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1.
Invest Ophthalmol Vis Sci ; 60(12): 3821-3829, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31529078

RESUMEN

Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.


Asunto(s)
Conexina 43/metabolismo , Conexinas/metabolismo , AMP Cíclico/metabolismo , Cristalino/metabolismo , Sistemas de Mensajero Secundario/fisiología , Colorantes Fluorescentes , Uniones Comunicantes/fisiología , Células HeLa , Humanos , Activación del Canal Iónico/fisiología , Técnicas de Placa-Clamp , Permeabilidad , Transfección
2.
FASEB J ; 33(3): 3404-3419, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30452880

RESUMEN

Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) has been shown to activate ADP-ribosyl cyclase (ARC), which produces the Ca2+ mobilizing second messenger, cyclic ADP-ribose (cADPR). In the present study, we examined how ROS activates cluster of differentiation (CD)38, a mammalian prototype of ARC. CD38 exists in type II and III forms with opposing membrane orientation. This study showed the coexpression of type II and III CD38 in lymphokine-activated killer (LAK) cells. The catalytic site of the constitutively active type II CD38 faces the outside of the cell or the inside of early endosomes (EEs), whereas the basally inactive type III CD38 faces the cytosol. Type III CD38 interacted with Nox4/phosphorylated-p22phox (p-p22phox) in EEs of LAK cells upon IL-8 treatment. H2O2 derived from Nox4 activated type III CD38 by forming a disulfide bond between Cys164 and Cys177, resulting in increased cADPR formation. Our study identified the mechanism by which type III CD38 is activated in an immune cell (LAK), in which H2O2 generated by Nox4 oxidizes and activates type III CD38 to generate cADPR. These findings provide a novel model of cross-talk between ROS and Ca2+ signaling.-Park, D.-R., Nam, T.-S., Kim, Y.-W., Bae, Y. S., Kim, U.-H. Oxidative activation of type III CD38 by NADPH oxidase-derived hydrogen peroxide in Ca2+ signaling.


Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Peróxido de Hidrógeno/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Línea Celular Tumoral , ADP-Ribosa Cíclica/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Estrés Oxidativo/fisiología , Sistemas de Mensajero Secundario/fisiología
3.
Am J Physiol Cell Physiol ; 316(1): C48-C56, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30404557

RESUMEN

Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in ß-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the ß-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca2+, although Ca2+ responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 ß-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to ß-cells.


Asunto(s)
Linfocitos B/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Receptores de Glucagón/metabolismo , Receptores de Sulfonilurea/metabolismo , Linfocitos B/efectos de los fármacos , Femenino , Gliburida/farmacología , Humanos , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Persona de Mediana Edad , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología
4.
J Neurosci ; 39(8): 1484-1504, 2019 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-30541912

RESUMEN

Serotonin (5-hydroxytryptamine, 5-HT) receptor agonists are neuroprotective in CNS injury models. However, the neuroprotective functional implications and synaptic mechanism of 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), a serotonin receptor (5-HT1A) agonist, in an adult male Wistar rat model of chronic glaucoma model remain unknown. We found that ocular hypertension decreased 5-HT1A receptor expression in rat retinas because the number of retinal ganglion cells (RGCs) was significantly reduced in rats with induced ocular hypertension relative to that in control retinas and 8-OH-DPAT enhanced the RGC viability. The protective effects of 8-OH-DPAT were blocked by intravitreal administration of the selective 5-HT1A antagonist WAY-100635 or the selective GABAA receptor antagonist SR95531. Using patch-clamp techniques, spontaneous and miniature GABAergic IPSCs (sIPSCs and mIPSCs, respectively) of RGCs in rat retinal slices were recorded. 8-OH-DPAT significantly increased the frequency and amplitude of GABAergic sIPSCs and mIPSCs in ON- and OFF-type RGCs. Among the signaling cascades mediated by the 5-HT1A receptor, the role of cAMP-protein kinase A (PKA) signaling was investigated. The 8-OH-DPAT-induced changes at the synaptic level were enhanced by PKA inhibition by H-89 and blocked by PKA activation with bucladesine. Furthermore, the density of phosphorylated PKA (p-PKA)/PKA was significantly increased in glaucomatous retinas and 8-OH-DPAT significantly decreased p-PKA/PKA expression, which led to the inhibition of PKA phosphorylation upon relieving neurotransmitter GABA release. These results showed that the activation of 5-HT1A receptors in retinas facilitated presynaptic GABA release functions by suppressing cAMP-PKA signaling and decreasing PKA phosphorylation, which could lead to the de-excitation of RGC circuits and suppress excitotoxic processes in glaucoma.SIGNIFICANCE STATEMENT We found that serotonin (5-HT) receptors in the retina (5-HT1A receptors) were downregulated after intraocular pressure elevation. Patch-clamp recordings demonstrated differences in the frequencies of miniature GABAergic IPSCs (mIPSCs) in ON- and OFF-type retinal ganglion cells (RGCs) and RGCs in normal and glaucomatous retinal slices. Therefore, phosphorylated protein kinase A (PKA) inhibition upon release of the neurotransmitter GABA was eliminated by 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), which led to increased levels of GABAergic mIPSCs in ON- and OFF-type RGCs, thus enhancing RGC viability and function. These protective effects were blocked by the GABAA receptor antagonist SR95531 or the 5-HT1A antagonist WAY-100635. This study identified a novel mechanism by which activation of 5-HT1A receptors protects damaged RGCs via the cAMP-PKA signaling pathway that modulates GABAergic presynaptic activity.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Proteínas del Ojo/metabolismo , Glaucoma/metabolismo , Receptor de Serotonina 5-HT1A/fisiología , Células Ganglionares de la Retina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Bucladesina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Isoquinolinas/farmacología , Masculino , Hipertensión Ocular/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Wistar , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Antagonistas del Receptor de Serotonina 5-HT1/farmacología , Sulfonamidas/farmacología
5.
J Leukoc Biol ; 104(6): 1187-1198, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30295956

RESUMEN

Reactive oxygen species (ROS) are electrophilic chemical species produced from incomplete oxidation. They have long been known as aggressive molecules that lead to direct tissue and cellular damage. Recent studies have reconsidered ROS as second messengers in the initiation and amplification of cell signaling, but how ROS regulate lung tissue and immune cell remain unknown. In this study, we used a LPS-induced acute lung injury (ALI) mouse model to observe disease, progression and determine ROS-related immune responses. We found that ROS play an essential pathogenic role in ALI, however, the major role of ROS in exacerbating ALI was increasing bronchoalveolar fluid (BALF) B cells rather than eliciting tissue damage. Moreover, these pathogenic B cells are FasL+ killer B cells, which reported to damage Fas-sensitive target cells including pulmonary epithelial cells. Furthermore, via in vitro transwell assays and in vivo treatment with neutralizing antibodies. ROS promoted pulmonary epithelial cells to produce CXCL9 and CXCL10, which recruited B cells into BALF. These results demonstrated that during lung injury, instead of causing oxidative damage, ROS mainly serve as second messengers, interacting with tissue and immune cells to enhance immune responses that lead to more severe disease.


Asunto(s)
Acetilcisteína/uso terapéutico , Lesión Pulmonar Aguda/inmunología , Células Epiteliales Alveolares/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Proteína Ligando Fas/análisis , Depuradores de Radicales Libres/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Sistemas de Mensajero Secundario/fisiología , Acetilcisteína/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Células Epiteliales Alveolares/metabolismo , Animales , Apoptosis , Líquido del Lavado Bronquioalveolar/citología , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL9/biosíntesis , Quimiotaxis de Leucocito/efectos de los fármacos , Citotoxicidad Inmunológica , Progresión de la Enfermedad , Femenino , Depuradores de Radicales Libres/farmacología , Lipopolisacáridos , Pulmón/inmunología , Pulmón/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/antagonistas & inhibidores
6.
7.
Mol Microbiol ; 109(5): 600-614, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29873124

RESUMEN

Cyclic di-GMP and cyclic di-AMP are second messengers produced by a wide variety of bacteria. They influence bacterial cell survival, biofilm formation, virulence and bacteria-host interactions. However, many of their cellular targets and biological effects are yet to be determined. A chemical proteomics approach revealed that Mycobacterium smegmatis RecA (MsRecA) possesses a high-affinity cyclic di-AMP binding activity. We further demonstrate that both cyclic di-AMP and cyclic di-GMP bind specifically to the C-terminal motif of MsRecA and Mycobacterium tuberculosis RecA (MtRecA). Escherichia coli RecA (EcRecA) was devoid of cyclic di-AMP binding but have cyclic di-GMP binding activity. Notably, cyclic di-AMP attenuates the DNA strand exchange promoted by MsRecA as well as MtRecA through the disassembly of RecA nucleoprotein filaments. However, the structure and DNA strand exchange activity of EcRecA nucleoprotein filaments remain largely unaffected. Furthermore, M. smegmatis ΔdisA cells were found to have undetectable RecA levels due to the translational repression of recA mRNA. Consequently, the ΔdisA mutant exhibited enhanced sensitivity to DNA-damaging agents. Altogether, this study points out the importance of sequence diversity among recA genes, the role(s) of cyclic di-AMP and reveals a new mode of negative regulation of recA gene expression, DNA repair and homologous recombination in mycobacteria.


Asunto(s)
AMP Cíclico/fisiología , Proteínas de Unión al ADN/metabolismo , Mycobacterium smegmatis/fisiología , Rec A Recombinasas/metabolismo , Sistemas de Mensajero Secundario/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/fisiología , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Rec A Recombinasas/genética , Reparación del ADN por Recombinación
8.
Proc Natl Acad Sci U S A ; 115(26): E6048-E6055, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29891656

RESUMEN

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.


Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Nucleótidos Cíclicos/metabolismo , Fosfolipasas/metabolismo , Sistemas de Mensajero Secundario/fisiología , Vibrio cholerae/enzimología , Proteínas Bacterianas/genética , Membrana Celular/genética , Nucleótidos Cíclicos/genética , Fosfolipasas/genética , Vibrio cholerae/genética
9.
Int J Biol Macromol ; 116: 633-647, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29723624

RESUMEN

Motility of spermatozoa is a crucial factor for determining semen quality. Here we report motility inhibitory factor (MIF-II) from goat epididymal plasma, revealing its structure, function, localization and motility inhibitory pathway. Structural characterization with MALDI revealed novelty of this protein while circular dichroism data confirmed its alpha helical nature. Higher dilutions of MIF-II antibody increased cauda sperm motility and induced immature/immotile caput sperm motility as tested microscopically. Higher number of sperm cells and lower dilutions of antibody induced agglutination in cauda sperm showing surface localization. Indirect immuno-fluorescence showed MIF-II localization throughout the caput sperm surface which relocated more towards acrosomal region with maturation. ELISA assay revealed gradual increase and decrease in concentration of MIF-II in epididymal plasma and plasma membrane respectively from caput to cauda. Signaling cascade that leads to sperm motility inhibition elevates nitric oxide levels through cAMP dependent pathway. MIF-II treatment doesn't alter sperm surface morphology. Expression pattern of MIF-II during epididymal maturation goes hand-in-hand with gaining motility potential as well as dormancy of spermatozoa before ejaculation. Both MIF-II and its antibody inhibit fertilization in-vitro thus expected to open new gateway for future male infertility and contraceptive development research.


Asunto(s)
Proteínas , Sistemas de Mensajero Secundario/fisiología , Motilidad Espermática/fisiología , Espermatozoides , Animales , AMP Cíclico/metabolismo , Epidídimo/química , Epidídimo/metabolismo , Humanos , Masculino , Proteínas/química , Proteínas/metabolismo , Conejos , Ratas , Espermatozoides/química , Espermatozoides/metabolismo
10.
PLoS Genet ; 14(4): e1007342, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29659565

RESUMEN

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.


Asunto(s)
Fosfatos de Dinucleósidos/metabolismo , Osmorregulación/fisiología , Streptococcus agalactiae/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Homeostasis/fisiología , Interacciones Huésped-Patógeno/fisiología , Humanos , Mutación , Osmorregulación/genética , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Potasio/metabolismo , Sistemas de Mensajero Secundario/fisiología , Streptococcus agalactiae/genética , Streptococcus agalactiae/crecimiento & desarrollo
11.
Psychopharmacology (Berl) ; 235(6): 1793-1805, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29663017

RESUMEN

Alcohol use disorder (AUD), which combines the criteria of both alcohol abuse and dependence, contributes as an important causal factor to multiple health and social problems. Given the limitation of current treatments, novel medications for AUD are needed to better control alcohol consumption and maintain abstinence. It has been well established that the intracellular signal transduction mediated by the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP) crucially underlies the genetic predisposition, rewarding properties, relapsing features, and systemic toxicity of compulsive alcohol consumption. On this basis, the upstream modulators phosphodiesterases (PDEs), which critically control intracellular levels of cyclic nucleotides by catalyzing their degradation, are proposed to play a role in modulating alcohol abuse and dependent process. Here, we highlight existing evidence that correlates cAMP and cGMP signal cascades with the regulation of alcohol-drinking behavior and discuss the possibility that PDEs may become a novel class of therapeutic targets for AUD.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Alcoholismo/tratamiento farmacológico , Alcoholismo/enzimología , Sistemas de Liberación de Medicamentos/tendencias , Inhibidores de Fosfodiesterasa/administración & dosificación , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Humanos , Nucleótidos Cíclicos/antagonistas & inhibidores , Nucleótidos Cíclicos/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
12.
Proc Natl Acad Sci U S A ; 115(17): 4471-4476, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29559526

RESUMEN

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.


Asunto(s)
Adhesión Bacteriana/fisiología , Biopelículas/crecimiento & desarrollo , AMP Cíclico/metabolismo , Fimbrias Bacterianas/fisiología , Pseudomonas aeruginosa/fisiología , Sistemas de Mensajero Secundario/fisiología
13.
PLoS One ; 13(3): e0194459, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29590153

RESUMEN

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans.


Asunto(s)
Proteínas de Artrópodos/biosíntesis , Proteínas Portadoras/metabolismo , Hepatopáncreas/metabolismo , Hormonas de Invertebrados/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Penaeidae/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transcripción Genética/fisiología , Vitelogeninas/biosíntesis , Animales , GMP Cíclico/metabolismo , Femenino
14.
Chem Pharm Bull (Tokyo) ; 66(2): 155-161, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29386466

RESUMEN

Cyclic ADP-ribose (cADPR), a general mediator involved in Ca2+ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxy groups of the riboses are linked by a pyrophosphate unit. This review focuses on chemical synthetic studies of cADPR analogues of biological importance. Although cADPR analogues can be synthesized by enzymatic and chemo-enzymatic methods using ADP-ribosyl cyclase, the analogues obtained by these methods are limited due to the substrate-specificity of the enzymes. Consequently, chemical synthetic methods providing a greater variety of cADPR analogues are required. Although early chemical synthetic studies demonstrated that construction of the large 18-membered ring structure is difficult, the construction was achieved using the phenylthiophosphate-type substrates by treating with AgNO3 or I2. This is now a general method for synthesizing these types of biologically important cyclic nucleotides. Using this method as the key step, the chemically and biologically stable cADPR mimic, cADP-carbocyclic-ribose (cADPcR) and -4-thioribose (cADPtR), were synthesized.


Asunto(s)
Calcio/metabolismo , ADP-Ribosa Cíclica/análogos & derivados , Sistemas de Mensajero Secundario/fisiología , Tioglicósidos/síntesis química , ADP-Ribosa Cíclica/síntesis química , Modelos Moleculares , Estructura Molecular , Nitrato de Plata/química
15.
Cell Rep ; 22(1): 255-268, 2018 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-29298426

RESUMEN

Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs) that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP), with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.


Asunto(s)
AMP Cíclico/metabolismo , Imagen Molecular , Neuronas/metabolismo , Optogenética , Receptores Acoplados a Proteínas G/metabolismo , Sistemas de Mensajero Secundario/fisiología , Animales , Ratones , Neuronas/citología
16.
Dev Biol ; 435(1): 6-14, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29341896

RESUMEN

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Hormona Luteinizante/metabolismo , Meiosis/fisiología , Folículo Ovárico/metabolismo , Sistemas de Mensajero Secundario/fisiología , Sustitución de Aminoácidos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Femenino , Hormona Luteinizante/genética , Ratones , Ratones Transgénicos , Mutación Missense , Folículo Ovárico/citología , Fosforilación/genética , Ratas
17.
Dev Biol ; 434(1): 196-205, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29274320

RESUMEN

Mammalian oocytes are arrested in meiotic prophase from around the time of birth until just before ovulation. Following an extended period of growth, they are stimulated to mature to the metaphase II stage by a preovulatory luteinizing hormone (LH) surge that occurs with each reproductive cycle. Small, growing oocytes are not competent to mature into fertilizable eggs because they do not possess adequate amounts of cell cycle regulatory proteins, particularly cyclin-dependent kinase 1 (CDK1). As oocytes grow, they synthesize CDK1 and acquire the ability to mature. After oocytes achieve meiotic competence, meiotic arrest at the prophase stage is dependent on high levels of cAMP that are generated in the oocyte under the control of the constitutively active Gs-coupled receptor, GPR3. In this study, we examined the switch between GPR3-independent and GPR3-dependent meiotic arrest. We found that the ability of oocytes to mature, as well as oocyte CDK1 levels, were dependent on follicle size, but CDK1 expression in oocytes from preantral follicles was not acutely altered by the activity of follicle stimulating hormone (FSH). Gpr3 was expressed and active in incompetent oocytes within early stage follicles, well before cAMP is required to maintain meiotic arrest. Oocytes from Gpr3-/- mice were less competent to mature than oocytes from Gpr3+/+ mice, as assessed by the time course of germinal vesicle breakdown. Correspondingly, Gpr3-/- oocytes contained significantly lower CDK1 levels than their Gpr3+/+ counterparts that were at the same stage of follicle development. These results demonstrate that GPR3 potentiates meiotic competence, most likely by raising cAMP.


Asunto(s)
Proteína Quinasa CDC2/biosíntesis , Puntos de Control del Ciclo Celular/fisiología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Profase Meiótica I/fisiología , Oocitos/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Sistemas de Mensajero Secundario/fisiología , Animales , Proteína Quinasa CDC2/genética , AMP Cíclico/genética , Femenino , Ratones , Ratones Noqueados , Oocitos/citología , Receptores Acoplados a Proteínas G/genética
18.
Artículo en Inglés | MEDLINE | ID: mdl-28787545

RESUMEN

Advances in high-resolution microscopy and other techniques have emphasized the spatio-temporal nature of information transfer through signal transduction pathways. The compartmentalization of signaling molecules and the existence of microdomains are now widely acknowledged as key features in biochemical signaling. To complement experimental observations of spatio-temporal dynamics, mathematical modeling has emerged as a powerful tool. Using modeling, one can not only recapitulate experimentally observed dynamics of signaling molecules, but also gain an understanding of the underlying mechanisms in order to generate experimentally testable predictions. Reaction-diffusion systems are commonly used to this end; however, the analysis of coupled nonlinear systems of partial differential equations, generated by considering large reaction networks is often challenging. Here, we aim to provide an introductory tutorial for the application of reaction-diffusion models to the spatio-temporal dynamics of signaling pathways. In particular, we outline the steps for stability analysis of such models, with a focus on biochemical signal transduction. WIREs Syst Biol Med 2018, 10:e1395. doi: 10.1002/wsbm.1395 This article is categorized under: Biological Mechanisms > Cell Signaling Analytical and Computational Methods > Dynamical Methods Models of Systems Properties and Processes > Mechanistic Models.


Asunto(s)
Modelos Biológicos , Transducción de Señal/fisiología , Bacterias/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Humanos , Sistemas de Mensajero Secundario/fisiología , Proteínas de Unión al GTP rho/metabolismo
19.
Neuron ; 96(3): 572-603, 2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-29096074

RESUMEN

The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications.


Asunto(s)
Fenómenos Fisiológicos Celulares/fisiología , Espacio Intracelular/genética , Neuronas/fisiología , Neurociencias/métodos , Optogenética/métodos , Animales , Humanos , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Neuronas/química , Neurociencias/tendencias , Optogenética/tendencias , Rodopsina/análisis , Rodopsina/genética , Sistemas de Mensajero Secundario/fisiología
20.
Adv Exp Med Biol ; 993: 213-216, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28900916

RESUMEN

In the title of this part of the book, the tail is wagging not just in a single dog but multiple dogs; in other words, a single process SOCE (tail) somehow involves a cross talk of (wagging) large and powerful organelle and cellular compartments (dogs). So how is this possible? Is this really necessary? Is the title actually appropriate?SOCE is a rather special process, it allows efficient signaling based on a ubiquitous second messenger (Ca2+) in multiple cell and tissue types, it has specific signaling modality (i.e., some downstream reactions depend specifically on SOCE and not just on global Ca2+ increase), it is vital for the normal functioning of multiple types of cells and tissues, and when misregulated it induces important pathological processes. The reader hopefully agree that such an important "tail" is more appropriate for a kangaroo than for a Chihuahua and that it has awesome wagging capacity.


Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Sistemas de Mensajero Secundario/fisiología , Animales , Humanos
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