The Distribution of the Number of Maxima in a Cyclic Sequence

A simple statistical method is presented which tests for non-randomness in a cyclic sequence. The distribution of the statistic is tabulated for short sequences, and a large sample approximation is derived. Significant values of the statistic are given for sequences of length up to 52. Easily used computer programs to carry out appropriate calculations are presented.     

Cyclic GMP-activated channels of the chick pineal gland: Effects of divalent cations,pH, and cyclic AMP

Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 M Ca²⁺ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca²⁺ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg²⁺ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 M, 500 M, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 M cyclic AMP decreased the probability of channel activation caused by 20–100 M cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca²⁺, pH, or cyclic AMP.     

Distribution of Cyclic Nucleotide Phosphodiesterase in Mouse Brain

Seventy-one regions of mouse brain, and many subdivisions of some of these, were analyzed for cyclic nucleotide phosphodiesterase. The samples were dissected from lyophilized frozen sections. Since the average sample weighed only 25 ng (20 X 75 X 75 mu3), regions as small as the locus ceruleus could be analyzed. Activities in gray areas ranged 40-fold from a high in the pars reticulata of the substantia nigra to a low in the deep cerebellar nuclei. The activity in fiber tracts also varied about 40-fold, and on a lipid-free dry weight basis was similar to the activity in the gray matter where the fibers originated. The rank order for gray regions was basal ganglia, amygdala, hippocampus, cerebral cortex, most of the diencephalic nuclei, nuclei of the pons, cerebellum, and nuclei of the medulla.     

Circadian Variation of Cyclic AMP in the Rat Pineal Gland

Abstract: This study was carried out to investigate circadian variation of cyclic AMP contents in the rat pineal glands, using the high-energy microwave radiation technique. The pattern of cyclic AMP concentration in the pineal gland showed a distinct circadian variation, with the maximum level at 0200 and the lowest at 1400. The administration of propranolol completely blocked the dark-induced increase in the pineal cyclic AMP level at 0200, and the administration of isoproterenol induced a threefold, rapid increase in the cyclic AMP level at 1400, although it did not change the level at 0200.     

Cyclic AMP Resets the Circadian Clock in Cultured Xenopus Retinal Photoreceptor Layers

Abstract: The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180° out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

Distribution of Calmodulin- and Cyclic AMP-Stimulated Protein Kinases in Synaptosomes

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.     

Cyclic AMP Accumulation Induces a Rapid Desensitization of the Cyclic AMP-Dependent Protein Kinase in Mouse Striatal Neurons

Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn(2+)-protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 microM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8-bromo-cAMP (8-Br-cAMP) at 100 microM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8-Br-cAMP treatment. Finally, not only 8-Br-cAMP- but also forskolin- and vasoactive intestinal peptide-induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation.     

Regulation of Tryptophan Hydroxylase by Cyclic AMP, Calcium, Norepinephrine, and Light in Cultured Chick Pineal Cells

Abstract: The level of ³⁵S incorporation into tryptophan hydroxylase (TPH) shows a circadian rhythm in cultured chick pineal cells. The TPH oscillation peaks in the early subjective night, persists in constant darkness, and can be phase shifted by light, in parallel to the effect of these treatments on melatonin synthesis. Using quantitative two-dimensional polyacrylamide gel electrophoresis, we have examined the regulation of TPH by agents known to affect melatonin synthesis in the chick pineal. We report here that ³⁵S incorporation into TPH is induced by cyclic AMP and calcium, and partially inhibited by acute exposure to light. Cyclic AMP also causes a proportional increase in the radiolabeling of one of the TPH isoforms and a concomitant decrease in another isoform, possibly reflecting a change in the phosphorylation state of TPH. This effect is reversed by treatments known to reduce intracellular cyclic AMP levels in the chick pineal. Cyclic AMP thus appears to be involved in both translational and posttranslational processes regulating the expression of TPH in chick pineal cells.     

Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP-Dependent Protein Kinase in Rat Frontal Cortex

The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by approximately 25%, whereas enzyme activity was increased in the particulate fractions by approximately 20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant drugs, because representatives of several other classes of psychotropic drugs-namely, haloperidol, morphine, and diazepam--failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the clinical actions of these treatments.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Cyclic ureide and imide metabolism in microorganisms producing a -hydantoinase useful for -amino acid production

The microbial transformation of -5-monosubstituted hydantoins has been applied to industrial scale production of optically active amino acids. Hydantoinase and N-carbamoyl amino acid amidohydrolase, which are the key enzymes in this transformation, from various microorganisms have been studied extensively. Blastobacter sp. A17p-4, which was isolated for -amino acid production through hydantoin transformation, shows not only diverse cyclic ureide-metabolizing activities including those of -hydantoinase and N-carbamoyl- -amino acid amidohydrolase, but also cyclic imide-metabolizing activities. A recent study revealed the participation of -hydantoinase in the metabolism of cyclic imides and the existence of novel enzymes, imidase and half-amidase, in this bacterium. -hydantoinase functions in the metabolism of bulky cyclic imides, while imidase functions in that of simple cyclic imides in combination with half-amidase, which functions in the hydrolysis of the imidase reaction products, half-amides. Imidase and half-amidase are different from reported cyclic-amide-metabolizing enzymes, and are widely found in bacteria, yeasts and molds.     

Capsaicin-Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture

Capsaicin, which induces fluxes of sodium, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 microM) to 10-20 pmol cGMP/mg protein in these cells. Cyclic AMP (cAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin-induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage-sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin-evoked calcium uptake through voltage-sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin-evoked ion fluxes or to their desensitisation.     

Cyclic Nucleotide Phosphodiesterase Activity in Bovine Brain Coated Vesicles

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat-inactivated, formaldehyde-fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP-sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1-methyl-3-isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 microM. These compounds also inhibited the cyclic GMP-stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 microM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.     

Cyclic nucleotide content of tobacco BY-2 cells

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.     

Cyclic GMP-Dependent Protein Kinase Substrates in Rat Brain

Abstract: Cyclic GMP (cGMP)-dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G-substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion-exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca²⁺-sensitive lipid-binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate—P34—was found. However, after chromatography we detected >40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP-dependent protein kinase or protein kinase C. Four PKG substrates—P140, P65, P32, and P18—were detected in the cytosol. Six PKG substrates—P130, P85 (doublet), P58, P54, and P38—were enriched from the Ca²⁺-sensitive lipid-binding protein fraction. In peripheral membrane fractions >30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.     

Cholecystokinin Stimulates Dopamine Synthesis in Synaptosomes by a Cyclic AMP-Dependent Mechanism

The effects of different fragments of cholecystokinin (CCK) on dopamine synthesis were studied in synaptosomal preparations from the striatum, substantia nigra, and frontal cortex. In striatal synaptosomes, dopamine synthesis rate measured by dopamine accumulation was 12.5% lower than that measured by 3,4-dihydroxyphenylalanine (DOPA) accumulation; however, K+-accelerated synthesis was the same for both methods. Synthesis rate was independent of exogenous tyrosine levels. In the three regions studied, the combined stimulatory effects of 8-Br-cyclic AMP and high K+ were additive. CCK-5, CCK-3, CCK-27-33, and CCK-8 (sulphated) enhanced synthesis, CCK-5 being the most potent fragment. The nonsulphated octapeptide had no effect. In all three regions, CCK-5 and high K+ had an additive effect on dopamine synthesis; CCK-5 and 8-Br-cyclic AMP together produced the same enhancement of synthesis as CCK-5 alone. CCK-5 produced similar dose-dependent increases in dopamine synthesis and cyclic AMP accumulation in striatal synaptosomes, and both effects were blocked by the CCK antagonist proglumide.     

The Estimation of the Number and the Length Distribution of Gene Conversion Tracts from Population DNA Sequence Data

DNA sequence variation studies report the transfer of small segments of DNA among different sequences caused by gene conversion events. Here, we provide an algorithm to detect gene conversion tracts and a statistical model to estimate the number and the length distribution of conversion tracts for population DNA sequence data. Two length distributions are defined in the model: (1) that of the observed tract lengths and (2) that of the true tract lengths. If the latter follows a geometric distribution, the relationship between both distributions depends on two basic parameters: ψ, which measures the probability of detecting a converted site, and , the parameter of the geometric distribution, from which the average true tract length, 1/(1 - ), can be estimated. Expressions are provided for estimating by the method of the moments and that of the maximum likelihood. The robustness of the model is examined by computer simulation. The present methods have been applied to the published rp49 sequences of Drosophila subobscura. Maximum likelihood estimate of for this data set is 0.9918, which represents an average conversion tract length of 122 bp. Only a small percentage of extant conversion events is detected.