THE EFFECTS OF CYCLIC AMP ON DIFFERENTIATION OF A MUTANT DICTYOSTELIUM DISCOIDEUM CAPABLE OF DEVELOPING WITHOUT MORPHOGENESIS

The dev 1510 mutant of Dictyostelium discoideum differs from the wild type in that unaggregated cells are capable of differentiating into either spores or stalk cells depending on the culture conditions (12). Taking advantage of this fact, the effects of cyclic AMP (cAMP) on differentiation of the mutant cells were examined under conditions that prevent normal morphogenesis. In the presence of low concentrations of exogenous cAMP, the cells differentiated into only stalk cells, whereas in the presence of high concentrations they differentiated into only spores. Untreated cells formed stalk cells, but this was inhibited by addition of phosphodiesterase, indicating that it was induced by a low concentration of cAMP which they produced themselves. Cyclic GMP and dibutyryl cAMP also induced spore formation though less effectively, while 5'AMP, ADP and ATP had no effect. During development, the cells increased in sensitivity to cAMP in that spore formation was induced at lower concentration of cAMP after 4 hr of starvation. Treatment of cells that had been starved for 6hr with 10⁻⁴M cAMP for as short a time as 30 min was enough to induce 8% of the cells to form spores.
The effects on cAMP-induced differentiation of chemicals that are known to influence development of the wild type were also examined. Both NH₄Cl and KCl inhibited cAMP-induced stalk formation, but had no effect on spore formation. In the presence of arginine, spore formation was induced at a lower concentration of cAMP with higher efficiency. CaCl₂, LiCl and KF had no effect on cAMP-induced differentiation.     

Cyclic GMP-dependent stimulation of the membrane-bound insulin-sensitive cAMP phosphodiesterase from rat adipocytes

The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Km enzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 microM cGMP for 5-20 min at 37 degrees C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki = 0.10 microM), griseolic acid (Ki = 0.19 microM), unlabeled cAMP (Km = 0.20 microM), and cGMP (Ki = 0.16 microM), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Km phosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 microM cGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.     

A plausible role for a membrane-bound cyclic AMP phosphodiesterase in cellular slime mold chemotaxis.

1. Kinetics of membrane-bound cyclic AMP phosphodiesterase of the cellular slime mold, Dictyostelium discoideum, were studied under two conditions: in the 27 000 times g sediment of cell homogenates (particle-bound phosphodiesterase) and in cell suspensions using external cyclic AMP as a substrate (cell-bound phosphodiesterase). Both methods revealed non-Michaelian kinetics with interaction coefficients less than 1. 2. The membrane-bound phosphodiesterase has a specificity different from that of the cyclic AMP receptor, also present at the cell surface. 3. The membrane-bound enzyme was solubilized by lithium 3, 5-diiodosalicylate and partially purified. In this state the non-linear kinetics were still retained; however, the enzyme was not inhibited by the D. discoideum inhibitor, unlike the cell-bound phosphodiesterase in vivo. This indicates that both enzymes share an inhibitor binding site and that this site is cryptic in the cell-bound state. 4. Production of periodic cyclic AMP pulses by centers, and their relay by other cells, is believed to occur during aggregation. It is suggested that the cell-bound enzyme determines a "time window" significantly smaller than the period of pulsing, and optimizes stimulation of the cyclic AMP receptors in chemotaxis and signal relaying.     

Enzyme activity changes during cyclic AMP-induced stalk cell differentiation in P4, a variant of Dictyostelium discoideum.

The P4 variant of Dictyostelium discoideum is characterized by the production of fruiting structures in which the overall proportion of stalk to spore material is increased, relative to the wild type. The altered morphology of the mutant is due to increased sensitivity to cyclic AMP which promotes stalk cell differentiation. In the presence of 10-4 M-cyclic AMP the entire population of P4 amoebae forms clumps of stalk cells on the surface of the dialysis membrane support. Measurement of changes in activity of a range of developmentally-regulated enzymes during the development of P4 in the presence and absence of cyclic AMP has allowed us to identify three classes of enzyme: (i) Those, such as beta-glucosidase II, trehalose-6-phosphate synthetase and uridine diphosphogalactose-4-epimerase, which are required for the production of spores. (ii) Enzymes, primarily but perhaps not exclusively, required during stalk cell formation. Typical of these are N-acetylglucosaminidase and alkaline phosphatase. (iii) General enzymes, such as threonine dehydrase, alpha-mannosidase and uridine diphosphoglucose pyrophyosphorylase, which are present inboth pre-stalk and pre-spore cells and appear to be necessary for the development of both cell types.     

A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP.

A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2'-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

Oscillations of Ca++ concentration during the cell differentiation of Dictyostelium discoideum: their relation to oscillations in cyclic AMP and other components

Periodic cyclic-AMP pulses control the cell aggregation and differentiation of Dictyostelium discoideum. Another component required for the aggregation and differentiation of these cells appears to be extracellular Ca+ +. Oscillations in extracellular Ca+ + concentration were investigated in suspensions of differentiating cells. We observed spike-shaped and sinusoidal Ca+ + oscillations. In the course of differentiation, spike-shaped Ca+ + oscillations preceded sinusoidal oscillations, and no phase change occurred at the transition from spike-shaped to sinusoidal Ca+ + oscillations. Spike-shaped and sinusoidal Ca+ + oscillations were related to oscillations in (1) the cyclic-AMP and cyclic-GMP content of cells, (2) the light-scattering properties of cells, and (3) the extracellular pH. Spikeshaped Ca+ + oscillations were observed together with cyclic-AMP oscillations. The minima of the extracellular Ca+ + concentration trailed the maxima of the cyclic-AMP concentration by about 30 s. Sinusoidal Ca+ + oscillations were not accompanied by measurable cyclic-AMP oscillations. The amplitudes of the sinusoidal Ca+ + oscillations were smaller than those of the spike-shaped Ca+ + oscillations. A Ca+ + oscillation of small amplitude (instead of a spike-shaped oscillation) was observed when one cyclic-AMP spike was skipped. Our results provide evidence for the existence of a sinusoidal cyclic-AMP-independent Ca+ + oscillation of small amplitude, and they also suggest that spike-shaped Ca+ + oscillations may be superimposed on such small-amplitude oscillations. When D. discoideum cells produce cyclic-AMP spikes, the uptake of additional Ca+ + is induced, resulting in Ca+ + oscillations of a large amplitude.     

Human thyroid cyclic nucleotide phosphodiesterase. Its characterization and the effect of several hormones on the activity.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were investigated in the human thyroid gland from patients with hyperthyroidism. Low substrate concentration (0.4 muM) was used. About 60% of the cyclic-AMP and 80% of the cyclic-GMP hydrolytic activities in the homogenate were obtained in the soluble fraction (105 000 X g supernatant). The thyroid gland contains two forms of cyclic-AMP phosphodiesterase, one with a Km of 1.3-10(-5) M and the second with a Km of 2-10(-6) M. Cyclic-AMP and cyclic-GMP phosphodiesterase were purified by gel filtration on a Sepharose-6B column. Cyclic-AMP phosphodiesterase activities were found in a broad area corresponding to molecular weights ranging from approx. 200 000 to 250 000 and cyclic-GMP phosphodiesterase activity was found in a single area corresponding to a molecular weight of 260 000. Cyclis-AMP phosphodiesterase activities were stimulated by the protein activator which was found in human thyroid and this stimulation was dependent on Ca2+. Stimulation of cyclic-AMP phosphodiesterase by the activator was not significant even in the presence of enough Ca2+. The effect of D,L-triiodothyronine, D,L-thyroxine, L-diiodotyrosine, L-monoiodotyrosine, L-thyronine, L-diiodothyronine, thyrotropin, hydrocortisone, adrenocorticotropin, cyclic-AMP and cyclic-GMP on the phosphodiesterase activities was studied. Cyclic-AMP, cyclic-GMP, D,L-triiosothyronine, D,L-thyroxine, adrenocorticotropin and hydrocortisone where found to inhibit the phophodiesterase. Triiodothyronine and thyroxine inhibited cyclic-AMP phosphodiesterase more effectively than cyclic-GMP phosphodiesterase. Thyroxine was a more potent inhibitor than triiodothyronine. The concentration of cyclic AMP producing a 50% inhibition of cyclic-GMP phosphodiesterase activity was 5-10(-5) M, while the concentration of cyclic GMP producing a 50% inhibition of cyclic-AMP phosphodiesterase was 3-10(-3) M. Both cyclic-AMP and cyclic-GMP phosphodiesterase activities in the homogenate of hyperthyroidism, thyroid carcinoma and adenoma were higher than in normal thyroid tissue, when assayed with a low concentration of the substrate (0.4 muM). When a higher concentration (1 mM) of cyclic nucleotides was used as the substrate, cyclic-AMP hydrolytic activity in adenoma tissue was similar to that of normal tissue, while the other activities were higher than normal.     

Mepacrine-induced inhibition of human platelet cyclic-GMP phosphodiesterase

The effect of mepacrine (DL-quinacrine-HCI), a specific inhibitor of phospholipase C, on cyclic-GMP levels in human platelets was investigated. The concentrations of mepacrine producing 50% inhibition of human platelet aggregation induced by 5 microM ADP and 3 micrograms/ml of collagen were 50 +/- 8 and 70 +/- 15 microM, respectively. Addition of mepacrine to human platelet suspension resulted in increases in cyclic GMP. In contrast to cyclic-GMP levels, cyclic-AMP content was not affected by mepacrine. Mepacrine did not stimulate guanylate cyclase, but did specifically inhibit human platelet cyclic-GMP phosphodiesterase, separated from cyclic-AMP phosphodiesterase or other forms of phosphodiesterase on DEAE-cellulose columns. Stimulation by cyclic GMP of human platelet cyclic-GMP-stimulated cyclic-AMP phosphodiesterase activity was not inhibited by mepacrine. The IC50 value of the drug for cyclic-GMP phosphodiesterase was 40 microM, and IC50 for cyclic-AMP phosphodiesterase was 1.2 mM. Mepacrine was 30-times more potent as an inhibitor of human platelet cyclic GMP than of cyclic-AMP phosphodiesterase. Mepacrine blocks arachidonate release from human platelets by inhibiting phosphatidylinositol-specific phospholipase C. The increase in cyclic-GMP levels produced by addition of mepacrine will explain part of the pharmacological action of this drug.     

Effect of endotoxin on lipid order and motion in erythrocyte membranes

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 μg/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes,using a rapid immunological procedure for the isolation of plasma membrane

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a ‘high K_m’ form, whereas the particulate activity had both ‘high K_m’ and ‘low K_m’ components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both ‘high K_m’ and ‘low K_m’ phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The ‘low K_m’ enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes.The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.     

An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium.

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km approximately 5 microM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominant-negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.     

Dynamics of cyclic-AMP efflux in relation to alpha-MSH secretion from melanotrope cells of Xenopus laevis.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP

We have examined the regulation of two key enzymes that control polyamine biosynthesis-L-ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) - by agents increasing cAMP in S49 lymphoma cells. Incubation of wild type S49 cells with beta-adrenergic agonists (terbutaline or isoproterenol) inhibited ODC and SAMDC activities rapidly (less than 2 hr). more quickly than these agents arrested the cells in the G1 phase of the cell cycle. The beta-adrenergic antagonist propranolol blocked inhibition of ODC activity produced by isoproterenol, but only if added simultaneously or less than 4 hr after the agonist. Incubation of wild type S49 cells with cholera toxin or PGE1 also inhibited ODC activity. Decreases in ODC activity produced by beta-adrenergic agonists, cholera toxin, PGE1 or dibutyryl cAMP were all enhanced by the phosphodiesterase inhibitor Ro 20-1724. Results of studies of ODC and SAMDC activity in S49 variants having lesions in the pathway of cAMP generation and action were as follows: kin- cells (which lack cAMP-dependent protein kinase activity) showed no inhibition of ODC by any agent; AC- cells (which have absent nucleotide coupling units in their adenylate cyclase system) only demonstrated inhibition in response to dibutyryl cAMP; UNC cells (which have deficient coupling of hormone receptors and adenylate cyclase) only demonstrated inhibition in response to dibutyryl cAMP and cholera toxin, and beta-depleted cells (which have a decreased number of beta-adrenergic receptors) responded as did wild type cells except for absent response to isoproterenol. We conclude that inhibition of ODC and SAMDC activity in S49 cells is an early response to agents that increase cAMP and that this action occurs via the "classical" pathways of activation of adenylate cyclase and protein kinase. These results in S49 cells contrast with evidence in other systems in which cAMP has been suggested to enhance polyamine biosynthesis, perhaps through alternative mechanisms.     

Secretion of fructosyltransferase by Streptococcus salivarius involves the sucrose-dependent release of the cell-bound form

Three strains of Streptococcus salivarius including a recent clinical isolate were found to possess Ca2(+)-dependent fructosyltransferase (FTF) activity. The extracellular FTF activity of cells grown on sucrose increased as much as 9-fold compared with cells grown on either glucose, fructose or galactose. This increase in activity was due not to induction of FTF by sucrose, but to the release of the cell-bound form of the enzyme. Studies with washed cells of S. salivarius ATCC 25975 showed that the extent of release of the cell-bound FTF activity was dependent upon the sucrose concentration up to 4 mM, at which concentration maximum release (95%) of cell-bound FTF occurred. Several lines of evidence suggested that either substrate binding or de novo synthesis of fructan is required for the release of the cell-bound FTF activity.     

Adenylyl cyclase and the control of cell differentiation in Dictyostelium dicoideum.

Adenylyl cyclase is part of a biochemical network that controls cell differentiation in Dictyostelium discoideum. At a certain stage of development the enzyme is rhythmically activated, with periods of about 8 min. These oscillations are superimposed upon an increase of the basal activity extending over a period of hours. The basal activity remains low in a mutant blocked at an early stage of development. In strain Ax-2 two periods of strongly increasing basal activity have been found: the first from 2 to 4 h after the end of the growth phase, the other beginning at about 8 h. Based on the periodic regulation of adenylyl cyclase, cyclic AMP is released into the extracellular space in the form of pulses. Application of cyclic-AMP pulses, but not its continuous influx, stimulates the increase of basal adenylyl cyclase activity. Two other constituents of the cyclic-AMP signal system cyclic-AMP receptors and cell-surface phosphodiesterase, are similarly controlled. The principal importance of positive feedback loops in a network controlling cell differentiation is discussed.