Influence Of Cyclic AMP Research Articles

Distinct pathways of adaptive evolution in Cryptococcus neoformans reveal a mutation in adenylyl cyclase with trade-offs for pathogenicity

Pathogenic fungi populate a wide range of environments and infect a diversity of host species. Despite this substantial biological flexibility, the impact of interactions between fungi and their hosts on the evolution of pathogenicity remains unclear. We studied how repeated interactions between the fungus Cryptococcus neoformans and relevant environmental and mammalian host cells-amoeba and mouse macrophages-shape the evolution of this model fungal pathogen. First, using a collection of clinical and environmental isolates of C.neoformans, we characterized a range of survival phenotypes for these strains when exposed to host cells of different species. We then performed serial passages of an environmentally isolated C.neoformans strain through either amoeba or macrophages for ∼75 generations to observe how these interactions select for improved replication within hosts. In one adapted population, we identified a single point mutation in the adenylyl cyclase gene, CAC1, that swept to fixation and confers a strong competitive advantage for growth inside macrophages. Strikingly, this growth advantage in macrophages is inversely correlated with disease severity during mouse infections, suggesting that adaptation to specific host niches can markedly reduce the pathogenicity of these fungi. These results raise intriguing questions about the influence of cyclic AMP (cAMP) signaling on pathogenicity and highlight the role of seemingly small adaptive changes in promoting fundamental shifts in the intracellular behavior and virulence of these important human pathogens.

Influence of cyclic AMP and serum factors upon expression of a retinoid-responsive gene in neuroblastoma cells.

Cyclic AMP can profoundly influence the growth and differentiation of neuronal cells in culture. In this study, the relationship between this second messenger signal transduction pathway, cell differentiation, and the expression of a retinoid-responsive, thymosin beta-10 gene was examined. Thymosin beta-10 and cognate mRNA were expressed at high levels in actively proliferating rat B104 neuroblastoma cells cultured in medium containing 10% FCS. These cells were induced to differentiate in the presence of the cAMP analog N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2-cAMP) (1 mM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 microM). Expression of thymosin beta-10 mRNA was markedly inhibited (greater than 90% and 70%, respectively) by these compounds. Addition of sodium butyrate (NaB, 1 mM) indicated that at least part of the inhibitory actions of Bt2-cAMP were due to esterase-induced release of butyrate from this compound. Adenosine (50 microM), a metabolic precursor to endogenous cyclic AMP, also inhibited accumulation of thymosin beta-10 mRNA (to less than 70% of control levels). The inhibitory action of Bt2-cAMP upon thymosin beta-10 mRNA levels was time dependent; levels were inhibited by greater than 50% 24 hours after addition of the cAMP analog and by greater than 90% after 72 hours. Serum starvation (0.2% FCS for seven days) provoked a marked increase in neurite out-growth; this morphological change was also accompanied by a modest inhibition of thymosin beta-10 mRNA accumulation. These findings together with previous observations imply that both cyclic AMP-dependent and retinoid-responsive mechanisms coordinate thymosin beta-10 gene expression during neuroembryogenesis.

Influence of cyclic AMP, agmatine, and a novel protein encoded by a flanking gene on speB (agmatine ureohydrolase) in Escherichia coli.

The speB gene of Escherichia coli encodes agmatine ureohydrolase (AUH), a putrescine biosynthetic enzyme. The speB gene is transcribed either from its own promoter or as a polycistronic message from the promoter of the speA gene encoding arginine decarboxylase. Two open reading frames (ORF1 and ORF2) are present on the strand complementary to speB; approximately 90% of ORF2 overlaps the speB coding region. Analysis of transcriptional and translational fusions of ORF1 or ORF2 to lacZ revealed that ORF1 encoded a novel protein while ORF2 was not transcribed. Deletion of ORF1 from a plasmid containing ORF1, ORF2, and speB reduced the activity of AUH by 83%. In contrast, the presence of plasmid-encoded ORF1 caused an 86% increase in chromosomally encoded AUH activity. ORF1 did not stimulate alkaline phosphatase expressed from a phi(speB-phoA) transcriptional fusion encoded on the same plasmid. Western analysis (immunoblot) of a phi(ORF1-lacZ) translational fusion revealed that ORF1 encodes a 25.3-kDa protein. Agmatine induced transcription of phi(speB-phoA) but not phi(speA-phoA) fusions. Consequently, agmatine affects selection between the monocistronic and the polycistronic modes of speB transcription. In contrast, cyclic AMP (cAMP) repressed AUH activity of chromosomally encoded AUH but had no effect on plasmid-borne speB nor phi(speB-phoA). It is concluded that ORF1 encodes a protein which is a posttranscriptional regulator of speB, agmatine induces speB independent of speA, and cAMP regulates speB indirectly.

The activities of aminoacyl-tRNA synthetase phosphatase and aminoacyl-tRNA synthetases in MPC-11 and Krebs II ascites cells

The activity of aminoacyl-tRNA synthetase phosphatase as well as the activities of aminoacyl-tRNA synthetases in Krebs II ascites cells and MPC-11 cells have been investigated. The activity of the phosphatase was greater in the tumor cells than in normal tissues. The aminoacyl-tRNA synthetase activities were 100–300 times higher than the activities found in the uterus of ovariectomized mice, but not very different from the activities found in the liver. The influence of cyclic AMP, 2-deoxyadenosine 3-phosphate and 2-deoxyguanosine 3-phosphate on the growth of MPC-11 cells, grown in suspension culture, was also investigated.

The influence of cyclic nucleotides on hybridoma growth and monoclonal antibody production

The influence of cyclic AMP and cyclic GMP, known regulatory mediators of cellular response, on hybridoma growth and monoclonal antibody production is studied. The cGMP-treated cells exhibited 41% higher specific antibody secretion rate, resulting in 52% higher antibody yields. Addition of 1 mM cAMP inhibited cellular growth but enhanced the specific production rate by 37%.

Influence of cAMP and calcium on [3H]inositol efflux, inositol phosphate accumulation, and insulin release from isolated rat islets.

The influence of cyclic AMP (cAMP) and extracellular calcium on phosphoinositide (PI) hydrolysis in isolated islets was assessed and related to insulin output. Three stimulants were chosen to activate the beta-cell: sulfated cholecystokinin (CCK-8S, 200 nM), high-level glucose (20 mM), and the sulfonylurea tolbutamide (200 microM). The insulin secretory response to all three agonists was amplified by forskolin (which increases cAMP levels) and reduced by nitrendipine (which decreases calcium influx). All three stimulants increased the hydrolysis of inositol-containing phospholipids, an event monitored by an increase in [3H]inositol efflux from [3H]inositol-prelabeled islets and by the accumulation of labeled inositol phosphates. Forskolin, despite its positive impact on insulin secretion, reduced [3H]inositol efflux and inositol phosphate accumulation in response to all agonists. A similar inhibitory effect on these parameters was noted with nitrendipine; however, nitrendipine abolished secretion in response to all agonists. These findings support the following conclusions: 1) an increase in cellular cAMP levels reduces the quantitative impact of various agonists on these indices of PI hydrolysis; 2) despite this inhibitory effect, cAMP amplifies the insulin secretory response to these agonists; and 3) extracellular calcium is a crucial determinant of both PI hydrolysis and the ensuing insulin secretory response.

Influence of cyclic AMP on the growth response and anaerobic metabolism of carbon monoxide in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus strain 1 (str. 1), a photoheterotrophic bacterium, used CO as an energy substrate under anaerobic CO/light conditions, and exhibited a diauxic growth response when CO was removed from the culture. Changes in the level of cyclic AMP which occurred in cells during diauxie suggested that the cyclic nucleotide operated as an intracellular control molecule. During CO/light-phase growth, intracellular cyclic AMP was 30 pmol/mg protein, and, as str. 1 adapted for photosynthetic growth after removal of CO, intracellular cyclic AMP levels decreased to 9 pmol/mg protein. Reexposure of a light culture to CO induced synthesis of CO oxidation activity (measured as CO:MV oxidoreductase). If 10 mM cyclic AMP was added with CO, the rate of synthesis of CO:MV oxidoreductase activity increased 25-fold, and str. 1 produced 1,230 units of activity (nmol CO oxidized min-1 mg-1 protein) after only 1 h. With cyclic AMP and no CO, no incerease in CO oxidation activity was seen. Appearance of CO oxidation activity in str. 1 represented de novo protein synthesis and was blocked with chloramphenicol. In addition to stimulating formation of CO oxidative activity, a high level of cyclic AMP in str. 1 during growth with CO appeared to influence photometabolism negatively by repressing bacteriochlorophyll formation.

Cyclic AMP-regulating agents inhibit endotoxin-mediated cartilage degradation

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.

Influence of cyclic AMP and hydrolysis products on cell type regulation in Dictyostelium discoideum

We describe the effect of cyclic AMP on regulation of the proportion of prespore and prestalk cells in Dictyostelium discoideum. Prespore and prestalk cells from slugs were enriched on Percoll density gradients and allowed to regulate in suspension culture under 100% oxygen. The transition of prespore to prestalk cells is blocked by cAMP, while cAMP phosphodiesterase and caffeine cause a decrease in the number of prespore cells. This suggests that extracellular cAMP plays a role in cell type proportioning by inhibiting the conversion of prespore to prestalk cells. Low concentrations of cAMP prevent the conversion of prestalk to prespore cells; the same effect is seen with hydrolysis products of cAMP, 5 AMP, adenosine and also adenine. We suggest that, when low concentrations of cAMP are added to regulating cells, the cAMP itself is quickly broken down and the breakdown products thereafter inhibit the prestalk-to-prespore conversion. The relevance of these findings is discussed in the context of an non-positional double-negative feedback model for cell type homeostasis.

Influence of cyclic AMP on photosynthetic development in Rhodospirillum rubrum.

During O2-free growth in the light and in medium with pyruvate, Rhodospirillum rubrum exhibits diauxic growth. The cells first fermented pyruvate and afterwards photometabolized. Exogenous cyclic AMP acted to prolong the lag period between fermentative and photosynthetic development, as well as to slow the light-dependent growth rate. This observation, and in situ changes in the cyclic AMP levels in cells undergoing biphasic growth, suggested that the cyclic nucleotide was involved in photosynthetic differentiation, perhaps by repressing the formation of the bacteriochlorophyll needed to support growth in the light.

Cell surface and cell division.

A mathematical model of the regulation of cell division is suggested. The model is based on the hypothesis that the process giving rhythm to cell division is located in the cell membrane: i.e., the process of free-radical oxidation of membrane lipids. Much depends on the physical state of the membrane. In the membrane, phase transitions take place because of the changes in lipid composition. These transitions differ in normal and tumor cells: in normal cells they are sharp and hysteretic owing to the presence of a framework (membrane skeleton) on the surface of the membrane, while in tumor cells the integrity of the surface is violated so that the transitions are smooth. This model makes it possible to explain differences in the regulation of normal and cancer cell proliferation. Within the limits of the model, such phenomena as density dependent inhibition of growth, reverse transformation, influence of cyclic AMP and ions of Ca2+ on the cell cycle, the actions of serum and of proteases on the cycle, and so on, are explained. A rational scheme for the appearance of the selective damage found in tumor cells is proposed.

Effect of cyclic AMP-dependent protein kinases on gene expression

Mechanisms for regulating activation of cyclic AMP-dependent protein kinase were investigated and a possible biological role of this enzyme was elucidated. Autophosphorylation of cyclic AMP-dependent protein kinase was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phosphoform of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and nonphosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cyclic AMP. By the method of circular dichroism it was also shown that cyclic AMP and its 8-Br-substituted analog were bound in the cyclic AMP-binding site of the protein kinase in anti-conformation. The data obtained suggest the change of conformation of the 8-Br-cyclic AMP molecule during activation of protein kinase: while having a preferential syn-conformation in the free state in solution, the molecule of 8-Br-cyclic AMP acquires anti-conformation in the fixed complex with the regulatory subunit. A study was made of nuclear translocation of cyclic AMP-dependent protein kinase and its subunits, as well as of the binding of these proteins to metaphase chromosomes. The CHO cell cultures were treated with 3H-labeled protein kinase and its subunits. The results indicate that cyclic AMP-dependent protein kinase becomes translocated into the nucleus in a dissociated state and that the subunits have specific binding sites on chromatin. Transformation of normal mouse fibroblasts by virus SV 40 interferes with the nuclear translocation of the regulatory subunit. The process is restored when the level of cyclic AMP in the system is increased. Binding of the regulatory subunit to metaphase chromosomes of cells transformed by virus SV 40 does not change. In the case of spontaneous cancer (KB cells) translocation of the regulatory subunit remains unaffected, whereas acceptance of the protein by the metaphase chromosomes is impeded. The results of this work suggest that compartmentalization of cyclic AMP-dependent protein kinase, and particularly of its regulatory subunit, in the cell is highly significant for cellular processes. Disorders arising as a result of neoplastic transformation involve changes in nuclear translocation of the regulatory subunit and in its binding to the structural elements of the genome. The effect of the regulatory subunit of cyclic AMP-dependent protein kinase on the process of transcription in vitro was shown. The regulatory subunit was found to increase the number of initiation sites on chromatin for RNA polymerase. In this connection, an attempt was made to detect more directly expression of the genome by the regulatory subunit and to identify the final protein product formed. The regulatory subunit induced a specific synthesis of new proteins, the direct and first response of 3T3 cells to the introduction of the regulatory subunit being the synthesis of protein P-15. The molecular weight of the protein was 15,000, the isoelectric point 6.3. The electrophoretic analysis of the cytosol of SV 40-3T3 cells revealed a general derepression of the genome of the virus-transformed cells. A protein identical with P-15 was detected to be present in SV 40-3T3 cells. The treatment of these cells with the regulatory subunit as well as with cyclic AMP separately did not affect the synthesis of P-15, whereas introduction of the cyclic AMP-regulatory subunit complex caused a significant expression of protein P-15. The data obtained indicate that the protein synthesis is dependent on nuclear translocation of the regulatory subunit.

Influence of cyclic AMP on the incorporation of [1-14C]acetate into lipids of whole cells and spheroplasts ofMycobacterium smegmatisATCC14468

Influence of cyclic AMP on the incorporation of [1-¹⁴C]acetate into lipids of whole cells and spheroplasts of<i>Mycobacterium smegmatis</i>ATCC14468

Influence of cyclic AMP on the incorporation of [1-14C]acetate into lipids of whole cells and spheroplasts of Mycobacterium smegmatis ATCC14468

Influence of cyclic AMP on the incorporation of [1-14C]acetate into lipids of whole cells and spheroplasts of Mycobacterium smegmatis ATCC14468

The autophosphorylation reaction in the mechanism of activation of pig brain cyclic AMP-dependent protein kinase

Autophosphorylation of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phospho form of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. The activation constant of phospho- and dephosphoprotein kinase under the influence of cyclic AMP and the dissociation constant of the cyclic AMP complex with phospho- and dephospho forms of the holoenzyme were estimated. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and non-phosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cyclic AMP. The anti-conformation of the cyclic nucleotide fixed in the complex with the phospho form of the regulatory subunit is suggested.

Regulation of phosphatidylcholine metabolism by cyclic AMP in a model alveolar type 2 cell line.

The influence of cyclic AMP on the metabolism of phosphatidylcholine, the major component of pulmonary surfactant was examined in a cell line (A549) with type 2 pneumonocyte characteristics. It was found that cyclic AMP increased both the total amount of phosphatidylcholine and disaturated phosphatidylcholine as well as the incorporation of [3H]choline into these fractions. The effect was specific for cyclic AMP since 5'-AMP, adenosine, and cyclic GMP did not alter phosphatidylcholine or disaturated phosphatidylcholine levels. Cyclic AMP had no effect on phosphatidylcholine and disaturated phosphatidylcholine metabolism in another non-type 2 human epithelial cell line (MA-160). Since the ability of various cyclic AMP analogs to increase phosphatidylcholine and disaturated phosphatidylcholine levels was correlated with their ability to activate protein kinase, it seems likely that a protein phosphorylation mechanism is involved in controlling phosphatidylcholine metabolism.

Influence of cyclic AMP on intracellular pH regulation and chloride fluxes in barnacle muscle fibers.

THE pH sensitivity of numerous intracellular processes requires the close regulation of internal pH (pHi). As the pHi of many cells is maintained at a higher value than it would have if H+ were at equilibrium across the cell membrane, it follows that the acidifying effects of cellular metabolism and of passive H+ influx and HCO−3 efflux must be balanced by active acid extrusion. It is now generally accepted that this extrusion is stimulated by decreases in pHi, and that, in several cells, it is accomplished by exchanging external HCO−3 for internal Cl− (refs 1–4). Recently, a role for cyclic 3′,5′-adenosine monophosphate (cyclic AMP) in pHi regulation was suggested by the observation that dibutyryl cyclic AMP attenuates the CO2-induced fall of pHi in mammalian cardiac muscle5. We now report experiments on the effects of cyclic AMP on acid extrusion and on accompanying Cl− fluxes in the barnacle muscle fibre, a preparation known to contain cyclic AMP (ref. 6). Our data show that internally applied cyclic AMP enhances the response of the pHi-regulating system to acid loads.

Disruption of multicellular organization in the cellular slime molds by cyclic amp

Addition of cyclic AMP causes disorder in the multicellular stage of a number of species of cellular slime molds. In those which produce fruits with cellular stalks, the addition of cyclic AMP stimulates prestalk cells to differentiate into mature stalk cells. Prespore cells do not differentiate into spores under the influence of cyclic AMP, most degenerate and seem to die. I hypothesize that the normal course of differentiation from vegetative cells is one leading to spores, but that cyclic AMP can divert this course to one leading to the stalk cell. Dibutyryl cyclic AMP, cyclic GMP and cyclic AMP disrupt slugs of Polysphondylium pallidum, while species of Dictyostelium are disrupted by only cyclic AMP. The multicellular stage of P. violaceum is unaffected by high concentrations of exogenous cyclic nucleotides. Cell organization of Acytostelium ellipticum, a species with an acellular stalk, was disrupted by cyclic AMP, but no stalk cells were formed; only spores.

Einfluss möglicher phosphodiesterase-inhibitoren und cAMP auf die betacyan-akkumulation

The influence of cyclic AMP, theophylline, papaverine and NH 4NO 3 on the accumulation of betacyanin in callus of Portulaca grandiflora, var. JR, were studied in relation to amounts of dihydroxyphenylalanine (DOPA), dopamine, phenylalanine, tyrosine, protein, ‘lipid’ and the nucleotides CMP, AMP, GMP and UMP present. Inhibition of betacyanin formation is characterized by reduced amounts of DOPA and dopamine and a constant rise of GMP and CMP (GMP/CMP = 8). Protein accumulation is also inhibited. The increase in pigment accumulation due to theophylline and NH 4NO 3 is characterized by a raised DOPA and protein concentration and a lower GMP/CMP ratio (=3). The increase in betacyanin accumulation is due to de novo synthesis of enzymes. Inhibition is probably due to the regulation of the callus phosphodiesterase by papaverine and theophylline (≦ 10 −5 M/1) which triggers a change in concentration of nucleotides, which eventually regulates tyrosinase biosynthesis.

Stimulation of cellular autophagy by parathyroid hormone and cyclic adenosine 3′,5′: Monophosphate in isolated tubular fragments from the rat’s kidney cortex

Tubular fragments isolated from the cortex of the rat's kidney were qualitatively and quantitatively investigated with the electron microscope. The tubules frequently burst open and became "inverted" in such a way that the rarefied brush border now formed the outer circumference. By morphometry a decrease of the average cell volume in the proximal tubular fragments was ascertained. This was mostly caused by a loss of cytoplasmic ground substance, endoplasmic reticulum and ribosomes. Cytoplasmic herniations of the basal surface, filled with free ribosomes, suggested a partial shedding of the protein synthesizing apparatus. The number of autophagic vacuoles (AV) per unit area of cytoplasm was determined in proximal tubular fragments. After isolation alone, without further incubation, the number of AV was as low as the number found in an earlier study in proximal tubular cells in situ during the diurnal minimum. After control incubation the number of AV increased to about the mean value of the AV found in cells in situ during the whole diurnal cycle. By comparison with the control incubation the number of AV increased by a factor of 1.6 to 1.7, if cyclic adenosine 3',5': monophosphate (cyclic AMP) or parathyroid hormone (PTH) were added to the incubation-medium; it now reached about the number of AV found in situ during the diurnal maximum. The increase in the number of AV paralleled that of the production of ammonia and glucose from endogenous sources under the influence of cyclic AMP and PTH. This suggests that the breakdown of cytoplasmic components by cellular autophagy could be functionally related to gluconeogenesis. A quantitative comparison between the measured production of ammonia and glucose indicates, however, that in the system of isolated tubular fragments there may exist other mechanisms of degradation, and of the provision of substrates for gluconeogenesis, than cellular autophagy only.