Cyclic Adenosine Monophosphate in the Nervous System of Aplysia californica : I. Increased synthesis in response to synaptic stimulation

In the isolated abdominal ganglion of Aplysia, previously incubated in adenine-³H, the amount of ³H-labeled adenosine-3',5' monophosphate (cAMP) doubled after electrical stimulation of nerves at a physiological rate (1/sec). No change was detected after 4 min of stimulation. An increase in cAMP was first seen after 15 min; lengthening the period of stimulation to 1 hr did not increase the extent of the effect. ATP contained 50% of the total radioactivity taken up from adenine-³H, cAMP about 0.1%. During stimulation both the total amount and the specific radioactivity of adenosine triphosphate (ATP) did not change. Thus, the increased amount of radioactivity found in cAMP after stimulation represented an increase in its rate of synthesis. During stimulation formation of cAMP-³H was not altered in nerves or in the cell body of an identified neuron (R2). In addition, no changes were detected in the total amounts of cAMP in the ganglion and in the cell body of R2. It seems likely that the increase was initiated by synaptic activity rather than by action potentials. It was blocked by elevating the concentration of Mg, which also blocks synaptic activity without impairing conduction of impulses. Moreover, impulse activity induced by ouabain and glutamate did not result in increased formation of cAMP.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Synthesis of γ-Glutamyldopamine and Other Peptidoamines in the Nervous System of Aplysia californica

The synthesis of a series of gamma-glutamyl amines (gamma-Glu-amines), including gamma-Glu-dopamine, gamma-Glu-5-hydroxytryptamine, gamma-Glu-octopamine, gamma-Glu-tryptamine, gamma-Glu-tyramine, and gamma-Glu-phenylethylamine, by nervous tissue of the marine mollusc Aplysia californica is described. After ganglia were incubated in vitro with 14C-amines, the unchanged amine and a new 14C-labeled product, identified as the gamma-Glu conjugate of the amine, were isolated from the tissue extracts. Identification was made by comparing the chromatographic properties (HPLC, TLC, and LC) of the isolated conjugates with chemically synthesized gamma-Glu-amines before and after acid hydrolysis.     

低聚磷酸盐与次黄嘌呤偶合添加提高环磷酸腺苷发酵性能

环磷酸腺苷(cAMP)在微生物细胞内由ATP直接环化形成,而ATP的合成需要能量与前体的持续供应。通过添加次黄嘌呤激活补救途径,促进了cAMP的合成,与对照批次相比生产效率提高了39. 1%,但发酵进行至51h产物不再生成,而且产量未能得到提高。偶合添加次黄嘌呤和2g/L-broth六聚偏磷酸钠发酵批次的cAMP产量达到7. 24g/L,比单独添加次黄嘌呤和六聚偏磷酸钠的批次产量分别提高了125. 5%和93. 5%,生产效率也显著提高,达到了0. 101g/(L·h)。六聚偏磷酸钠和次黄嘌呤偶合添加工艺将低聚磷酸盐和补救途径的优势相结合,有效促进了cAMP合成与积累。     

Effect of Electric Convulsion Therapy on Urinary Excretion of 3′, 5′ Cyclic Adenosine Monophosphate

Electric convulsion therapy (E.C.T.) was used in the treatment of 13 women inpatients suffering from depressive symptoms. Twelve of the patients showed a significant increase in urinary excretion of 3′, 5′ cyclic adenosine monophosphate (cAMP) on the day of treatment, whereas four controls who received all or part of the preliminary treatment but no electric shock showed a reduction. The results of this study are consistent with the hypothesis that the antidepressant action of E.C.T. is mediated through an increased production of cAMP in brain tissue.     

Development of neurons in the abdominal ganglion of Aplysia californica. II. Nonneural support cells.

The release by nonneural support cells of a diffusable chemical substance into the local environment in which sympathetic neurons develop is thought to play a crucial role in their differentiation. In this paper, we describe a novel class of nonneural support cells associated with a central ganglion of Aplysia californica during the premetamorphic stages of development. These support cells contain secretory granules whose contents are primarily released at metamorphosis. The release of these contents may signal the burst of neuronal growth and maturation that occurs following metamorphosis. The evidence in support of this notion is the following: (1) Spontaneous release of the granule material at metamorphosis coincides with an increase in cell body growth and a more marked increase in the density of synapses of the abdominal ganglion. (2) Premature release of the granule material before metamorphosis with artificial seawater containing a high concentration of potassium results in a burst in cell body growth and a premature increase in synapse density. (3) Premature release of granule material also results in a precocious increase in the number of spines formed and synaptic contacts received by specific identified cells. Based on the findings in this and the preceding paper, we propose a two-stage model of the developmental program for differentiation of neurons in the abdominal ganglion. First, axosomatic contacts trigger axonal outgrowth. Second, material released from the granules of the support cells stimulates further steps in neuronal differentiation, including cell growth, spine development, and synapse formation.     

DNA sequence organization in the mollusc Aplysia californica.

The sequence organization of the DNA of the mollusc Aplysia californica has been examined by a combination of techniques. Close-spaced interspersion of repetitive and single copy sequences occurs throughout the majority of the genome. Detailed examination of the DNA of this protostome reveals great similarities to the pattern observed in the two deuterostome organisms previously examined in detail in this laboratory, Xenopus laevis and Strongylocentrotus purpuratus. Labeled and unlabeled Aplysia DNA were prepared from developing embryos and sheared to a fragment length of 400 nucleotides. The kinetics of reassociation were studied by means of hydroxyapatite chromatography, single-strand-specific S1 nuclease, and optical methods of assay. Aplysia DNA of this fragment length contains at least five resolvable kinetic fractions. One classification of these fractions, listed with their reassociation rate constants (l M-1 sec-1) is: single copy (0.00057), slow (0.047), fast (2.58), very fast (4000), and foldback (greater than 10(5)). Sequence arrangement was deduced from: the kinetics of reassociation of DNA fragments of length 400 or 2000 nucleotides; the hyperchromicity of reassociated fragments containing duplex regions; the size of duplex regions resistant to S1 nuclease; and the reassociation of labeled fragments of various lengths with short driver fragments. More than 80% of the single copy DNA sequences are interspersed with repetitive sequences. The maximum spacing of the repeats is about 2000 nucleotides, and the average less than 1000. The very fast fraction does not show interspersion with single copy sequences or with other kinetic fractions. The foldback fraction sequences are fairly widely interspersed. The slow fraction sequences are interspersed with the fast fraction, and possibly also with the single copy DNA. The fast fraction is the dominant interspersed repetitive fraction. Its sequences are adjacent to the great majority of the single copy sequences and have an average length of about 300 nucleotides.     

Development of embryonic cells containing serotonin, catecholamines, and FMRFamide-related peptides in Aplysia californica

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.     

Serotonin catabolism depends upon location of release: characterization of sulfated and gamma-glutamylated serotonin metabolites in Aplysia californica

Serotonin is a vital neurotransmitter for the functioning of the nervous system in species throughout the animal phyla. Despite its ubiquitous nature, the metabolism of this molecule has yet to be completely elucidated in even the most basic of organisms. Two novel serotonin catabolites, serotonin-O-sulfate and gamma-glu-serotonin-O-sulfate, are chemically characterized using capillary electrophoresis with wavelength-resolved fluorescence detection and electrospray mass spectrometry, and the formation of gamma-glu-serotonin in Aplysia californica is confirmed. These novel compounds appear to be synthesized enzymatically, and known mammalian enzymes exist for all serotonin transformations observed here. The pathway of serotonin inactivation depends upon the type of neuronal tissue subjected to neurotransmitter incubation, with assorted serotonin products observed in distinct locations. Initially demonstrated to be in the metacerebral cell (MCC) soma, the new serotonin metabolite serotonin-O-sulfate may contribute to important functions in the serotonergic system beyond simple serotonin inactivation.     

A Cyclic Adenosine Monophosphate Link in the Catecholamine Enhancement of Transmitter Release at the Neuromuscular Junction

The frequency of miniature endplate potentials (mepps) in rat diaphragms was markedly increased by epinephrine and norepinephrine in preparations exposed to 15 mM K⁺. The effect was rapid in onset but gradually declined during continued exposure to the catecholamines. N⁶, O^2'-dibutyryl adenosine 3',5'-monophosphate (dibutyryl-cAMP) also caused transient frequency increases resembling in time-course those observed with catecholamines. Contrary to previous reports, catecholamines and dibutyryl-cAMP had little effect on mepp frequency in preparations not treated with K⁺. Sustained increases with theophylline and decreases with adenosine were found in both K⁺-treated and untreated preparations. Analysis of the data obtained with catecholamines showed the intensity of the response to be a function of nerve terminal polarization. The inability of catecholamines and dibutyryl-cAMP to affect mepp frequency of untreated preparations argues against an obligatory role for cAMP in the neurosecretory mechanism. The findings are consistent with an action of catecholamines and cAMP in the regulation of transmitter release at fatigued preparations.     

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

Background

Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection.

Objective

This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.

Study Design

Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined.

Results

Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4⁺CD25⁺Foxp3⁺ and CD4⁺Foxp3⁺ T cells compared with patients with HFMD or BE. The expression frequency of CD4⁻CD8⁻ was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4⁺Foxp3⁺ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.

Conclusions

These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.     

Effect of Inhibitors of Ribonucleic Acid and Protein Synthesis on the Cyclic Adenosine Monophosphate Stimulation of Plasmid ColE1 Replication

Addition of cyclic adenosine 3'-5'-monophosphate (c-AMP) to growing Escherichia coli cells, colicinogenic for the plasmid ColE1, results in a fourfold stimulation in the rate of synthesis of the plasmid deoxyribonucleic acid (DNA). The stimulation is transient (30 min) and is succeeded by a brief period (30 min) of cessation of plasmid DNA replication. The stimulation of ColE1 DNA replication also occurs in chloramphenicol-treated cells. Rifampin inhibits ColE1 DNA replication in the presence or absence of c-AMP. Employing thymine starvation conditions to stop ColE1 DNA synthesis, it was found that c-AMP, added during the period of thymine starvation, effected a stimulation in the amount of subsequent replication which took place when replicating conditions were restored. The stimulatory effect of c-AMP under these conditions was not prevented by chloramphenicol but was completely eliminated when rifampin was present. Under these conditions, when rifampin was added after the effect of c-AMP was allowed to occur, subsequent replication of the plasmid could take place, but only one round of replication occurred. A model to account for the c-AMP effects is presented.     

Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica

The distribution of (Na+ + K+) ATPase over the plasma membranes of the proximal convoluted tubule from canine renal cortex has been determined. Ultrathin frozen sections of this tissue were stained with rabbit antibodies to this enzyme and ferritin-conjugated goat antirabbit gamma-globulin. It is demonstrated that high concentrations of this enzyme uniformly line the intercellular spaces of this epithelium. The consequences of this observation are discussed in terms of the low resistant tight junctions of these tubules and the isotonic fluid transport which they support. Furthermore, antibodies to (Na+ + K+) ATPase recognize an antigen on the luminal surfaces of the tubules within the brush border. It is proposed that the enzyme is present in this region of the plasma membrane as well, although at much lower concentration. To further substantiate this conclusion, a brush border fraction has been purified from rabbit kidney and been shown to contain significant (Na+ + K+) ATPase. These results contradict earlier conclusions about the location of (Na+ + K+) ATPase in this tissue.     

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ~60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.     

Distribution and coexistence of urotensin I and urotensin II peptides in the cerebral ganglia of Aplysia californica.

Urotensin I (UI) and urotensin II (UII) were demonstrated in the cerebral ganglia of Aplysia californica by applying immunocytochemical and radioimmunoassay procedures. Sequential analysis of adjacent sections of the cerebral ganglia of Aplysia demonstrated that the UI-immunoreactive (UI-IR) neurons of the F cluster of the cerebral ganglia also contained UII immunoreactivity (UII-IR). Both UI-IR and UII-IR were also observed in a cuff-like arrangement of fibers surrounding the proximal portion of the supralabial nerve, as well as in a few fibers in the anterior tentacular nerves. The UI-IR perikarya of the cerebral ganglia appeared to project to the entire CNS of Aplysia, but the UII-IR fibers appeared only in the neuropile and commissure of the cerebral ganglia. The UI-IR staining was abolished by previous immunoabsorption of the UI antiserum with sucker (Catastomus commersoni) UI, but not with ovine corticotropin-releasing factor (CRF), rat/human CRF, or goby (Gillichthys mirabilis) UII. Immunostaining with UII antiserum was quenched by goby UII, but not by sucker UII-A, UII-B, UII-A(6-12), or carp (Cyprinus carpio) UII-alpha and UII-gamma. The UII staining was not abolished by UI or somatostatin. The F cluster was not stained when a somatostatin antiserum was applied. Radioimmunoassay of dilutions of cerebral ganglia extract, using UII antiserum, revealed a parallel displacement curve to synthetic goby UII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cyclic AMP in heart and gill of Aplysia by the putative neurotransmitters dopamine and serotonin.

Serotonin (5-HT) and dopamine (DA), but not several other putative neurotransmitters, stimulate cyclic adenosine-3',5'-monophosphate (cAMP) production in slices of $\text{Aplysia}$ gill. Furthermore, 5-HT but not DA increases cAMP in slices of the heart of $\text{Aplysia}$. Several lines of evidence indicate that the receptors are distinct entities; however, no drugs were found to block one receptor without affecting the other.     

Stages in the post-hatching development of Aplysia californica

In order to study the development of the nervous system of the marine mollusc, Aplysia californica, it is necessary objectively to assess the maturity of individual specimens. This can be done by defining stages in the life cycle. The post-hatching development can be divided into four phases: planktonic, metamorphic, juvenile, and adult. These phases can be further subdivided into 13 stages on the basis of behavioral and morphological characteristics visible in living specimens: Stage 1, newly hatched; Stage 2, eyes develop; Stage 3, the larval heart beats; Stage 4, maximum shell size is reached; Stage 5, the propodium develops; Stage 6, red spots appear; Stage 7, the velum is shed; Stage 8, eyebrows appear; Stage 9, pink color develops; Stage 10, white spots appear; Stage 11, rhinophores grow; Stage 12, the genital groove forms; Stage 13, egg laying begins. Reconstructions from serial sections taken from specimens fixed at each of these stages reveal the sequence of formation of the major organ systems. The nervous system develops gradually. The cerebral and pedal ganglia are present at Stage 1, the optic ganglia develop at Stage 2, the abdominal, pleural, and osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and the genital ganglion at Stage 13. Because Aplysia develops gradually, it is possible to analyze the contribution which gastropod torsion makes to the different phases of the life cycle. The Aplysia embryo undergoes 120 degrees torsion prior to Stage 1. The major visceral organs, the digestive system, heart, gill, and visceral nervous system, develop sybsequently in their post-torsional positions. After metamorphosis, there is a partial de-torsion which involves only the digestive system. Torsion of the digestive system may therefore be beneficial only to the pre-metamorphic larva, and not to the postmetamorphic juvenile.