Author: neuroscience research

Indeed, genetic or chemical disruption of the intestinal barrier associated with inflammation produces systemic DNA damage and genotoxic stress, including in circulating leukocytes. Here, barrier disruption and hyperpermeability produced by silencing GUCY2C was associated with elevated levels of oxidative DNA damage in circulating leukocytes. Conversely, defending barrier integrity and restricting permeability by activating GUCY2C with oral ST decreased systemic genotoxicity in hepatocytes produced by DSS. Further, systemic genotoxicity produced by barrier disruption reflecting GUCY2C silencing was associated with increased spontaneous and carcinogen-induced extra-intestinal tumorigenesis, specifically in lung, liver and lymph nodes. In the context of the role of GUCY2C as a tissue-specific tumor suppressing receptor, the present observations expand that function beyond transformation in the colorectum, to neoplasia in lymph nodes, liver, and lung through maintenance of epithelial barrier integrity. Moreover, it is tempting to speculate that loss of paracrine hormone expression and silencing of GUCY2C in inflammatory bowel disease produces systemic genotoxicity which contributes to the pattern of extra-intestinal cancer in these patients specifically in lymph nodes, liver,Herbacetin and lung. The present study further clarifies the evolving role of the GUCY2C paracrine hormone system in modulating the intestinal barrier. A role for GUCY2C in regulating epithelial barrier function was recently demonstrated, and the present work confirms that phenomenon. Those studies revealed that silencing GUCY2C disrupted the barrier and induced inflammation, associated with production of interferon gamma and IL-12. In turn, those events were associated with increased expression of epithelial cell myosin light chain kinase, an established regulator of tight junction structure and function, and suppression of JAM-A and claudin 2 expression. These phenomena were presumed to be the molecular mechanisms mediating GUCY2C-dependent barrier disruption. However, canonical regulation of myosin light chain phosphorylation by cGMP is mediated by myosin light chain phosphatase, rather than myosin light chain kinase. Moreover, these observations are complicated by the contribution of inflammatory cytokines,Diosgenin-glucoside including interferon gamma, to regulation of expression of myosin light chain kinase in intestinal epithelial cells in vivo and in vitro. Thus, the contribution of these components may not reflect the primary mechanism underlying GUCY2C regulation of barrier integrity but, rather, an epiphenomenon related to the associated inflammation. In contrast, the present study demonstrates a mechanistic role for GUCY2C as a primary modulator of the intestinal epithelial barrier, mediated by AKT1, occludin and claudin 4, but not JAM-A and claudin 2, in mouse models in vivo, and in human intestinal cell monolayers in vitro in the absence of confounding by inflammation. Paradoxically, subsequent work from the same group suggested that eliminating GUCY2C signaling protects the intestinal mucosa from inflammatory injury. Beyond proliferation, metabolism, and chromosomal integrity, the present studies reveal an additional dimension of AKT1dependent epithelial homeostatic regulation by GUCY2C. Indeed, it is tempting to speculate that one essential function of GUCY2C signaling is maintenance of intestinal barrier integrity. In that context, regulation of epithelial tight junctions contributing to macromolecular permeability described here can be added to other established GUCY2C functions, including fluid and electrolyte secretion accelerating lumenal clearance; differentiation of Paneth cells producing antimicrobial peptides; and differentiation of goblet cells producing intestinal mucus that contribute to the separation of systemic and environmental compartments across the mucosal interface.

The present studies extend this homeostatic role of GUCY2C beyond proliferation, differentiation, migration, DNA damage sensing and repair, and metabolism to maintenance of macromolecular permeability and the intestinal barrier. Elimination of GUCY2C signaling produced basal macromolecular hyperpermeability and potentiated barrier disruption resulting in colitis induced by DSS. Conversely, genetic or pharmacological induction of GUCY2C signaling reduced basal permeability and DSS-induced hyperpermeability and inflammation. Results in mouse models were recapitulated in monolayers of human intestinal epithelial cells in vitro, in the absence of other confounding physiological mechanisms like inflammation,Epigoitrin demonstrating a primary mechanistic role for GUCY2C in regulating the epithelial barrier and macromolecular permeability. Moreover, protection of the epithelial barrier by GUCY2C in vivo and in vitro was associated with alterations in steady state concentrations of tight junction proteins, including occludin and claudin 4. Regulation of steady-state concentrations of these components are one established mechanism modulating the dynamic assembly and deployment of tight junction complexes, barrier integrity and macromolecular permeability. Integration of crypt-villus homeostatic programs mediated by GUCY2C is coordinated through AKT1. Ligand activation of GUCY2C and accumulation of cGMP produces dephosphorylation of AKT1, modulating downstream signaling circuits regulating proliferation and metabolism. In the present study,Isoforskolin homeostatic regulation of epithelial barrier function and permeability by GUCY2C was mediated by AKT1. In that context, specifically reducing AKT1, but not AKT2, blocked alterations in occludin and claudin 4, but not JAM-A and claudin 2, and macromolecular hyperpermeability induced by eliminating GUCY2C signaling in mice. Further, in monolayers of human intestinal epithelial cells in vitro, eliminating AKT1 expression induced steady state levels of occludin and claudin 4, but not JAMA and claudin 2, and barrier function to levels achieved by ST activation of GUCY2C. Moreover, a constitutively activated form of AKT1 produced resistance to, while eliminating AKT1 expression from these cells mimicked, the effects of GUCY2C activation on epithelial cell permeability. Together, these results demonstrate a key mechanistic role for suppression of AKT1 signaling in the regulation of epithelial cell barrier function by GUCY2C. These results reinforce the established role of AKT in regulating the expression of junctional proteins, disrupting epithelial cell membrane complexes, and inducing barrier dysfunction. Further, they identify occludin and claudin 4, but not JAM-A and claudin 2, as specific downstream targets of AKT1 contributing to the regulation of epithelial barrier permeability in vitro and in vivo. They underscore the integrated homeostatic role of GUCY2C signaling, centrally coordinated through AKT, in regulating intestinal epithelial structure and function. There is an established mechanistic relationship between disruption of the intestinal epithelial barrier, inflammation resulting from systemic immune exposure to normally compartmentalized lumenal antigens, and the generation of reactive oxygen species producing DNA damage and genotoxic stress. In turn, reactive oxygen species producing DNA damage comprise one mechanism contributing to tumorigenesis generally, and to the evolution of colon cancer in the context of inflammatory bowel disease, specifically. Importantly, there is emerging recognition that this oxidative stress extends beyond the primary site of barrier disruption and inflammation, and is disseminated systemically.

These observations suggest that bacterial cells might sense the molecular ratio between UMP and intermediates in the de novo UMP biosynthesis such as carbamoyl-L-aspartate, which accumulates in the pyrC mutant strain, as a signal of the relative balance between the two UMP biosynthetic pathways. An unbalance towards UMP biosynthesis via the SKI II pyrimidine salvage pathway triggers cellulose production, and this effect relies on the activity of the diguanylate cyclase YedQ. The interplay between nucleotide salvage pathway and cellulose production might be connected to the role of cellulose and other EPS in the response to environmental stresses such as desiccation and resistance to bacteriophages. In bacterial biofilms, events leading to extensive cell lysis, such as exposure to antibiotics or attack by bacteriophages, would release cell components into the local environment: thus, a sudden increase in concentrations of exogenous nucleotides due to bacterial lysis might function as an ����alarm signal���� to neighboring cells, which would react by producing EPS as a defense mechanism against environmental stresses. For intracellular pathogenic enterobacteria, sensing an increase of exogenous nucleotide concentration might instead signal Qingyangshengenin-B stress events in the host cell, such as leakage of nucleotides from the nuclear compartment. Consistent with our observations, it has been reported that allosteric inhibition of the CarB protein by exogenous uracil strongly influences production of extracellular structures and negatively affects expression of type III secretion systems in the intracellular pathogen Shigella flexneri. In Pseudomonas fluorescens, a spontaneous mutation in the carB gene affects the proportion of capsulated and non-capsulated subpopulations via yet unknown molecular mechanisms. Our results complement and expand these observations, and underline the importance of the interplay linking biofilm formation, bacterial virulence, production of extracellular structures, and nucleotide biosynthetic pathways: better understanding of these connections at the molecular level will allow us to improve our strategies in preventing bacterial biofilms. In this perspective, our results provide strong evidence to confirm previous findings suggesting that drugs targeting nucleotide biosynthetic pathways have a strong potential as antibiofilm agents. The mammalian placenta is the first organ to be developed during gestation and carries out multiple functions required for normal embryonic development in the uterine environment. Impaired placental development is associated with many complications to both moms and babies during pregnancy, including preeclampsia, intrauterine growth retardation and fetal loss.

By regulatory proteins directly involved in sensing intracellular pyrimidine concentrations. In this work, we have shown that mutations in genes belonging to de novo nucleotide biosynthetic pathways strongly affect csgDEFG expression and curli production in E. coli. Interplay between nucleotide metabolism and biofilm appears to be conserved in different bacteria; however, specific effects and mechanism may vary substantially. Indeed, although our results are consistent with PYR-41 previous findings showing that active de novo UMP biosynthesis is necessary for biofilm formation in P. aeruginosa, in this bacterium inhibition of purine biosynthesis through inactivation of the purH gene does not affect adhesion factors�� production, in contrast to what observed in E. coli. Likewise, pyrimidines appear to control EPS production and biofilm formation in V. cholerae through the dedicated regulator CytR, which does not appear to play a direct role in curli regulation in E. coli. Despite these differences, it seems that absence of de novo pyrimidine biosynthesis can act as a signal for severe nutrient starvation, which can in turn prevent biofilm formation and promote biofilm dispersal. In E. coli, the effects of mutations in the de novo UMP biosynthesis on curli production can be complemented by supplementing growth medium with uracil, thus suggesting that pyrimidine nucleotide availability, regardless whether it is achieved via de novo UMP biosynthesis or the pyrimidine salvage pathway, allows efficient csgDEFG transcription and expression of the CsgD regulon. Regulation of csgDEFG expression by intracellular nucleotide concentrations might take place by direct modulation of transcription initiation by RNA polymerase, similar to transcription control by GTP availability described for ribosomal promoters, or through not yet identified nucleotide-sensing regulatory proteins. Alternatively, perturbations in nucleotide pools might affect accumulation of cdi-GMP, a signal molecule necessary for csgDEFG expression, possibly by impairing Bulleyaconi-cine-A Diguanylate cyclases�� enzymatic activity. Diguanylate cyclases play a role in pyrimidine-dependent regulation of cellulose production. Cellulose production is regulated by a more complex mechanism since, in addition to pyrimidine availability, it seems to respond to the relative activity of the two UMP biosynthetic pathways. Indeed, MG1655 produces twice as much cellulose when grown in the presence of exogenous uracil, i.e., in conditions in which UMP biosynthesis is mostly carried out via the pyrimidine salvage pathway and de novo UMP biosynthesis is inhibited.

The normal functions of progranulin in the central nervous system remain to be defined, although studies have suggested a role of progranulin in promoting neuronal survival and regulating inflammation in the CNS. Moreover, mechanisms that regulate progranulin levels and the receptor and signaling pathways involved in progranulin action remain to be defined. Tolterodine tartrate Sortilin was recently identified as a progranulin binding partner in an expression cloning screen. Sortilin is a type I single pass transmembrane protein in the VPS10 family which regulates intracellular protein trafficking and acts as a cell surface receptor that mediates pro-NGF and pro-BDNF mediated cell death when coupled with p75/NTR. Sortilin mediates progranulin endocytosis and regulates the level of progranulin in the brain. The level of secreted progranulin is dramatically increased in sortilin knockout mice. Furthermore, ablation of sortilin is able to correct the decreased progranulin level in mice heterozygous for PGRN deletion. A genome wide association study has also found two SNPs close to sortilin that affect sortilin expression associated with PGRN level in the plasma. Thus progranulin-sortilin interaction is a major determinant of progranulin level in vivo. Here we report the mapping of the binding sites between progranulin and sortilin. We show that progranulin binds to the beta propeller region of sortilin through its C-terminal tail. The crystal structure of the VPS10 domain of sortilin was recently determined in a complex with another sortilin ligand, neurotensin. Neurotensin, a brain-gut tridecapeptide, interacts with the sortilin beta-propeller domain via its extreme carboxyl terminus. Our data suggests that progranulin and neurotensin interact with sortilin in a similar fashion. Since progranulin haplo-insufficiency is a leading cause of FTLD and sortilin is a main determinant of progranulin level, reagents that modulate progranulin-sortilin interaction and thus help restore progranulin levels in the brain might be of high therapeutic interest. Our study demonstrated a critical role of the progranulin Cterminal tail in mediating its interaction with sortilin and thus an important Naringin dihydrochalcone function of this fragment in controlling progranulin trafficking. This result will impact future experiments with progranulin gene therapy or protein administration to treat FTLD. By deleting the last 3 residues of progranulin, higher levels of progranulin in the brain could be possibly achieved by bypassing sortilin mediated regulation of progranulin trafficking and lysosomal degradation. However, it is still not clear whether the progranulinsortilin interaction also plays a role in progranulin signaling. Future studies are required to determine whether sortilin mediates the reported effects of progranulin on neuronal survival and inflammation. We have also found that the C-terminal carboxylate of progranulin in crucial in mediating the interaction between progranulin and sortilin.