Pharmacogenetics of cyclic guanylate, antioxidants, and antioxidant enzymes in Saccharomyces

Supplements of antioxidants, superoxide dismutase (SOD), catalase, cyclic guanylate (cGMP), and theophylline, or omission of iron and copper from the medium are therapeutic for the inferior growth and viability of yeast mutants doubly deficient in mitochondrial and exocellular SOD isozymes under oxidative stresses. Cyclic adenylate tends to be ineffective or counterproductive. Oxy-stress resistant revertants are cross-resistant to other oxy-stresses and acquire one, the other, or both isozymes. The principal conclusions are: i) a genetic defect in cGMP metabolism probably compromises regulation of the enzymes' synthesis; ii) the enzymes are only essential for growth and viability under oxidative stresses; iii) oxidative toxicity is mediated by both exo- and endocellular oxy-radicals, particularly hydroxyl radicals; and iv) the pharmacogenetic features and the mutants' phenotypes are quite similar to those of negative antioxidant enzyme regulatory mutants of the related ascomycete Neurospora.     

Selection and analysis of superoxide dismutase mutants of Neurospora.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.     

Chemoattractant-mediated increases in cGMP induce changes in Dictyostelium myosin II heavy chain-specific protein kinase C activities

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP (Abu- Elneel et al. 1996. J. Biol. Chem. 271:977- 984). Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization (Liu, G., and P. Newell. 1991. J. Cell. Sci. 98: 483-490). This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane association, phosphorylation, and activity (hereafter termed MHC-PKC activities). cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP- induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows nearly normal cAMP-induced cGMP response but has aberrant cGMP binding activity, show no changes in MHC- PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC.     

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

Purification and characterization of a human platelet cyclic nucleotide phosphodiesterase

A cyclic nucleotide phosphodiesterase was extensively purified from the 100000g supernatant fraction of human platelets. The purification was 2500-3000-fold with 30% recovery of activity. The enzyme was isolated by DEAE-cellulose chromatography followed by adsorption to blue dextran-Sepharose and elution with cAMP. The protein has a molecular weight of 140 000 as determined by gel filtration. On NaDodSO4-containing polyacrylamide gels the major band is at 61 000 daltons, suggesting that the enzyme may exist as a dimer in solution under nondenaturing conditions. The enzyme requires Mg2+ or Mn2+ for activity. The calcium binding protein calmodulin does not stimulate hydrolysis of cAMP by this enzyme. The purified enzyme hydrolyzes both cAMP and cGMP with normal Michaelis-Menten kinetics with Km values of 0.18 microM and 0.02 microM, respectively. The hydrolysis of cGMP, however, is only one-tenth as rapid as the hydrolysis of cAMP. Cyclic GMP does not stimulate cAMP hydrolysis but instead is a potent competitive inhibitor of cAMP hydrolysis. The enzyme is also competitively inhibited by the phosphodiesterase inhibitors papaverine, 3-isobutyl-l-methylxanthine, and dipyridamole. The enzyme did not cross-react with an antibody raised to a cAMP phosphodiesterase isolated from dog kidney, indicating that the enzymes are not immunologically related. The inhibition of cAMP hydrolysis by cGMP suggests a possible regulatory link between these two cyclic nucleotides. One of the roles of cGMP in platelets may be to potentiate increases in intracellular cAMP by inhibiting the hydrolysis of cAMP by this enzyme.     

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.     

New messages from old messengers: cAMP and mycobacteria

Cyclic nucleotides are ancient second messengers, and the enzymes that synthesize cAMP and cGMP [cyclic nucleotide monophosphates (cNMPs)] are encoded in the genomes of several bacteria. We focus here on recent biochemical and structural information on the proteins that make and break cyclic nucleotides in mycobacteria, namely the nucleotide cyclases and phosphodiesterases, respectively. The presence of these enzymes along with putative cNMP-binding proteins suggests an intricate regulation of cAMP metabolism and utilization by these organisms. It is anticipated that future research will be directed towards identifying cellular processes that are regulated by cAMP in mycobacteria and deciphering the cross-talk between mycobacterial pathogens and their eukaryotic host.     

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.     

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.     

Characterization of revertants of stmF mutants of Dictyostelium discoideum: evidence that stmF is the structural gene of the cGMP-specific phosphodiesterase

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm+) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm+ revertants carried phenotypic suppressors unlinked to stmF. One Stm+ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.     

The epidermal growth factor-induced cell migration and expression of the 47,000 Mr secreted glycoprotein EIP-1 of rat liver epithelial cells are down-modulated by cyclic AMP

The migration of rat liver epithelial cells induced by epidermal growth factor (EGF) was inhibited by cyclic AMP (cAMP) and cholera toxin, but not by cGMP, cAMP and cholera toxin also inhibited the expression of the EGF/transforming growth factor (TGF) alpha-inducible protein EIP-1 (Mr 47,000), but not that of other proteins induced by the growth factor. cAMP therefore specifically and selectively represses the EGF-induced expression of this protein, which by synthesis in the presence of tunicamycin and by enzymatic treatments was shown to be N-glycosylated and sialylated. The close correlation of the expression of EIP-1 with the growth factor-induced migration suggests that this glycoprotein is involved in the cellular translocation process. Modulation of cell migration and of EIP-1 expression through increased intracellular concentrations of cAMP indicate that factors operating through this signal system can modulate the phenotypic and gene expression changes mediated by the EGF-receptor. Identification of the ligand(s) that can cause the cAMP-mediated effects might be an important step towards understanding the regulation of liver cell migration in vivo.     

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.     

Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.     

Mutations in chemosensory cilia cause resistance to paraquat in nematode Caenorhabditis elegans

The relationship between oxidative stress and longevity is a matter of concern in various organisms. We isolated mutants resistant to paraquat from nematode Caenorhabditis elegans. One mutant named mev-4 was long-lived and showed cross-resistance to heat and Dyf phenotype (defective in dye filling). Genetic and sequence analysis revealed that mev-4 had a nonsense mutation on the che-11 gene, homologues of which are involved in formation of cilia and flagella in other organisms. The paraquat resistance was commonly observed in various Dyf mutants and did not depend on the daf-16 gene, whereas the extension of life span did depend on it. Expression of antioxidant enzyme genes seemed normal. These results suggest that chemosensory neurons are a target of oxidative stress and influence longevity dependent on the daf-16 signaling in C. elegans.     

Bonsaï, a ribosomal protein S15 homolog, involved in gut mitochondrial activity and systemic growth

The regulation of cellular growth is crucial in the control of cell proliferation. While most of the metabolic energy necessary to sustain growth is produced in mitochondria, the regulation of mitochondrial activity and its implications for growth have remained unexplored. Here, a gene named bonsa? is described, which is essential for normal growth in Drosophila. The Bonsa? protein bears strong homology to prokaryotic ribosomal protein S15 and localizes to mitochondria, suggesting a role in mitochondrial protein translation. Accordingly, bonsa? mutants have defective mitochondrial activity, but surprisingly, only the gut appears affected. Consistent with these observations, bonsa? is predominantly expressed in the gut. These results show that bonsa? plays a preponderant role in gut mitochondria. Although gut mitochondrial respiration is altered in bonsa? mutants, the digestive process appears normal, suggesting that a gut function other than digestion is impaired in the mutants. Cytochrome c oxidase, a respiratory chain enzyme partly encoded by the mitochondrial genome, is found to be active in bonsa? mutants. This suggests that mitochondrial translation is not abolished in the mutants. Altogether, these observations suggest that mitochondrial activity is regulated at the tissue-specific level and that this regulation has profound implications for growth and development.     

Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.     

3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.