use of Acetyl Bromide for the Multigram Synthesis of the Anti-HIV Agent 2′,3′-Didehydro-2′,3′-Dideoxycytidine (d4C)

Abstract

Treatment of uridine (1) with acetyl bromide produced bromoacetate 2 which was reduced with Zn/Cu to give the 2′,3′-unsaturated uridine (d4U; 3). Conversion of the uracil moiety of 3 to thioamide 7 with Lawesson's reagent, followed by amination and deprotection with methanolic ammonia, afforded d4C (9). This multigram scale process for the synthesis of d4C proceeded in 20% yield from uridine.     

A continuous spectrophotometric assay for cyclic 3′,5′-nucleotide phosphodiesterase

In the course of studies on cyclic nucleotide phosphodiesterase, the need for an assay which was both sensitive and continuous was realized. Most of the methods available for monitoring phosphodiesterase either depend on the use of accessory enzymes, and are accordingly subject to intrinsic limitations, or are not capable of continuously monitoring the enzymatic reaction. The present paper describes a new spectrophotometric assay for cyclic nucleotide phosphotidesterase which is highly reproducible, rapid, simple, and more sensitive than many of the previously published assays for this enzyme. The method is sensitive enough to detect the enzymatic conversion of 75 pmol of cAMP to 5′-AMP per minute. This assay is based on the fact that the hydrolysis of cyclic nucleotides to the corresponding 5′-phosphate ester by phosphodiesterase makes available an additional titratable proton of the 5′-phosphate group. By incorporating phenol red into the assay mixture, the rate of proton production can be continuously measured by monitoring the decrease in absorbance of the basic chromophore of phenol red at 560 nm. The primary advantages of the spectrophotometric assay described here are: (a) it provides initial velocity measurements, and thus is ideally suited to studying the kinetic properties of partially purified preparations of enzyme, and (b) it does not require the tedious and time-consuming purification of commercially available substrates which is often required when radioisotopic assays are used in detailed kinetic studies. The chief limitations are: (a) the sensitivity is not sufficient to accurately monitor the “low K_m” enzyme, and (b) the use of the assay to quantitate revoveries throughout extensive purification, where different buffer salts at different pH values are used, would require that the enzyme be dialyzed prior to assay.     

Synthesis of 2′,3′-Dideoxyribavirin

Abstract

A synthesis of 1-(2,3-dideoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2′,3′-dideoxyribavirin, ddR) is described. Glycosylation of the sodium salt of 1,2,4-triazole-3-carbonitrile (5) with 1-chloro-2-deoxy-3,5-di-0-p-toluoyl-α-D-erythro-pentofuranose (1) gave exclusively the corresponding N-1 glycosyl derivative with β-anomeric configuration (6), which on ammonolysis provided a convenient synthesis of 2′-deoxyribavirin (7). Similar glycosylation of the sodium salt of methyl 1,2,4-triazole-3-carboxylate (2) with 1 gave a mixture of corresponding N-1 and N-2 glycosyl derivatives (3) and (4), respectively. Ammonolysis of 3 furnished yet another route to 7. A four-step deoxygenation procedure using imidazolylthiocarbonylation of the 3′-hydroxy group of 5′-0-toluoyl derivative (9a) gave ddR (11). The structure of 11 was proven by single crystal X-ray studies. In a preliminary in vitro study ddR was found to be inactive against HIV retrovirus.