Exogenous Cyclic AMP Research Articles

Exogenous cyclic AMP, cholera toxin, and endotoxin induce expression of the lipopolysaccharide receptor CD14 in murine bone marrow cells: role of purinoreceptors.

Little is known about the mechanisms of lipopolysaccharide (LPS) signaling in immature cells that do not express the LPS receptor CD14 yet. Bone marrow granulocytes do not constitutively express CD14 but can be stimulated by low doses of LPS in the absence of serum and then express an inducible form of LPS receptor (iLpsR). We show that in addition to LPS, cholera toxin (CT) and various cyclic AMP (cAMP) analogs can also induce the expression of iLpsR, which was identified as CD14. Induction was independent of intracellular cAMP. The hypothesis that cAMP analogs act via a cell surface receptor was suggested by the unresponsiveness of trypsin-treated cells to these inducers and by the specific binding of [(3)H]cAMP to the cells. This binding was not inhibited by LPS or CT but was inhibited by various purine derivatives. However, the receptor involved is not a conventional purinoreceptor since both an agonist and an antagonist of such receptors were able to induce iLpsR expression. The results suggest that cAMP analogs and other purine derivatives induce iLpsR after interaction with an unconventional, trypsin-sensitive, purinoreceptor distinct from LPS and CT receptors.

Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum.

Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.

Cyclic AMP-adenosine pathway induces nitric oxide synthesis in aortic smooth muscle cells.

The main purpose of this investigation was to evaluate whether the cyclic AMP-adenosine pathway, ie, the conversion of cAMP to AMP and, hence, to adenosine, is involved in the regulation of nitric oxide (NO) synthesis by vascular smooth muscle cells (SMCs). Treatment of confluent monolayers of SMCs with adenosine, 2-chloroadenosine (stable analog of adenosine), and agents that elevate endogenous (SMC-derived) adenosine (EHNA and iodotubericidin) increased nitrite/nitrate (stable metabolites of NO) levels in the medium and enhanced the conversion of 3H-L-arginine to 3H-L-citrulline by cytosolic extracts obtained from the pretreated SMCs. The stimulatory effects of adenosine were not mimicked by low (1 to 100 nmol/L) concentrations of CGS21680, an A2A receptor agonist, or CPA, a selective A1 receptor agonist. The stimulatory effects of 2-chloroadenosine and EHNA plus iodotubericidin were significantly inhibited by KF17837, a selective A2 receptor antagonist, and by DPSPX, an A1/A2 receptor antagonist, but not by DPCPX, a selective A1 receptor antagonist. DDA (adenylyl cyclase inhibitor) and Rp-cyclic AMP (protein kinase A inhibitor) did not block the effects of adenosine on NO synthesis. Incubation of SMCs with exogenous cyclic AMP, at concentrations previously shown to elevate levels ofadenosine in the medium, also increased nitrite/nitrate levels and 3H-L-citrulline formation, and the effects of cyclic AMP on NO synthesis were blocked by DPSPX and KF17837, but not by DPCPX. These findings provide evidence that exogenous and SMC-derived adenosine induce NO synthesis via A2B receptors linked to a pathway not involving adenylyl cyclase/protein kinase A. Moreover, extracellular cyclic AMP induces NO synthesis via conversion to adenosine and activation of A2B adenosine receptors. The cyclic AMP-adenosine pathway may be importantly involved in the vascular production of NO.

Modulatory role of 1,25 dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP pathway in the rat.

1. Previous studies have shown that vitamin D3 deficiency impairs the insulin response to glucose via an alteration of signal transduction pathways, such as Ca2+ handling and the phosphoinositide pathway. In the present study the adenylyl cyclase pathway was examined in islets from 3 independent groups: normal rats, 4 weeks-vitamin D3 deficient rats and one week-1,25 dihydroxyvitamin D3 (1,25(OH)2D3) treated rats. 2. We found that the very low rate of insulin release observed in vitamin D3 deficient rats could be restored in vitamin D3 deficient islets only with high concentrations of dioctanoyl-cyclic AMP (DO-cyclic AMP), whereas 1,25(OH)2D3 improved the sensitivity of the islets to this exogenous cyclic AMP analogue. 3. The beneficial effect of 1,25(OH)2D3 observed with or without DO-cyclic AMP was protein kinase A-dependent, since the addition of N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H-89), a specific inhibitor of cyclic AMP-dependent protein kinases, decreased the insulin release of treated rats back to the level seen in vitamin D3 deficient islets. 4. The low rate of insulin release could not be consistently related to an alteration in cyclic AMP content of the islets. Indeed, low insulin response to a barium+theophylline stimulus observed in vitamin D3 deficient islets was paradoxically associated with a supranormal cyclic AMP content in the islets. 5. This paradoxical increase in cyclic AMP observed in these conditions could not be attributed to a lower total phosphodiesterase (PDE) activity, although the portion of Ca(2+)-calmodulin-independent PDE was predominant in islets from vitamin D3 deficient rats. 6. On the other hand, the higher cyclic AMP content of vitamin D3 deficient islets could be related to an increase in glucagon-induced cyclic AMP synthesis in relation to the hyperglucagonaemia previously observed in vitamin D3 deficient rats. Since higher concentrations of exogenous glucagon and higher endogenous cyclic AMP concentrations were required in vitro to restore insulin release to normal values, the cyclic AMP-dependent pathways that usually potentiate insulin secretion appeared to be less efficient in relation to an alteration in the post cyclic AMP effector system. 7. 1,25(OH)2D3 exerted a stimulating effect on insulin release via protein kinase A activation but reduced the supranormal cyclic AMP synthesis, thus exerting a differential modulatory influence on biochemical disturbances in islets induced by vitamin D3 deficiency.

Regulation of the Salmonella typhimurium pepT gene by cyclic AMP receptor protein (CRP) and FNR acting at a hybrid CRP-FNR site.

The Salmonella typhimurium pepT gene is induced nearly 30-fold in response to anaerobiosis. Anaerobic expression is dependent on the transcriptional regulator encoded by fnr (previously oxrA). Primer extension analysis and site-directed mutagenesis experiments show that pepT is transcribed from two sigma 70 promoters. One promoter (P1) is FNR dependent and anaerobically induced, while the other (P2) appears to be constitutive. The potABCD operon is divergently transcribed from a promoter near pepT P2. Sequence analysis of pepT promoter mutations which either elevate anaerobic expression or confer constitutive expression revealed that these mutations affect the -10 region of the P1 or P2 promoter, respectively. The pepT200 mutation, which changes the -10 region of the FNR-dependent P1 promoter to the consensus, has the surprising effect of allowing five- to sevenfold anaerobic induction in the absence of FNR. We have shown that the anaerobic induction of pepT-lacZ in a pepT200 fnr strain is dependent on wild-type alleles of both crp and cya. In a pepT200 pepT-lacZ strain, beta-galactosidase activity was elevated aerobically in the presence of exogenous cyclic AMP (cAMP) and was elevated also in succinate minimal medium relative to its level in glucose minimal medium. Primer extension analysis confirmed that P1 is the cAMP receptor protein (CRP)-dependent promoter. Site-directed mutagenesis experiments indicated that a hybrid CRP-FNR binding site positioned at -41 of the P1 promoter is utilized by both FNR and CRP. CRP-cAMP also appeared to repress FNR-dependent transcription of pepT under anaerobic conditions in both the pepT+ and pepT200 backgrounds. Although both CRP and FNR are capable of binding the hybrid site and activating transcription of pepT, CRP requires the consensus -10 sequence for efficient activation.

Identification and Characterization of Basal and Cyclic AMP Response Elements in the Promoter of the Rat Hexokinase II Gene

Hexokinases catalyze the phosphorylation of glucose and initiate cellular glucose metabolism. Hexokinase II (HKII) is the principal hexokinase isoform in skeletal muscle, heart, and adipose tissue. Isoproterenol and exogenous cyclic AMP (cAMP) increase HKII gene transcription in L6 myotubes. Various segments of the HKII promoter that direct the expression of the chloramphenicol acetyltransferase reporter gene were transfected into L6 myotubes to identify basal and cAMP response elements. The 5'-flanking region that extends 90 base pairs upstream of the transcription start site includes a CCAAT box and a cAMP response element (CRE); both contribute to basal promoter activity and each provides an independent, maximal response to cAMP. An inverted CCAAT motif, or Y box, located just upstream of the CCAAT box, contributes to basal promoter activity but is not involved in the cAMP response. Homo- and heterodimers composed of the CRE-binding protein and activating transcription factor-1 bind specifically to the CRE. The Y box and the CCAAT box specifically bind the factor NF-Y (also known as CBF).

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphosphate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 microM veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylyl cyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.

Elevated intracellular cyclic AMP inhibits chemotaxis in human eosinophils

Elevated intracellular cyclic AMP is associated with the inhibition of many inflammatory cellular responses. In this study, we examined the effect of cyclic AMP on eosinophil chemotaxis. Eosinophils were isolated from healthy human volunteers using an immunomagnetic method. Eosinophils were treated with agents that elevate intracellular cyclic AMP and evaluated for chemotactic responses to platelet-activating factor (PAF; 10 −6 M) and to complement factor 5a (C5a; 10 −8 M) in microchemotaxis chambers. Forskolin, prostaglandin E 1 (PGE 1), and a phosphodiesterase (PDE) IV-selective inhibitor inhibited eosinophil chemotactic responses. The mean per cent inhibition of eosinophil chemotaxis in response to PAF by forskolin, PGE 1, and the PDE IV-selective inhibitor (10 −5 M) was 16.8 ± 5.3, 26.6 ± 9.5, and 35.1 ± 6.1%, respectively ( n = 5). The corresponding values for C5a were 17. 5 ± 7.9, 20.8 ± 10.7, and 39.5 ± 5.0%. An exogenous cyclic AMP analogue (dibutyryl cyclic AMP, 10 −3 M) also inhibitied eosinophil chemotaxis by 69.4 ± 12.8 and 66.9 ± 11.6% in response to PAF and C5a, respectively ( n = 5). We conclude that elevated intracellular cyclic AMP inhibits eosinophil chemotaxis.

Association with MDCK epithelial cells by Salmonella typhimurium is reduced during utilization of carbohydrates.

Association of Salmonella typhimurium with MDCK epithelial cells in monolayers, represented primarily by intracellular bacteria after 30 min of contact, with centrifugation followed by vigorous washing, was measured during aerobic and anaerobic growth of the bacteria in brain heart infusion broth. Cell association was greatest during a short period in the late log phase of growth under aerobic conditions. At this time, the pH of the growth medium was changing from acid to alkaline and glucose (0.2% initially) was exhausted. Addition of excess glucose (0.5%) to brain heart infusion broth, which was not exhausted before the bacteria entered the stationary phase of growth, in which cell association dropped sharply, resulted in repression of cell association by the bacteria. The repressive effect of glucose on cell association could not be reversed by exogenous cyclic AMP in the bacterial growth medium. Under anaerobic conditions, the effect of glucose on cell association by the bacteria was not as great and the glucose was not exhausted before the bacteria entered the stationary phase. When S. typhimurium was grown in a rich but carbohydrate-free medium, cell association by the bacteria increased earlier in the growth cycle under both aerobic and anaerobic conditions. The addition of glucose and certain other utilizable carbohydrates to this medium caused a repression of cell association by S. typhimurium that was greater under aerobic growth conditions. These results show that cell association by S. typhimurium, which is accompanied by rapid internalization (cell invasion), is the same under aerobic and anaerobic conditions if the bacteria are grown to the log phase in a carbohydrate-free medium. This suggests that prior reports of greater cell invasion by S. typhimurium during anaerobic growth may have arisen from the use of media containing carbohydrates which were found to be more repressive during aerobic growth of the bacteria.

Antagonism by forskolin of the 5-HT 3 receptor-mediated current in nodose ganglion neurons is independent of cyclic AMP

The effect of forskolin on the inward current mediated by 5-HT 3 receptors (5-HT current) was investigated in rat nodose ganglion neurons. Forskolin inhibited the peak amplitude of the 5-HT current and increased current desensitization in a dose-dependent manner. Dideoxyforskolin, which does not stimulate adenylate cyclase, had a similar inhibitory effect on the 5-HT current. The effect of forskolin was neither mimicked by intracellular application of exogenous cyclic AMP (0.5 to 4 mM) nor occluded by intracellular forskolin (30 μM) or protein kinase inhibitor H-7 (100 μM). Intracellular applications of forskolin, cyclic AMP or H-7 had no effect on 5-HT current. Data suggest that forskolin acted at an extracellular site on 5-HT 3 receptors and this effect of forskolin was not mediated by cyclic AMP-dependent processes

Unaffected nodulation and nitrogen fixation in carbohydrate pleiotropic mutants ofRhizobium meliloti

Pleiotropic mutants were isolated fromRhizobium meliloti M5N1 with the resistance transposon Tn5. They failed to use carbohydrates as carbon source, but not dicarboxylic acids. Diauxic growth properties of the wild type and uptake assays in both M5N1 and a mutant strain 2.10 showed that the mutant was affected in the active transport of sugars. Exogenous cyclic AMP failed to affect the phenotype of the mutant, and a relatively high frequency of reversion was observed. This suggests that the mutant 2.10 was probably altered in acrp-like gene. However, its symbiotic properties remained unaffected. Thus, symbiosis does not depend on sugar utilization.

Protein phosphatase 1 and 2A in tick salivary glands as assessed by responses to okadaic acid

Crude protein phosphatase acitivity was inhibited 80% by nanomolar okadaic acid (OA) in salivary glands of unfed ticks but only 40% in salivary glands of feeding ticks. An additional 40% of protein phosphatase was inhibited by micromolar OA in the salivary glands of feeding ticks but only 10% in salivary glands of unfed ticks. Cyclic AMP and OA alone or together increased the phosphorylation of multiple proteins in a plasma membrane-enriched 900 g supernatant fraction of tick salivary glands. Exogenous cyclic AMP stimulated increased incorporation of phosphate into proteins with approximate molecular weights of 109, 70, 64, 51, 48, 42 and 18.5 kDa. Micromolar OA in the absence of exogenous cyclic AMP stimulated increased incorporation of phosphate into proteins with apparent molecular weights of 109, 93, 74.5, 70, 51, 48, 42 and 18.5 kDa. Cyclic AMP and OA (10 −6 and 10 −9M) stimulated significantly greater phosphorylation of an 18.5 kDa mol. wt protein above that observed in response to stimulation by OA (10 −6 and 10 −9 M) or exogenous cyclic AMP alone. Micromolar okadaic acid inhibited the amount and number of proteins but not volume of saliva secreted by whole ticks in response to stimulation by DA and theophylline. However, micromolar and nanomolar okadaic acid inhibited the ability of dopamine to stimulate fluid secretion by isolated salivary glands. Overall, the data support the existance of type 1 and 2A protein phosphatases in tick salivary glands and a role for protein phosphatases in modulating tick salivary secretion.

Oscillator control of cell division in Euglena: cyclic AMP oscillations mediate the phasing of the cell division cycle by the circadian clock

The achlorophyllous ZC strain of Euglena gracilis exhibits a circadian rhythm of cell division in constant darkness (DD). Mitosis occurs during a restricted part of the circadian cycle, corresponding to the dark intervals in a light-dark cycle comprising 12 h of light and 12 h of darkness. We have demonstrated that division-phased cultures also exhibit bimodal, circadian changes of cyclic AMP level. Maximum cyclic AMP levels occurred at the beginning of the light period (CT (circadian time) 00-02), and at the beginning of darkness (CT 12-14). These variations persisted in cultures that had been transferred into DD and appeared to be under the control of the circadian oscillator rather than to be cell division cycle (CDC)-dependent, since they continued in cultures that had reached the stationary phase of growth. In the experiments reported in this paper, we tested for the possible role of this periodic cyclic AMP signal in the generation of cell division rhythmicity by examining the effects of exogenous cyclic AMP signals and of forskolin, which permanently increased the cyclic AMP level, on the cell division rhythm. Perturbations of the cyclic AMP oscillation by exogenous cyclic AMP resulted in the temporary uncoupling of the CDC from the circadian timer. The addition of cyclic AMP during the subjective day resulted in delays (up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cyclic AMP was administered in the middle of the subjective night. Measurement of the DNA content of cells by flow cytometry indicated that cyclic AMP injected at CT 06-08 delayed progression through S phase, and perhaps also through mitosis. When added at CT 18-20, cyclic AMP accelerated the G2/M transition. The circadian oscillator was not perturbed by the addition of exogenous cyclic AMP: the division rhythm soon returned to its original phase. On the other hand, the permanent elevation of cyclic AMP levels in the presence of forskolin induced a rapid loss of cell division rhythmicity. These findings are consistent with the hypothesis that cyclic AMP acts downstream from the oscillator and that the cyclic AMP oscillation is an essential component of the signaling pathway for the control of the CDC by the circadian oscillator. The receptors for cyclic AMP in Euglena have been shown to be two cyclic AMP-dependent kinases (cPKA and cPKB). Pharmacological studies using cyclic AMP analogs suggested that cPKA mediates cyclic AMP effects during the subjective day, and cPKB during the subjective night.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased activity of the sarcoplasmic reticular calcium pump in porcine stunned myocardium

The aim was to determine whether changes in sarcoplasmic reticular Ca2+ transport activity and the degree of phosphorylation of phospholamban of "stunned" myocardium are involved in the reversible depression of contractile function. In anaesthetised open chest swine, stunning was induced by subjecting the myocardium perfused by the left anterior descending coronary artery to two cycles of 10 min of occlusion and 30 min of reperfusion. Before and after stunning, systemic haemodynamic variables and regional myocardial function and perfusion were determined, while biopsies were taken for determination of the content of high energy phosphate compounds. Sarcoplasmic reticular function (ATP dependent Ca2+ transport and phosphorylation of phospholamban) of the stunned and control myocardium was determined at the end of the stunning protocol. In the stunned myocardium the segment length shortening decreased from 17.4(SD 4.0)% to 3.5(4.4)%, while perfusion was 38% less than at baseline. ATP and total adenine nucleotide levels of the stunned myocardium were about 35% lower than in the control myocardium, but the energy charge was normal as creatine phosphate levels had increased by 66% over the content determined at baseline. Ca2+ uptake by the sarcoplasmic reticulum isolated from the stunned region was 17% (p < 0.05) higher than Ca2+ uptake from the control region [1240(303) and 1450(280) nmol.min-1.mg-1 protein, respectively]. In the presence of exogenous cyclic AMP dependent protein kinase the amount of 32P incorporated into phospholamban was similar for both myocardial regions. In this model of stunned porcine myocardium, the phosphorylation state of phospholamban was unchanged, but Ca2+ uptake by the sarcoplasmic reticulum was slightly increased. The results indicate that a change in active Ca2+ transport by the sarcoplasmic reticulum is most likely not to be the principal cause of contractile dysfunction of stunned myocardium.

Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.

Evidence of a stimulatory effect of cyclic AMP on pancreatic lipase and colipase synthesis in rats.

The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of 35S-cystine. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cystine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 microM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2 h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat.

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP.

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.

Regulation mechanisms of phenolic production in the lichen Himantormia lugubris, as deduced from the analysis of metabolite accumulation

HPLC analysis of the phenolic fraction of the lichen Himantormia lugubris reveals that atranorin, barbatolic acid and methyl β- orcinol carboxylate are mainly accumulated in the thallus. Samples floated in the dark on acetate actively synthesize barbatolic acid from the beginning of the incubation, and atranorin after an initial loss during the first hour of incubation. The amount of the atranorin precursor, methyl β-orcinol carboxylate, remains unchanged after an initial decrease similar to that found for atranorin. Active production of both atranorin and barbatolic acid is inhibited by white light and recovered by adding 3-(3′-dichlorophenyl)-1,1-dimethylurea (DCMU) or ATP to the incubation media. The supply of exogenous ATP strongly activates the accumulation of methyl β-orcinol carboxylate. This possibly indicates that ATP behaves as a limiting factor for the steps catalyzed by acetyl-CoA carboxylase and methionine adenosyltransferase, related to the production of this last phenolic. A glucose supply in the dark assures the ability of lichen thalli for phenolic synthesis whereas this process is strongly decreased by exogenous cyclic AMP.

Characterization and complementation analysis of sporogenous mutants ofDictyostelium discoideum

Sporogenous mutants of the cellular slime mold Dictyostelium discoideum are defined as mutants which are able to undergo terminal differentiation into spores in monolayer cultures in the presence of millimolar amounts of exogenous cyclic AMP. We describe the morphological development and cellular differentiation of a collection of 12 independently isolated sporogenous mutants of strain V12 M2. All mutants develop more rapidly than do wild-type at an air-water interface, display aberrant morphogenesis, and show overt spore and stalk differentiation as soon as 4 hr after starvation. All mutants differentiate in submerged monolayer culture in the presence of cAMP into variable proportions of spores and stalk cells. A number of the mutants also form both stalk cells and spores in submerged culture in the absence of exogenous cAMP. The spores formed by many of the mutants have a greatly reduced viability. Using parasexual genetics, we have found that two of the 12 mutants analyzed are dominant to wild-type and the remaining ten fall into a minimum of four complementation groups, the overall analysis thus yielding a minimum of four and a maximum of seven complementation groups. Intracellular cAMP levels in vegetative cells are significantly elevated in the two dominant mutants but are similar to wild type in all the other mutants.