Changes in the rate of chitin-plus-chitosan synthesis accompany morphogenesis of Mucor racemosus.

The in vivo differential rates of chitin-plus-chitosan biosynthesis in Mucor racemosus were determined under a variety of conditions, leading to yeast cell or mycelial morphology. Chitin-chitosan was determined as hot NaOH-insoluble radioactivity derived from N-acetyl-D-[1-3H]glucosamine in the medium. Control experiments demonstrated that the labeled material possessed the properties of chitin-plus-chitosan. Our results indicate that Mucor yeasts have a relatively low differential rate of chitin-plus-chitosan synthesis and that mycelial cells have a threefold-elevated differential rate. Treatment of aerobic cells with exogenous N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate, an agent which induces yeast cell morphology, also results in a lowered rate of chitin-plus-chitosan synthesis. Control experiments eliminated the possibility that the observed rate changes were due to changes in endogenous pool size, uptake of exogenous N-acetyl-p-[1-3H]glucosamine, or alterations in growth rate. Therefore, the changes are seemingly linked to morphogenesis. These results strengthen the idea that cyclic adenosine 3',5'-monophosphate plays an important role in dimorphism in Mucor. In addition, pulse-chase experiments suggest that considerable modification of newly synthesized chitin plus chitosan in both yeast cells and mycelia occurs in vivo.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans.

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

Hyperkalemia, hyperglycemia, and plasma levels of cyclic AMP and cyclic GMP induced by portal vein injection of cyclic nucleotides and their butyryl derivatives into dogs

The levels of serum potassium, blood glucose, and plasma adenosine cyclic 3':5'-monophosphate (cAMP) and guanosine cyclic 3':5'-monophosphate (cGMP) were studied after the portal vein injection of cyclic nucleotides and their derivatives, (cAMP, cGMP, N6, O2'-dibutyryl adenosine 3':5'-monophosphate (DBcAMP), N6-monobutyryl adenosine cyclic 3':5'-monophosphate (NMBcAMP), and O2'-monobutyryl adenosine cyclic 3':5'-monophosphate (OMBcAMP), into dogs. Dose-related hyperglycemic responses were observed after the injection of DBcAMP (1-8 mg/kg). Transient and prominent hyperkalemia and hyperglycemia were caused by the injection of DBcAMP, NMBcAMP, and OMBcAMP (4 mg/kg). The hyperkalemic response was highest with NMBcAMP (1.22 mequiv./L), followed by OMBcAMP (0.64), DBcAMP (0.54), cGMP (0.47), and cAMP (0.41), whereas the hyperglycemic response was highest with NMBcAMP (146 mg/100 mL), followed by DBcAMP (93.6), OMBcAMP (77.1), and cAMP (56.0), and there was only a slight change with cGMP (28.4) compared with the control. The plasma level of cAMP was maximal with DBcAMP (1.92 nmol/mL), followed by NMBcAMP (1.28) and OMBcAMP (0.76), whereas the plasma levels of cGMP showed no evident change, except that caused by DBcAMP (0.27). Of the cyclic nucleotides tested, NMBcAMP was found to be most potent in causing both hyperkalemia and hyperglycemia. Based on these results, possible correlations between hyperkalemia, hyperglycemia, and plasma levels of cAMP and cGMP are discussed.     

Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.     

Role of Cyclic Adenosine 3′,5′-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.     

Glucose metabolism and dimorphism in Mucor.

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.     

Control of adenosine 3',5'-monophosphate level and protein phosphorylation by depolarizing agents in Coprinus macrorhizus

The treatment of mycelial cells with membrane-active antibiotics, uncouplers of oxidative phosphorylation and KCl leads to a transient increase in adenosine 3',-5'-monophosphate (cyclic AMP) levels in Coprinus macrorhizus. The maximal values and duration of increase in the cyclic AMP level depended on the kind and amount of these drugs. The treatment with these drugs simultaneously resulted in a rapid increase in the phosphorylation of three cellular proteins. The levels and time course of phosphorylation of these proteins were paralleled with the increase of cyclic AMP level in response to the drugs used. Thus, the treatment of these drugs causes the transient increase of cyclic AMP level and cyclic AMP stimulates the phosphorylation of particular proteins by activating protein kinases.     

The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells.

The possibility that hormones control hepatic gluconeogenesis via the regulation of the rate of mitochondrial pyruvate carboxylation was investigated with the use of suspensions of liver cells isolated from fasted rats. The mitochondria prepared from liver cells were judged in good condition as they exhibited satisfactory phosphorus-oxygen and respiratory control ratios and transported Ca2+ and K+ ions in an energy-dependent manner. Addition of glucagon, epinephrine, or cyclic adenosine 3':5'-monophosphate to liver cells caused a 50 to 80% increase in the rate of glucose synthesis from lactate. When mitochondria were isolated from the cells after treatment with these agonists, they displayed 2- to 3-fold increases in the rate of pyruvate carboxylation, pyruvate decarboxylation, and pyruvate uptake. These mitochondrial changes are similar to those obtained in hepatic mitochondria prepared from intact, hormone-treated rats. The mitochondrial responses were specific for agents that stimulated gluconeogenesis; no response occurred with 5'-AMP or cyclic adenosine 2':3'-monophosphate. In the cell suspensions, the dose response curves for the activation of mitochondrial pyruvate metabolism and for increased glucose synthesis from L-lactate were coincident with four different agonists. The mitochondrial changes resulting from stimulation with glucagon developed in 1 to 2 min after the rise in cyclic adenosine 3':5'-monophosphate and occurred at least as early as the increase in the rate of gluconeogenesis. When the intracellular level of cyclic adenosine 3':5'-monophosphate returned to basal values, the rates of mitochondrial pyruvate carboxylation and glucose synthesis also declined to control levels. It is concluded that the rate of mitochondrial pyruvate metabolisms can be increased by hormones and cyclic nucleotides and that control of mitochondrial pyruvate carboxylation is an important regulatory site of hepatic gluconeogenesis.     

Control of Neurospora crassa morphology by cyclic adenosine 3'', 5''-monophosphate and dibutyryl cyclic adenosine 3'', 5''-monophosphate.

The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.     

Variations in levels of cAMP, DNA and RNA in Streptomyces alboniger under conditions of aerial mycelium formation and repression

Abstract The relationship between endogenous levels of cyclic adenosine 3',5'-monophosphate (cAMP) and the formation of aerial mycelia was investigated in Streptomyces alboniger under conditions of aerial mycelium formation and repression. The relationship between cellular levels of DNA and RNA and aerial mycelium formation was also investigated. In contrast to cellular differentiation in other Streptomyces , neither variations in cAMP, DNA or RNA levels were found to be associated with the development of aerial mycelia in S. alboniger . The regulation of adenylate cyclase in S. alboniger , however, was found to differ from that of Escherichia coli and related organisms in that glucose raised, rather than lowered, endogenous cAMP levels.     

Control of cyclic adenosine 3'',5''-monophosphate levels by depolarizing agents in fungi.

It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Possible role of adenosine cyclic 3':5'-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6,O2-dibutyryl adenosine cyclic 3':5"-monophosphate.

We have demonstrated that in Chinese hamster ovary (CHO) cells, N6,O2'-dibutyryl adenosine cyclic 3':5'-monophosphate (dibutyryl cyclic AMP) has a remarkable morphogenetic effect in converting cells of a compact, epithelial-like morphology into a spindle-shaped, fibroblast-like form. Homogenates of CHO cells were found to contain two adenosine cyclic 3':5'-monophosphate (cyclic AMP) phosphodiesterase (EC 3.1.4.c) activities, which differ in apparent Km with respect to their substrate, cyclic AMP. These were designated cyclic AMP phosphodiesterase I, with a low Km of 2 to 5 muM and cyclic AMP phosphodiesterase II, with a high Km of 1 to 3 mM. Cyclic AMP phosphodiesterase I was competitively inhibited by N6-monobutyryl and dibutyryl cyclic AMP, with apparent Ki values of 40 to 60 muM and 0.25 to 0.35 mM, respectively. Experimental evidence demonstrates that the effect of exogenous dibutyryl cyclic AMP on cell morphology is a result of an increase in the endogenous level of cyclic AMP. This increase appears to be due largely to the inhibitory action of intracellular N6-monobutyryl cyclic AMP on cyclic AMP phosphodiesterase I, which results in a decreased rate of degradation of intracellular cyclic AMP.