Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 µm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 µm from the surface of a coverglass.

Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line.

A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.

Atrial natriuretic factor reduces cyclic adenosine monophosphate content of human fibroblasts by enhancing phosphodiesterase activity.

Radioligand binding studies disclosed one class of high affinity atrial natriuretic factor (ANF) receptors on human fibroblast membranes (Kd = 66 pM; maximum number of binding sites [Bmax] = 7,000 sites/cell). ANF increased cellular cyclic guanosine monophosphate (cGMP) content and suppressed isoproterenol- and PGE1-elevated, but not basal, cAMP content. Pertussis toxin pretreatment, which maximally ADP-ribosylated Gi, the guanine nucleotide-binding protein that couples inhibitory receptors to adenylate cyclase and blocks receptor-mediated inhibition of adenylate cyclase, did not interfere with ANF suppression of isoproterenol- or PGE1-elevated cellular cAMP content. Preliminary incubation of fibroblasts with 8-bromo cGMP or phosphodiesterase inhibitors, including 3-isobutyl-1-methylxanthine, Ro 20-1724, and cilostamide, however, prevented the ANF suppression of cAMP. MB 22948, an inhibitor that is partially selective for cGMP phosphodiesterase, did not block the effect of ANF. We conclude that in these cells, unlike other systems, ANF reduces cAMP content by activating a phosphodiesterase rather than by inhibiting adenylate cyclase.

Changes in cyclic adenosine 3':5'-monophosphate-dependent protein kinases during the progression of urethan-induced mouse lung tumors.

The cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases in lung adenomas are functionally different from those of normal lung. The relevance of this change to neoplastic conversion was examined by comparing tumor kinases with those obtained from the normal cell of origin and by studying the kinases at different stages of tumor growth. Lung tumors were collected from A strain mice at different times after a single injection of urethan. These tumors are predominantly of alveolar type two cell origin, and cAMP-binding proteins in extracts from isolated type two cells and from lung adenomas at various stages of tumor progression were compared. Both the incorporation of the cAMP photoaffinity analogue, cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP), into the regulatory subunits of the type I (RI) and type II (RII) cAMP-dependent protein kinases and the autophosphorylation of RII were similar in extracts from whole normal lung and from type two cells. Altered protein kinases are thus not characteristic of normal type two cells. Lung tumors showed a decrease in photodetectable RII which correlated in degree with tumor size and extent of anaplasticity. This decreased RII photolabeling during tumor growth was associated with increased RII autophosphorylation. In contrast, decreased RII photolabeling in extracts from neonatal lung is accompanied by a substantial decrease in RII autophosphorylation. The characteristics of RII during normal development thus clearly differ from those during neoplastic development. An increase in the amount of an Mr 37,000 proteolytic fragment derived from R-subunits was also noted as a function of tumor progression. DEAE-cellulose chromatography of tumor cytosol showed that the increase in the amount of Mr 37,000 protein was accompanied by increased subunit dissociation of the type I isozyme. The dissociated RI subunit has been shown to be more sensitive to cleavage by a Ca2+-dependent neutral protease than when RI was in the holoenzyme form. This protease is present in both normal lung and lung adenomas, and its activity increases during the later stages of tumor progression. A comparison of cAMP binding and the light-induced covalent incorporation of 8-N3-[32P]cAMP showed that, for both RI and RII, photoincorporation was about 75% as efficient as noncovalent binding. In contrast, although the Mr 37,000 fragment can be photolabeled with low concentrations of 8-N3-[32P]cAMP, noncovalent cAMP binding to the endogenous Mr 37,000 fragment could not be demonstrated with a standard filtration assay. Such altered cAMP binding characteristics following Ca2+-dependent proteolysis of R-subunits would all

Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues.

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J. Cyclic Nucleotide Res., 6: 163, 1980). We now shown that the site-selective cAMP analogues, which are manyfold more active in binding to the cAMP receptor protein than previously studied analogues, demonstrate a potent growth inhibition of seven breast and three colon human cancer cell lines. The cAMP receptor protein has two different cAMP binding sites, and cAMP analogues that selectively bind to either one of the two binding sites are known as either site 1 selective (C-8 analogues) or site 2 selective (C-6 analogues). Nineteen site-selective analogues, C-6 and C-8 monosubstituted and C-6,-8 disubstituted, were tested for their growth regulatory effect. The majority of these analogues demonstrated an appreciable growth inhibition, with no sign of toxicity in all 10 cancer lines at micromolar concentrations. The three most potent inhibitors were 8-Cl-, N6-benzyl-, and N6-phenyl-8-thio-p-chlorophenyl-cAMP, demonstrating 50% growth inhibition at 5-25 microM concentrations (IC50). Furthermore, N6-analogues, in combination with halogen or thio derivatives of C-8 analogues, demonstrated synergistic enhancement of growth inhibition. The growth inhibition paralleled a change in cell morphology, an augmentation of the RII cAMP receptor protein, and a reduction in p21 ras protein. The growth inhibition by 8-Cl-cAMP was not due to its metabolite, 8-Cl-adenosine, since: (a) the growth inhibition by 8-Cl-cAMP was released upon cessation of treatment, whereas that by 8-Cl-adenosine was not released; (b) 8-Cl-cAMP treatment did not affect cell cycle progression, whereas 8-Cl-adenosine brought about G1 synchronization; (c) 8-Cl-cAMP treatment caused reduction of p21 ras protein, whereas 8-Cl-adenosine did not affect p21 levels; and (d) 8-Cl-adenosine was not detected in either cell extracts or medium from the cells treated with 8-Cl-cAMP for 48-72 h. Site-selective cAMP analogues thus provide a new physiological means to control the growth of breast and colon human cancer cells.

[Modification of red cell membranes with perftoran in papaine emphysema in rats].

Papaine emphysema model on 75 mongrel mature white male rats (10 intact rats were control) was used to study the size, form, surface architechtonics, deformability and state of membrane-receptor erythrocyte complex before and after perftoran intraperitoneal administration. Perftoran emulsion produced a membrane-modulating effect with recovery of hormonal reception sensitivity, PHA-, cAMP-receptor systems as well as restoration of erythrocytic normocytosis and diskocytosis.

Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli.

The fnr gene is essential for the expression of anaerobic respiratory metabolism in Escherichia coli. Genetic and biochemical studies support the view that its product. Fnr, is a transcriptional regulatory protein specific for genes encoding anaerobic respiratory functions (fumarate, nitrate and nitrite reductases, hydrogenase, etc.). In this respect Fnr may be considered analogous to the well-characterized catabolite gene activator protein (CAP), which mediates the control of catabolite-sensitive gene transcription. With a view to identifying its function, the fnr gene has recently been cloned and the primary structure of the Fnr protein deduced from the nucleotide sequence. This has revealed the presence of three regions of sequence homology with CAP. One corresponds to the DNA-binding site, a region of about 20 highly conserved amino acids that is believed to form a characteristic three-dimensional structure in several transcriptional regulators. The other regions of homology are in the nucleotide binding domain of CAP but the residues that interact with cAMP are not identical in Fnr. These homologies suggest that Fnr and CAP may have similar three-dimensional structures and that the regulation of anaerobic energy metabolism may involve interaction between Fnr and an unidentified effector molecule.

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism.

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.

High-resolution measurements of cyclic adenosine monophosphate signals in 3D microdomains.

A large number of hormones, neurotransmitters, and odorants exert their effects on cells by triggering changes in intracellular levels of cyclic adenosine monophosphate (cAMP). Although the effector proteins that bind cAMP have been identified, it is not known how this single messenger can differentially regulate the activities of hundreds of cellular proteins. It has been clear, for some time, that compartmentation of cAMP signals must be taking place, but the physical basis for compartmentation and the nature of local cAMP signals are mostly unknown. We present here a high-resolution method for measuring cAMP signals near the membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We outline how these channels can be used in electrophysiological experiments to measure accurately changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We also describe how the method has been employed to dissect the roles of diffusion barriers and differential phosphodiesterase activity in creating distinct cAMP signals. This approach has much greater spatial and temporal resolution than other methods for measuring cAMP and should help to unravel the complexities of signaling by this ubiquitous messenger.

Localization of cyclic-AMP receptors with acidosomes in Dictyostelium discoideum.

Earlier studies have shown that in Dictyostelium discoideum, a buoyant membrane fraction contained approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity, leading to its designation acidosomes. It was proposed that acidosomes may be involved in endocytosis, specially in the acidification of endosomes. In this study we further investigated the putative function(s) of acidosomes. The findings suggest that acidosomes contain abundant receptors for cyclic AMP (CAR1) and that it may be the site for recycling of internalized receptors. Acidosomes also contain an abundance of Rab4 (Bush et al. 1994), a marker for early endosomes. By these criteria, we suggest that the acidosomes are analogous to early or recycling endosome present in mammalian cells. These findings suggest that the structure earlier defined biochemically, morphologically and immunologically as acidosomes may represent early and/or recycling endosomes in this protist.

Types of cyclic AMP binding proteins in human breast cancers.

Total level and type of cyclic AMP binding proteins have been measured in 117 breast cancers. Six major molecular species of binding proteins were detected. The pattern and relative proportion of binding proteins varied between individual tumours. However, there were highly significant correlations between the expression of different binding proteins, including positive relationships between 52 and 67 kD proteins, 43 kD and both 39 and 37 kD proteins and inverse correlations between 48 and 52 kD, 37 and 67 kD proteins. The expression of three binding proteins (48, 43 and 39 kD) was also positively related to total binding whereas that of the remaining three bindings proteins (67, 52 and 37 kD) was negatively correlated with total levels. It may be that differential expression of certain types of binding protein are the underlying rationale for our previously published finding that tumours with high levels of high binding protein are associated with poor prognosis.

A unique type of cyclic AMP-binding protein of Trypanosoma cruzi.

On centrifugation on sucrose density gradients, the cyclic AMP-receptor protein of Trypanosoma cruzi was clearly resolved from the type II regulatory subunit of protein kinase from bovine heart (S20,W = 8.25 and 4.1, respectively). The binding of cyclic [3H]AMP to these two proteins was affected to different extents by several cyclic AMP analogues. Such differences between the cyclic AMP-receptor protein of T. cruzi and cyclic AMP-binding proteins of other eukaryotes might be exploitable by chemotherapy.

Subcellular distribution and partial characterization of the cyclic AMP-binding proteins of Trypanosoma brucei.

We assayed the cyclic AMP-binding activity of Trypanosoma brucei by two well established methods, such as the one of Gilman (Proc. Natl. Acad. Sci. U.S.A. 67, 305-312, 1970) and Ueland & Doskeland (Biochem. J., 157, 117-126, 1976). The results indicate that the former technique underestimated the total amount of cyclic AMP bound by T. brucei homogenates by up to 7.5 fold. Similar results were obtained with other Trypanosomatidae such as Leishmania tropica, and Crithidia luciliae. The bulk of the cyclic AMP-binding proteins of T. brucei appeared soluble after centrifugation of post-large-granule extracts in isopycnic sucrose-gradients. Upon fractionation by DEAE-Sephacel column chromatography, two peaks of activity eluted which were responsible for all the specific cyclic AMP-binding activity present in the cytosol of T. brucei. These two activities, which we denominated as Peak 'a' and Peak 'b' respectively, differed in a number of properties such as sensitivity to proteases, stability to storage at -20 degrees C, displacement of cyclic AMP bound by adenine analogues, and coefficients of sedimentation.

Expression and distribution of parotid secretory proteins in experimental diabetes.

Previous studies of experimental diabetes have demonstrated changes in the levels of specific salivary proteins. The present study is part of a larger effort aimed at elucidating the mechanism(s) by which insulin regulates salivary protein expression in the rat parotid gland. Diabetes was induced in 2-3-month-old male Fischer 344 rats by injection of streptozotocin (STZ). After 30 days one group of rats was given insulin for 7 days. Untreated rats served as controls. As previously observed, parotid acinar cells from diabetic rats accumulated lipid and contained occasional crystalloid lysosomes. Quantitative immunogold labeling of secretory granules in diabetic glands revealed decreases of 30-60% for proline-rich-proteins (PRPs), amylase and parotid secretory protein (PSP), but labeling for acidic epididymal glycoprotein (AEG) was unchanged. The response to insulin treatment was variable: amylase and PSP labeling were partly restored, but PRP and AEG labeling showed little change. Photoaffinity labeling of cyclic AMP receptor proteins (cARP) showed changes in several tissues including a consistent increase in the diabetic parotid gland. Immunogold labeling of secretory granules with antibody to cARP was similar in control and diabetic parotids, but nuclear and cytoplasmic label was decreased in diabetic acinar cells. These results indicate that STZ-diabetes and insulin reconstitution cause variable changes in the expression of parotid secretory proteins. Changes in cARP levels suggest that the insulin and cyclic AMP pathways may interact in regulating expression of salivary secretory proteins.

[Changes in the receptor link during the development of renal sensitivity to aldosterone and antidiuretic hormone]. / Izmeneniia retseptoronogo zvena v protsesse razvitiia chuvstvitel'nosti pochki k al'dosteronu i antidiureticheskomu gormonu.

The aldosterone, cAMP and ADH receptors were studied in kidneys of 10-14- and 60-day old rats. Concentrations of aldosterone and cAMP receptors were reduced, while ADH binding was increased during the period of maturation of kidney functions. The investigation of the affinity and molecular weights of cAMP and ADH receptors suggests their complex nature and altering of their individual properties during ontogenesis. Aldosterone and cAMP with their receptors seem to participate in the regulation of gene activity in ontogenesis and ADH seems to participate in the formation of its own receptors.

[ADH and cAMP receptor binding in the kidney medulla of mice insensitive to ADH]. / Retseptsiia ADG i tsAMF v medulliarnoi chasti pochki myshei, nechuvstvitel'nykh k ADG.

Specific binding of ADH by the membrane fraction of the kidney medulla was lower in the normal CBA mice than in mutant mice with nephrogenic diabetes. Gel filtration of the solubilized ADH receptors of mutants revealed the presence of an unidentified factor which caused cooperative binding of ADH. DEAE-chromatography revealed no difference between cytosolic cAMP receptors in normal and mutant animals. Assay of GTP-ase activity of the membrane fraction revealed that ADH increased this parameter in CBA mice but not in mutant animals. Cholera toxin significantly diminished membrane ATP-ase activity whereas membrane preparations from mutant mice developed a reactivity to ADH. GTP binding ability in these preparations was higher than inn intact ones. In CBA mice this ability increased dramatically. HPLC profiles of G-protein complexes with GNP were very different in CBA and mutant mice. Mutation seems to cause changes both in binding and in "cross-talk" link op-complex membrane receptor of ADH.

[Method of measuring binding constants and the number of specific cyclic AMP receptor sites in tissue extracts]. / Metod izmereniia konstant sviazyvaniia i kolichestva spetsificheskikh uchastkov retseptsii tsAMF v ékstraktakh tkani.

A method for evaluation of binding constants and of amount of cAMP binding sites in crude tissue extracts was developed. The method is based on equilibrium binding of 3H-cAMP by proteins with subsequent ultrafiltration. Hydrolysis of cAMP and its unspecific sorption by proteins were eliminated under the conditions selected. Rat spleen cytosole contained 3.57 +/- 0.34 pmol of cAMP binding sites per mg of protein with dissociation constant of protein-cAMP complex (1.68 +/- 0.28).10(-8) M. As shown by studies on kinetics, binding constants and specificity of binding, the method permitted to evaluate quantitatively cAMP-dependent protein kinases in crude tissue extracts and to estimate their affinity to cAMP.

Modificações pós-traducionais durante o processo de amastigogênese do Trypanosoma cruzi / Post-translational modifications during the amastigogenesis of Trypanosoma cruzi

A doença de Chagas é uma doença negligenciada causada pelo protozoário Trypanosoma cruzi constituindo-se em um problema de saúde pública em vários países da América Latina. No seu complexo ciclo de vida, o protozoário passa por quatro estágios diferentes: tripomastigota metacíclica, amastigota, tripomastigota sanguíneo e epimastigota, que permitem sua sobrevivência nos diferentes ambientes com os quais o parasita entra em contato. A diferenciação dos tripomastigotas de T. cruzi em amastigotas (amastigogênese) ocorre com grandes mudanças morfológicas, estruturais e metabólicas no parasita e pode ser reproduzido in vitro por exemplo, pela acidificação do meio extracelular. Apesar dos vários trabalhos descritos na literatura, o processo ainda não é totalmente compreendido. A participação de NO na transdução de sinal durante a amastigogênese, sugerida por dados não publicados de nosso grupo, assim como a via de sinalização dependente de AMPc, foram o foco do presente estudo. A indução da amastigogênese foi obtida por incubação de tripomastigotas em meio de cultura acidificado (pH 6,0) e os parâmetros estudados comparados com parasitas controle (meio de cultura, pH 7,4). Estudamos a variação no perfil de nucleotídios cíclicos (AMPc, GMPc), de quinases (PKA, MAPK- ERK1/2), de uma fosfatase (PP2A), assim como o perfil de proteínas fosforiladas, S-nitrosiladas e nitradas até 6 h do início da amastigogênese. O processo foi dividido nas etapas: inicial (até 60 minutos) e tardio (em torno de 3-4 h), caracterizados por um aumento de formas amastigotas na etapa tardia. Houve um aumento de aproximadamente 17 vezes no nível de AMPc nos primeiros 15 minutos da amastigogênese (meio pH 6,0), seguido por aumento discreto no nível de PKA fosforilada, utilizado como indicador de atividade enzimática, este mais evidente na etapa tardia (360 minutos). Quanto à subunidade catalítica fosforilada da MAPK (ativa), há uma aparente diminuição no nível de fosforilação na fase inicial (30 minutos) e aumento na etapa tardia (120 minutos) do processo de amastigogênese. Quanto ao perfil geral de fosforilação de proteínas, há uma diminuição de fosforilação em torno de 30 minutos, seguida de aumento de fosforilação em proteínas de aproximadamente 5 e 100 kDa, mas de maneira geral, não se observaram grandes mudanças nesse perfil com a metodologia utilizada. Quanto às modificações por NO e seus derivados, foram observadas modificações por S-nitrosilação e nitração das proteínas, além do aumento de GMPc em torno de 60 minutos. Embora essas modificações modulem a atividade biológica de uma grande diversidade de proteínas, seu papel biológico não foi explorado.8 Em resumo, nossos resultados apontam para uma variação no perfil de fosforilação, S-nitrosilação e nitração de proteínas, além do aumento de AMPc e GMPc ao longo do processo de amastigogênese in vitro, com a via de sinalização dependente de quinases/ fosfatases e de óxido nítrico ocorrendo ao longo do processo de amastigogênese

Chagas disease is a neglected disease caused by the parasite Trypanosoma cruzi and is a public health problem in several Latin American countries. In its complex life cycle, the protozoan goes through four different stages: metacyclic trypomastigote, amastigote, blood trypomastigote and epimastigote, which allow its survival in the different environments which the parasite comes into contact. The differentiation of T. cruzi trypomastigotes into amastigotes (amastigogenesis) occurs with large morphological, structural and metabolic changes in the parasite and can be reproduced in vitro by, for example, acidification of the extracellular medium. Despite the many data described in the literature, the process is not yet fully understood. The participation of NO in signal transduction during amastigogenesis, suggested by unpublished data from our group, as well as the cAMP-dependent signaling pathway, were the focus of the present study. The induction of amastigogenesis was obtained by incubating trypomastigotes in acidified culture medium (pH 6.0) and the studied parameters compared with control parasites (culture medium, pH 7.4). We studied the variation in the profile of cyclic nucleotides (cAMP, cGMP), kinases (PKA, MAPK-ERK1 / 2), phosphatase (PP2A), as well as the profile of phosphorylated, S-nitrosylated and nitrated proteins up to 6 h. onset of amastigogenesis. The process was divided into early (up to 60 minutes) and late (around 3-4 hours), characterized by an increase in amastigote forms in the late stage. There was an approximately 17-fold increase in cAMP level in the first 15 minutes of amastigogenesis (pH 6.0 medium), followed by a slight increase in phosphorylated PKA level, most evident in the late stage (360 minutes). As for the phosphorylated catalytic subunit of MAPK (active), there is an apparent decrease in the phosphorylation level in the early phase (30 minutes) and increase in the late stage (120 minutes) of the amastigogenesis process. As for the general protein phosphorylation profile, there is a decrease in phosphorylation around 30 minutes, followed by an increase in phosphorylation of proteins (approximately 5 and 100 kDa), but overall, no major changes were observed in this profile with the methodology used. As for modifications by NO and its derivatives, modifications were observed by S-nitrosylation and protein nitration, besides the increase of cGMP around 60 minutes. Although these modifications modulate the biological activity of a wide range of proteins, their biological role has not been explored. In summary, our results point to a variation in phosphorylation, S-nitrosylation and nitration profile of proteins, as well as an increase in cAMP and cGMP along the amastigogenesis process, implicating kinases / phosphatases and nitric oxide dependent signaling pathways in this differentiation

Characterization and localization of adenosine 3':5'-monophosphate-binding proteins in the nervous system of Aplysia.

Earlier work in Aplysia californica has indicated that sensitization of the gill reflex, a simple form of learning, is produced by cAMP-dependent protein phosphorylation which regulates the flux of ions in sensory neurons of the abdominal ganglion. These changes in ion flux result in the enhanced release of neurotransmitter from synapses of the sensory neurons which, in turn, mediate the behavior. Because it can be presumed that protein phosphorylation regulates the functioning of ion channel proteins, we have characterized cAMP-binding proteins photoaffinity labeled with 8-N3-cAMP and have found that, unlike other tissues, the nervous system contains a great variety of binding species. Also unlike other tissues, several of the binding proteins in neurons are associated with membrane, and these components are concentrated in fractions enriched in nerve endings. Selectivity of phosphorylation, not only between substrates in cytosol and membrane but also between different regions of the cell, is thus possible because of the variety of cAMP-binding proteins in neurons. We think that these membrane-associated binding proteins are the most likely candidates for the regulatory subunits of the cAMP-dependent kinases that control the functioning of ion channel proteins at the synapse.

Immunological analyses of the chemotactic receptor of Dictyosteleum discoideum. Identification of cDNA clones.

The cell surface cAMP receptor was excised from preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to generate a polyclonal antiserum. The antiserum immunoprecipitates the two molecular weight forms of the cAMP receptor. Both forms are phosphorylated. Western blot analyses show that the antiserum is highly specific and recognizes only the two molecular weight forms of the cAMP receptor. Immunological studies indicate that both forms of the receptor are phosphorylated. Vegetative amoebae possess low levels of the cAMP receptor. Levels of the antigen increase in differentiated cells which express high cell surface cAMP binding activity. The antiserum was also used to isolate 6 lambda gt11 cDNA clones. One of those clones contains a 1.1-kilobase pair cDNA fragment which encodes for a protein of approximately 30,000-35,000 daltons. The antibody which binds to the fusion protein also recognizes the two molecular weight forms of the receptor.