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Sample records for secretion activated signaling

  1. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

  2. Epithelial Cell–Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

    Science.gov (United States)

    Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna C.; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.

    2016-01-01

    Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia. PMID:27064756

  3. Epithelial Cell-Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia.

    Science.gov (United States)

    Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A; Zabinski, Mary C; Yuen, Constance K; Lung, Wing Yi; Gower, Adam C; Belkina, Anna C; Ramirez, Maria I; Deng, Jane C; Quinton, Lee J; Jones, Matthew R; Mizgerd, Joseph P

    2016-09-01

    Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6G(bright)CD11b(bright) neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.

  4. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    International Nuclear Information System (INIS)

    Ye, R.D.; Wun, T.C.; Sadler, J.E.

    1988-01-01

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  5. Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

    Directory of Open Access Journals (Sweden)

    Jessica Wahlgren

    Full Text Available It has previously been shown that nano-meter sized vesicles (30-100 nm, exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.

  6. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    Science.gov (United States)

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

  7. Panax ginseng total protein promotes proliferation and secretion of collagen in NIH/3T3 cells by activating extracellular signal-related kinase pathway

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    Xuenan Chen

    2017-07-01

    Conclusion: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

  8. Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

    Science.gov (United States)

    Wanka, Franziska; Arentshorst, Mark; Cairns, Timothy C; Jørgensen, Thomas; Ram, Arthur F J; Meyer, Vera

    2016-08-20

    The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency. In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein. Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.

  9. Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt

    Science.gov (United States)

    Rhee, Man Hee; Sung, Yoon-Young; Yang, Won-Kyung; Kim, Seung Hyung; Kim, Ho-Kyoung

    2014-01-01

    Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin α IIb β 3 and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P < 0.001), 28.3% (P < 0.01), and 19.1% (P < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent. PMID:24701244

  10. Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

    Science.gov (United States)

    Chadha, Sonia; Tati, Swetha; Conti, Heather R.; Hube, Bernhard; Cullen, Paul J.; Edgerton, Mira

    2012-01-01

    Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence. PMID:23139737

  11. Secreted and Transmembrane Wnt Inhibitors and Activators

    Science.gov (United States)

    Cruciat, Cristina-Maria; Niehrs, Christof

    2013-01-01

    Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand–receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

  12. Importance of a diffusion-dominant small volume to activate cell-secreted soluble factor signaling in embryonic stem cell culture in microbioreactors: a mathematical model based study.

    Science.gov (United States)

    Chowdhury, Mohammad Mahfuz; Fujii, Teruo; Sakai, Yasuyuki

    2013-07-01

    In our previous studies, we observed that cell-secreted BMP4 had a prominent influence on mouse embryonic stem cell (mESC) behaviors in a membrane-based two-chambered microbioreactor (MB), but not in a macro-scale culture (6-well plate/6WP). In this study, we investigated how the physical aspects of these cultures regulated BMP4 signaling by developing mathematical models of the cultures. The models estimated signaling activity in the cultures by considering size of the undifferentiated mESC colonies and their growth, diffusion of BMP4, and BMP4 trafficking process in the colonies. The models successfully depicted measured profile of BMP4 concentration in the culture medium which was two times higher in the MB than that in the 6WP during 5-day culture. The models estimated that, owing to the small volume and the membrane, cells were exposed to a higher BMP4 concentration in the top chamber of the MB than that in the 6WP culture. The higher concentration of BMP4 induced a higher concentration of BMP4-bound receptor in the colony in the MB than in the 6WP, thereby leading to the higher activation of BMP4 signaling in the MB. The models also predicted that the size of the MB, but not that of the 6WP, was suitable for maximizing BMP4 accumulation and upregulating its signaling. This study will be helpful in analyzing culture systems, designing microfluidic devices for controlling ESC or other cell behavior. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

    Science.gov (United States)

    Ng, Daphne T W; Sarkar, Casim A

    2013-01-01

    Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

  14. Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

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    Carla Emiliani

    2013-03-01

    Full Text Available Exosomes are small extracellular vesicles (30–100 nm derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated.

  15. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

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    Annika Osswald

    Full Text Available Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

  16. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

    Science.gov (United States)

    Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U.

    2015-01-01

    Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

  17. HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway.

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    Ding, Chenyue; Zou, Qinyan; Wang, Fuxin; Wu, Huihua; Wang, Wei; Li, Hong; Huang, Boxian

    2018-01-01

    Human adipose-derived stem cells (hADSCs) are a potential therapeutic option for clinical applications because of their ability to produce cytokines and their capacity for trilineage differentiation. To date, few researchers have investigated the effects of hADSCs on natural ovarian aging (NOA). An NOA mouse model and human ovarian granule cells (hGCs) collected from individuals with NOA were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing NOA. Enzyme-linked immunosorbent assay was used to detect the serum levels of sex hormones and antioxidative enzymes. The proliferation rate and marker expression level of hGCs were measured by flow cytometry (FACS). Cytokines were measured by a protein antibody array methodology. Western blot assays were used to determine the protein expression levels of SIRT1 and FOXO1. Our results showed that hADSCs displayed therapeutic activity against ovarian function in an NOA mouse model, increasing the proliferation rate and marker expression level of hGCs. Furthermore, the yields of hADSC-secreted HGF and bFGF were higher than those of other growth factors. FACS showed that combination treatment with the growth factors HGF and bFGF more strongly promoted proliferation and inhibited apoptosis in hGCs than HGF or bFGF treatment alone. FACS and ELISA revealed that the combination treatment with both growth factors inhibited oxidative stress more forcefully than treatments with only one of these growth factors. In addition, protein assays demonstrated that combination treatment with both growth factors suppressed oxidative stress by up-regulating the expression of SIRT1 and FOXO1. These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which HGF and bFGF derived from hADSCs improved ovarian function during natural aging via reduction of oxidative stress by activating the SIRT1/FOXO1 signaling pathway. © 2018 The Author

  18. Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

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    Yuko Nakagawa

    Full Text Available BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+ ([Ca(2+](c and cAMP ([cAMP](c were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+](c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+](c response. The effect of sucralose on [Ca(2+](c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q inhibitor. Sucralose also induced sustained elevation of [cAMP](c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+ and cAMP-dependent mechanisms.

  19. Wnt secretion and signaling in Caenorhabditis elegans

    NARCIS (Netherlands)

    Yang, P.T.

    2008-01-01

    Wnt proteins are a highly conserved family of signaling molecules that play a central role during development and in adult tissue homeostasis. Wnt proteins regulate a variety of biological processes, ranging from cell proliferation and cell fate determination to cell migration, axon guidance and

  20. Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

    Science.gov (United States)

    Houtz, Jessica; Borden, Philip; Ceasrine, Alexis; Minichiello, Liliana; Kuruvilla, Rejji

    2016-11-07

    Insulin secretion by pancreatic islet β cells is critical for glucose homeostasis, and a blunted β cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet β cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate β cell dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation.

    Science.gov (United States)

    Roehrich, A Dorothea; Guillossou, Enora; Blocker, Ariel J; Martinez-Argudo, Isabel

    2013-02-01

    Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation. © 2013 Blackwell Publishing Ltd.

  2. Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

    Science.gov (United States)

    Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

    1993-03-10

    Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

  3. Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.

    Science.gov (United States)

    Pan, Qing; Zhang, Qiang; Chu, Jun; Pais, Roshan; Liu, Shanshan; He, Cheng; Eko, Francis O

    2017-01-01

    The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly ( p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine

  4. Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Qing Pan

    2017-12-01

    Full Text Available The polymorphic membrane protein D (Pmp18D is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1 as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88, nuclear factor kappa beta (NF-κB p50/p65, and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001 enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1

  5. Pathogen-secreted proteases activate a novel plant immune pathway.

    Science.gov (United States)

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  6. The glycogen metabolism via Akt signaling is important for the secretion of enamel matrix in tooth development.

    Science.gov (United States)

    Ida-Yonemochi, Hiroko; Otsu, Keishi; Ohshima, Hayato; Harada, Hidemitsu

    2016-02-01

    Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals

    DEFF Research Database (Denmark)

    Epifano, Carolina; Megias, Diego; Perez-Moreno, Mirna

    2014-01-01

    The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic...... migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell...... motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results...

  8. Neuropeptide delivery to the brain: a von Willebrand factor signal peptide to direct neuropeptide secretion

    Directory of Open Access Journals (Sweden)

    de Backer Marijke WA

    2010-08-01

    Full Text Available Abstract Background Multiple neuropeptides, sometimes with opposing functions, can be produced from one precursor gene. To study the roles of the different neuropeptides encoded by one large precursor we developed a method to overexpress minigenes and establish local secretion. Results We fused the signal peptide from the Von Willebrand Factor (VWF to a furin site followed by a processed form of the Agouti related protein (AgRP, AgRP83-132 or α-melanocyte stimulating hormone. In vitro, these minigenes were secreted and biologically active. Additionally, the proteins of the minigenes were not transported into projections of primary neurons, thereby ensuring local release. In vivo administration of VWF-AgRP83-132 , using an adeno-associated viral vector as a delivery vehicle, into the paraventricular hypothalamus increased body weight and food intake of these rats compared to rats which received a control vector. Conclusions This study demonstrated that removal of the N-terminal part of full length AgRP and addition of a VWF signal peptide is a successful strategy to deliver neuropeptide minigenes to the brain and establish local neuropeptide secretion.

  9. Delta-like Ligand-4-Notch Signaling Inhibition Regulates Pancreatic Islet Function and Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Fabienne Billiard

    2018-01-01

    Full Text Available Although Notch signaling has been proposed as a therapeutic target for type-2 diabetes, liver steatosis, and atherosclerosis, its direct effect on pancreatic islets remains unknown. Here, we demonstrated a function of Dll4-Notch signaling inhibition on the biology of insulin-producing cells. We confirmed enhanced expression of key Notch signaling genes in purified pancreatic islets from diabetic NOD mice and showed that treatment with anti-Dll4 antibody specifically abolished Notch signaling pathway activation. Furthermore, we showed that Notch inhibition could drive proliferation of β-islet cells and confer protection from the development of STZ-induced diabetes. Importantly, inhibition of the Dll4 pathway in WT mice increased insulin secretion by inducing the differentiation of pancreatic β-islet cell progenitors, as well as the proliferation of insulin-secreting cells. These findings reveal a direct effect of Dll4-blockade on pancreatic islets that, in conjunction with its immunomodulatory effects, could be used for unmet medical needs hallmarked by inefficient insulin action.

  10. Application of native signal sequences for recombinant proteins secretion in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Do, Duy Duc; Eriksen, Jens C.

    Background Methylotrophic yeast Pichia pastoris is widely used for recombinant protein production, largely due to its ability to secrete correctly folded heterologous proteins to the fermentation medium. Secretion is usually achieved by cloning the recombinant gene after a leader sequence, where...... alpha‐mating factor (MF) prepropeptide from Saccharomyces cerevisiae is most commonly used. Our aim was to test whether signal peptides from P. pastoris native secreted proteins could be used to direct secretion of recombinant proteins. Results Eleven native signal peptides from P. pastoris were tested...... by optimization of expression of three different proteins in P. pastoris. Conclusions Native signal peptides from P. pastoris can be used to direct secretion of recombinant proteins. A novel USER‐based P. pastoris system allows easy cloning of protein‐coding gene with the promoter and leader sequence of choice....

  11. A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

    Science.gov (United States)

    Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

    2017-04-24

    Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

  12. Activated platelets enhance IL-10 secretion and reduce TNF-α secretion by monocytes

    DEFF Research Database (Denmark)

    Gudbrandsdottir, Sif; Hasselbalch, Hans C; Nielsen, Claus H

    2013-01-01

    ), Escherichia coli LPS, or intact Porphyromonas gingivalis. Addition of platelets activated by thrombin-receptor-activating peptide enhanced IL-10 production induced by LPS (p gingivalis (p ....05), and P. gingivalis (p gingivalis-stimulated cultures (p ... of activated platelets. Adherence of platelets increased TG- and TT-induced IL-10 secretion by monocytes (p gingivalis (p

  13. Signal transduction pathways involved in intestinal salt and water secretion

    NARCIS (Netherlands)

    W. van den Berghe (Nina)

    1992-01-01

    textabstractThis thesis describes some novel aspects of the regulation of salt and water secretion in the intestinal epithelium. This process is not unique for the intestine, but a common and necessary function of many other organs, including the stomach (gastric juice), kidney (urine), sweatglands

  14. Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion

    Directory of Open Access Journals (Sweden)

    Stanko S. Stojilkovic

    2017-06-01

    Full Text Available Gonadotrophs are basophilic cells of the anterior pituitary gland specialized to secrete gonadotropins in response to elevation in intracellular calcium concentration. These cells fire action potentials (APs spontaneously, coupled with voltage-gated calcium influx of insufficient amplitude to trigger gonadotropin release. The spontaneous excitability of gonadotrophs reflects the expression of voltage-gated sodium, calcium, potassium, non-selective cation-conducting, and chloride channels at their plasma membrane (PM. These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the PM, as well as GABAA, nicotinic, and purinergic P2X channels gated by γ-aminobutyric acid (GABA, acetylcholine (ACh, and ATP, respectively. Activation of these channels leads to initiation or amplification of the pacemaking activity, facilitation of calcium influx, and activation of the exocytic pathway. Gonadotrophs also express calcium-conducting channels at the endoplasmic reticulum membranes gated by inositol trisphosphate and intracellular calcium. These channels are activated potently by hypothalamic gonadotropin-releasing hormone (GnRH and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate cyclase-activating peptides, endothelins, ACh, vasopressin, and oxytocin. Activation of these channels causes oscillatory calcium release and a rapid gonadotropin release, accompanied with a shift from tonic firing of single APs to periodic bursting type of electrical activity, which accounts for a sustained calcium signaling and gonadotropin secretion. This review summarizes our current understanding of ion channels as signaling molecules in gonadotrophs, the role of GnRH and paracrine agonists in their gating, and the cross talk among channels.

  15. Antifungal activity of epithelial secretions from selected frog species ...

    African Journals Online (AJOL)

    This study aimed to investigate the antifungal activity of skin secretions from selected frogs (Amietia fuscigula, Strongylopus grayi and Xenopus laevis) and one toad (Amietophrynus pantherinus) of the south Western Cape Province of South Africa. Initially, different extraction techniques for the collection of skin secretions ...

  16. Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

    International Nuclear Information System (INIS)

    Zhang, Yi; Gonzalez, Rachel M; Zangar, Richard C

    2011-01-01

    Protein secretion by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a limited understanding of the cellular processes that regulate protein secretion. In this study, we utilize human epithelial mammary cell (HMEC) lines that were engineered to express different levels of HER1, HER2 and HER3. Using an ELISA microarray platform, we evaluate the effects of epidermal growth factor family receptor (HER) expression on protein secretion in the HMEC lines upon initiation of HER1 receptor activation. The secreted proteins include three HER1 ligands, interleukins 1α and 18, RANTES, vascular-endothelial and platelet-derived growth factors, matrix metalloproteases 1, 2 and 9, and the extracellular portion of the HER1 and HER2 proteins. In addition, we investigate whether MAPK/Erk and PI3K/Akt signaling regulate protein secretion in these cell lines and if so, whether the involvement of HER2 or HER3 receptor alters their response to MAPK/Erk and PI3K/Akt signal pathway inhibition in terms of protein secretion. Differential expression of HER2 and HER3 receptors alters the secretion of a variety of growth factors, cytokines, and proteases. Some alterations in protein secretion are still observed when MAPK/Erk or PI3K/Akt signaling is inhibited. This study suggests that HER overexpression orchestrates broad changes in the tumor microenvironment by altering the secretion of a diverse variety of biologically active proteins

  17. Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat

    Directory of Open Access Journals (Sweden)

    Groneberg David A

    2008-02-01

    Full Text Available Abstract Background Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity. Methods The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique. Results Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity. Conclusion Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.

  18. Gq and Gs signaling acting in synergy to control GLP-1 secretion

    DEFF Research Database (Denmark)

    Pedersen, Maria Hauge; Ekberg, Jeppe Hvidtfeldt; Engelstoft, Maja Storm

    2017-01-01

    GPR40 is generally known to signal through Gq. However, in transfected cells, certain synthetic agonists can make the receptor signal also through Gs and cAMP (Hauge et al., 2015). Here we find that, in colonic crypt cultures, the GLP-1 secretion induced by such Gq + Gs GPR40 agonists is indeed i...

  19. p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland.

    Science.gov (United States)

    Carlsson, Stina K; Gierow, J Peter

    2012-03-01

    The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and β, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

  20. WISP3 (CCN6 Is a Secreted Tumor-Suppressor Protein that Modulates IGF Signaling in Inflammatory Breast Cancer

    Directory of Open Access Journals (Sweden)

    Celina G. Kleer

    2004-03-01

    Full Text Available Inflammatory breast cancer (IBC is the most lethal form of locally advanced breast cancer. We have found that WISP3 is lost in 80% of human IBC tumors and that it has growth- and angiogenesis-inhibitory functions in breast cancer in vitro and in vivo. WISP3 is a cysteine-rich, putatively secreted protein that belongs to the CCN family. It contains a signal peptide at the N-terminus and four highly conserved motifs. Here, for the first time, we investigate the function of WISP3 protein in relationship to its structural features. We found that WISP3 is secreted into the conditioned media and into the lumens of normal breast ducts. Once secreted, WISP3 was able to decrease, directly or through induction of other molecule(s, the IGF-1-induced activation of the IGF-IR, and two of its main downstream signaling molecules, IRS1 and ERK-1/2, in SUM149 IBC cells. Furthermore, WISP3 containing conditioned media decreased the growth rate of SUM149 cells. This work sheds light into the mechanism of WISP3 function by demonstrating that it is secreted and that, once in the extracellular media, it induces a series of molecular events that leads to modulation of IGF-IR signaling pathways and cellular growth in IBC cells.

  1. Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain.

    Science.gov (United States)

    Scherer, Thomas; Lindtner, Claudia; O'Hare, James; Hackl, Martina; Zielinski, Elizabeth; Freudenthaler, Angelika; Baumgartner-Parzer, Sabina; Tödter, Klaus; Heeren, Joerg; Krššák, Martin; Scheja, Ludger; Fürnsinn, Clemens; Buettner, Christoph

    2016-06-01

    Hepatic steatosis is common in obesity and insulin resistance and results from a net retention of lipids in the liver. A key mechanism to prevent steatosis is to increase secretion of triglycerides (TG) packaged as VLDLs. Insulin controls nutrient partitioning via signaling through its cognate receptor in peripheral target organs such as liver, muscle, and adipose tissue and via signaling in the central nervous system (CNS) to orchestrate organ cross talk. While hepatic insulin signaling is known to suppress VLDL production from the liver, it is unknown whether brain insulin signaling independently regulates hepatic VLDL secretion. Here, we show that in conscious, unrestrained male Sprague Dawley rats the infusion of insulin into the third ventricle acutely increased hepatic TG secretion. Chronic infusion of insulin into the CNS via osmotic minipumps reduced the hepatic lipid content as assessed by noninvasive (1)H-MRS and lipid profiling independent of changes in hepatic de novo lipogenesis and food intake. In mice that lack the insulin receptor in the brain, hepatic TG secretion was reduced compared with wild-type littermate controls. These studies identify brain insulin as an important permissive factor in hepatic VLDL secretion that protects against hepatic steatosis. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  2. The secrets of highly active older adults.

    Science.gov (United States)

    Franke, Thea; Tong, Catherine; Ashe, Maureen C; McKay, Heather; Sims-Gould, Joanie

    2013-12-01

    Although physical activity is a recognized component in the management of many chronic diseases associated with aging, activity levels tend to progressively decline with increasing age (Manini & Pahor, 2009; Schutzer & Graves, 2004). In this article we examine the key factors that facilitate physical activity in highly active community-dwelling older adults. Using a strengths based approach, we examined the factors that facilitated physical activity in our sample of highly active older adults. Twenty-seven older adults participated in face-to face interviews. We extracted a sub-sample of 10 highly active older adults to be included in the analyses. Based on a framework analysis of our transcripts we identified three factors that facilitate physical activity in our sample, these include: 1) resourcefulness: engagement in self-help strategies such as self-efficacy, self-control and adaptability; 2) social connections: the presence of relationships (friend, neighborhood, institutions) and social activities that support or facilitate high levels of physical activity; and 3) the role of the built and natural environments: features of places and spaces that support and facilitate high levels of physical activity. Findings provide insight into, and factors that facilitate older adults' physical activity. We discuss implications for programs (e.g., accessible community centers, with appropriate programming throughout the lifecourse) and policies geared towards the promotion of physical activity (e.g., the development of spaces that facilitate both physical and social activities). © 2013.

  3. Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

    DEFF Research Database (Denmark)

    Finnie, Christine; Andersen, Birgit; Shahpiri, Azar

    2011-01-01

    molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted...... analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms....... to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling...

  4. Activity of secreted amylases in Aspergillus

    Energy Technology Data Exchange (ETDEWEB)

    Bocheva, S.S.; Kurnitskaya, L.N.

    1981-01-01

    When A. oryzae was cultivated in a synthetic liquid medium containing maltose as a sole source of C, the activity of extracellular amylase was 8.43-11.92 units/100 mL. Addition of 1.0% and 2.0% NaCl to the medium increased the amylase activity approximately 5- and 10-fold, respectively.

  5. The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

    Science.gov (United States)

    Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

    2016-11-15

    It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

  6. BDNF-induced nitric oxide signals in cultured rat hippocampal neurons: time course, mechanism of generation, and effect on neurotrophin secretion.

    Science.gov (United States)

    Kolarow, Richard; Kuhlmann, Christoph R W; Munsch, Thomas; Zehendner, Christoph; Brigadski, Tanja; Luhmann, Heiko J; Lessmann, Volkmar

    2014-01-01

    BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous application of the NO donor sodium-nitroprusside markedly inhibited neurotrophin secretion. However, endogenously generated NO in response to depolarization and neurotrophin stimulation, both did not result in a negative feedback on neurotrophin secretion. These results suggest that a negative feedback of NO signaling on synaptic secretion of neurotrophins operates only at high intracellular levels of nitric oxide that are under physiological conditions not reached by depolarization or BDNF signaling.

  7. High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

    Science.gov (United States)

    Savijoki, K; Kahala, M; Palva, A

    1997-02-28

    A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

  8. Application of near-infrared spectroscopy to measurement of hemodynamic signals accompanying stimulated saliva secretion.

    Science.gov (United States)

    Sato, Hiroki; Obata, Akiko N; Moda, Ichiro; Ozaki, Kazutaka; Yasuhara, Takaomi; Yamamoto, Yukari; Kiguchi, Masashi; Maki, Atsushi; Kubota, Kisou; Koizumi, Hideaki

    2011-04-01

    We aim to test the feasibility of using near-infrared spectroscopy (NIRS) for indirect measurement of human saliva secretion in response to taste stimuli for potential application to organoleptic testing. We use an NIRS system to measure extracranial hemodynamics (Hb-signals around the temples) of healthy participants when taste stimuli are taken in their mouths. First, the Hb-signals and volume of expelled saliva (stimulated by distilled-water or sucrose-solution intake) are simultaneously measured and large Hb-signal changes in response to the taste stimuli (Hb-responses) are found. Statistical analysis show that both the Hb response and saliva volume are larger for the sucrose solution than for the distilled water with a significant correlation between them (r = 0.81). The effects of swallowing on the Hb-signals are investigated. Similar Hb responses, differing from the sucrose solution and distilled water, are obtained even though the participants swallow the mouth contents. Finally, functional magnetic resonance imaging is used to identify possible sources of the Hb signals corresponding to salivation. Statistical analysis indicates similar responses in the extracranial regions, mainly around the middle meningeal artery. In conclusion, the identified correlation between extracranial hemodynamics and the saliva volume suggests that NIRS is applicable to the measurement of hemodynamic signals accompanying stimulated saliva secretion.

  9. The signaling cascades of Ganoderma lucidum extracts in stimulating non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y cell lines.

    Science.gov (United States)

    Pinweha, Sirinthorn; Wanikiat, Payong; Sanvarinda, Yupin; Supavilai, Porntip

    2008-12-19

    Ganoderma lucidum (GL) is a medicinal mushroom that possesses various pharmacological properties which are also documented in the ancient reports where GL is praised for its effects on the promotion of health and longevity. In this study, we have investigated the effect of GL mycelia extracts on the non-amyloidogenic protein secretion (sAPPalpha) and the amyloid precursor protein (APP) expression in SH-SY5Y neuroblastoma cells. In order to characterize the signaling pathway which mediates GL-enhanced sAPPalpha secretion, we used inhibitors of nerve growth factor (NGF) signaling pathways, phosphatidylinositol 3 kinase (PI3K), phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK1/2), to block GL-mediated sAPPalpha secretion as well as ERK1/2 and PKC activation by using Western blot analysis. Our results provided for the first time evidence that GL mycelia extracts increased APP expression and promoted sAPPalpha secretion. In addition, GL extracts activated ERK1/2 and PKC phosphorylation. The complex signaling cascades of PI3K and ERK may be responsible for GL-mediated sAPPalpha secretion.

  10. Reactive oxygen species as a signal in glucose-stimulated insulin secretion.

    Science.gov (United States)

    Pi, Jingbo; Bai, Yushi; Zhang, Qiang; Wong, Victoria; Floering, Lisa M; Daniel, Kiefer; Reece, Jeffrey M; Deeney, Jude T; Andersen, Melvin E; Corkey, Barbara E; Collins, Sheila

    2007-07-01

    One of the unique features of beta-cells is their relatively low expression of many antioxidant enzymes. This could render beta-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H2O2. In both models, insulin secretion could be stimulated by provision of either exogenous H2O2 or diethyl maleate, which raises intracellular H2O2 levels. Provision of exogenous H2O2 scavengers, including cell permeable catalase and N-acetyl-L-cysteine, inhibited glucose-stimulated H2O2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H2O2, had no effect on GSIS. Because oxidative stress is an important risk factor for beta-cell dysfunction in diabetes, the relationship between glucose-induced H2O2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H2O2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.

  11. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

    Science.gov (United States)

    Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

    2012-01-01

    Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

  12. Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

    Science.gov (United States)

    Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

    2016-05-17

    Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

  13. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    Science.gov (United States)

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Active and separate secretion of fiber and penton base during the early phase of Ad2 or Ad5 infection

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Yuhua; Zhang, Bo; Hou, Weihong; Lin, Hongyu [Beijing Key Laboratory of Gene Resource and Molecular Development, Beijing Normal University, Beijing (China); Rebetz, Johan [The Rausing Laboratory, Department of Neurosurgery, Lund University, Lund (Sweden); Hong, Saw-See [Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1, Lyon Cedex 07 (France); Wang, Youjun; Ran, Liang [Beijing Key Laboratory of Gene Resource and Molecular Development, Beijing Normal University, Beijing (China); Fan, Xiaolong, E-mail: XFan@bnu.edu.cn [Beijing Key Laboratory of Gene Resource and Molecular Development, Beijing Normal University, Beijing (China)

    2017-05-15

    Fiber and penton base overproduced in adenovirus (Ad) infected cells can be secreted prior to progeny release and thereby regulate progeny spread. We aimed to investigate the mechanisms of fiber and penton base secretion in Ad2- or Ad5-infected A549 cells. Our flow cytometry analyses detected abundant surface fiber molecules, but little penton base molecules at 12 h post infection. Immunogold staining combined with transmission electron microscopic analyses revealed separate, non-co-localized release of fiber and penton base in the proximity of the plasma membrane. Depolymerization of microtubule and actin cytoskeletons, and inhibition of Rock kinase and myosin II activity together demonstrated cytoskeletal network-dependent fiber secretion. Inhibition of intracellular calcium [Ca{sup 2+}]{sub i} signaling caused diminished fiber secretion, which was associated with diminished progeny production. Thus, fiber and penton base are actively and separately secreted during the early stages of Ad2 or Ad5 infection, their secretion may play important role in Ad life cycle. - Highlights: •Excessive production of structural proteins is common to viral infection, which may regulate the host-virus equilibrium and the spreading of viruses. •The adenovirus (Ad) structural proteins, fiber and penton base, are respectively important for Ad binding to its receptor and subsequent internalization in host cells. In Ad infected cells, these two structural proteins are excessively produced. •The mechanisms underlying the release of fiber and penton base molecules at the early phase of Ad infection is yet poorly understood. •Our studies show that in Ad5 or Ad2 infected A549 cells, fiber and penton base molecules are actively and separately secreted. •Fiber secretion is dependent on cytoskeleton-mediated protein traffic. •Inhibition of myosin II motor and Ca{sup 2+} signaling activity significantly diminishes fiber secretion. •These findings could contribute to our

  15. Active and separate secretion of fiber and penton base during the early phase of Ad2 or Ad5 infection

    International Nuclear Information System (INIS)

    Yan, Yuhua; Zhang, Bo; Hou, Weihong; Lin, Hongyu; Rebetz, Johan; Hong, Saw-See; Wang, Youjun; Ran, Liang; Fan, Xiaolong

    2017-01-01

    Fiber and penton base overproduced in adenovirus (Ad) infected cells can be secreted prior to progeny release and thereby regulate progeny spread. We aimed to investigate the mechanisms of fiber and penton base secretion in Ad2- or Ad5-infected A549 cells. Our flow cytometry analyses detected abundant surface fiber molecules, but little penton base molecules at 12 h post infection. Immunogold staining combined with transmission electron microscopic analyses revealed separate, non-co-localized release of fiber and penton base in the proximity of the plasma membrane. Depolymerization of microtubule and actin cytoskeletons, and inhibition of Rock kinase and myosin II activity together demonstrated cytoskeletal network-dependent fiber secretion. Inhibition of intracellular calcium [Ca 2+ ] i signaling caused diminished fiber secretion, which was associated with diminished progeny production. Thus, fiber and penton base are actively and separately secreted during the early stages of Ad2 or Ad5 infection, their secretion may play important role in Ad life cycle. - Highlights: •Excessive production of structural proteins is common to viral infection, which may regulate the host-virus equilibrium and the spreading of viruses. •The adenovirus (Ad) structural proteins, fiber and penton base, are respectively important for Ad binding to its receptor and subsequent internalization in host cells. In Ad infected cells, these two structural proteins are excessively produced. •The mechanisms underlying the release of fiber and penton base molecules at the early phase of Ad infection is yet poorly understood. •Our studies show that in Ad5 or Ad2 infected A549 cells, fiber and penton base molecules are actively and separately secreted. •Fiber secretion is dependent on cytoskeleton-mediated protein traffic. •Inhibition of myosin II motor and Ca 2+ signaling activity significantly diminishes fiber secretion. •These findings could contribute to our understanding of Ad

  16. Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

    Science.gov (United States)

    Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

    2015-05-01

    In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Antifungal activity of lectins against yeast of vaginal secretion

    Directory of Open Access Journals (Sweden)

    Bruno Severo Gomes

    2012-06-01

    Full Text Available Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

  18. Secreted Frizzled-related protein-2 (sFRP2) augments canonical Wnt3a-induced signaling

    Energy Technology Data Exchange (ETDEWEB)

    Marschall, Zofia von [Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, DHHS, Bethesda, MD (United States); Fisher, Larry W., E-mail: lfisher@dir.nidcr.nih.gov [Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, DHHS, Bethesda, MD (United States)

    2010-09-24

    Research highlights: {yields} sFRP2 enhances the Wnt3a-induced {beta}-catenin stabilization and its nuclear translocation. {yields} sFRP2 enhances LRP6 phosphorylation and Wnt3a/{beta}-catenin transcriptional reporter activity. {yields} Dickkopf-1 (DKK1) fully antagonizes both Wnt3a/sFRP2-induced LRP6 phosphorylation and transcriptional activity. {yields} sFRP2 enhances expression of genes known to be regulated by Wnt3a signaling. -- Abstract: Secreted Frizzled-related proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic {beta}-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1, and Naked cuticle-1 homolog (NKD1). These results support sFRP2's role as an enhancer of Wnt/{beta}-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.

  19. Secreted Frizzled-related protein-2 (sFRP2) augments canonical Wnt3a-induced signaling

    International Nuclear Information System (INIS)

    Marschall, Zofia von; Fisher, Larry W.

    2010-01-01

    Research highlights: → sFRP2 enhances the Wnt3a-induced β-catenin stabilization and its nuclear translocation. → sFRP2 enhances LRP6 phosphorylation and Wnt3a/β-catenin transcriptional reporter activity. → Dickkopf-1 (DKK1) fully antagonizes both Wnt3a/sFRP2-induced LRP6 phosphorylation and transcriptional activity. → sFRP2 enhances expression of genes known to be regulated by Wnt3a signaling. -- Abstract: Secreted Frizzled-related proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic β-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1, and Naked cuticle-1 homolog (NKD1). These results support sFRP2's role as an enhancer of Wnt/β-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.

  20. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  1. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  2. Expressed prostatic secretion biomarkers improve stratification of NCCN active surveillance candidates: performance of secretion capacity and TMPRSS2:ERG models.

    Science.gov (United States)

    Whelan, Christopher; Kawachi, Mark; Smith, David D; Linehan, Jennifer; Babilonia, Gail; Mejia, Rosa; Wilson, Timothy; Smith, Steven S

    2014-01-01

    Active surveillance is a viable patient option for prostate cancer provided that a clinical determination of low risk and presumably organ confined disease can be made. To standardize risk stratification schemes the NCCN (National Comprehensive Cancer Network®) provides guidelines for the active surveillance option. We determined the effectiveness of expressed prostatic secretion biomarkers for detecting occult risk factors in NCCN active surveillance candidates. Expressed prostatic secretion specimens were obtained before robot-assisted radical prostatectomy. Secretion capacity biomarkers, including total RNA and expressed prostatic secretion specimen volume, were measured by standard techniques. RNA expression biomarkers, including TXNRD1 mRNA, prostate specific antigen mRNA, TMPRSS2:ERG fusion mRNA and PCA3 mRNA, were measured by quantitative reverse-transcription polymerase chain reaction. Of the 528 patients from whom expressed prostatic secretions were collected 216 were eligible for active surveillance under NCCN guidelines. Variable selection on logistic regression identified 2 models, including one featuring types III and VI TMPRSS2:ERG variants, and one featuring 2 secretion capacity biomarkers. Of the 2 high performing models the secretion capacity model was most effective for detecting cases in this group that were up-staged or up-staged plus upgraded. It decreased the risk of up-staging in patients with a negative test almost eightfold and decreased the risk of up-staging plus upgrading about fivefold while doubling the prevalence of up-staging in the positive test group. Noninvasive expressed prostatic secretion testing may improve patient acceptance of active surveillance by dramatically reducing the presence of occult risk factors among those eligible for active surveillance under NCCN guidelines. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  3. Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

    Science.gov (United States)

    Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2011-10-20

    Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. H2O2-Activated Mitochondrial Phospholipase iPLA2 gamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin; Ježek, Petr

    2015-01-01

    Roč. 23, č. 12 (2015), s. 958-972 ISSN 1523-0864 R&D Projects: GA ČR(CZ) GPP303/11/P320; GA ČR(CZ) GA13-02033S; GA ČR(CZ) GA13-06666S; GA ČR GA15-02051S Institutional support: RVO:67985823 Keywords : mitochondrial phospholipase iPLA2 gamma * uncoupling protein UCP2 * G-protein coupled receptor - 40 * glucose-stimulated insulin secretion * pancreatic beta cells Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 7.093, year: 2015

  5. JNK mitogen-activated protein kinase limits calcium-dependent chloride secretion across colonic epithelial cells.

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

    Neuroimmune agonists induce epithelial Cl(-) secretion through elevations in intracellular Ca2+ or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to Ca2+-dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating Cl(-) secretion in T(84) colonic epithelial cells. Western blot analysis revealed that a prototypical Ca2+-dependent secretagogue, carbachol (CCh; 100 microM), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular Ca2+, but not by forskolin (FSK; 10 microM), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 microM). Pretreatment of voltage-clamped T(84) cells with SP600125 (2 microM), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized T(84) cell monolayers, SP600125 potentiated CCh-induced K+ conductances but not Na+\\/K+ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating Ca2+ but not cAMP-dependent epithelial Cl(-) secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral K+ channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders.

  6. O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

    Science.gov (United States)

    Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M; Maitra, Sushmit; Thomas, Stephen G; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E; Sun, Tai-Ping

    2016-01-15

    The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. © 2016 Zentella et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Directory of Open Access Journals (Sweden)

    Andrea Kerekes

    Full Text Available Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  8. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Science.gov (United States)

    Kerekes, Andrea; Hoffmann, Orsolya Ivett; Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A; Bősze, Zsuzsanna; Hiripi, László

    2017-01-01

    Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  9. Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia.

    Science.gov (United States)

    Norris, J W; Pombo, M; Shirley, E; Blevins, G; Tablin, F

    2015-01-01

    Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. Two affected and 6 clinically normal horses. Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1). Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia. Copyright © The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

  10. N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

    NARCIS (Netherlands)

    Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

    OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

  11. 77 FR 34037 - Agency Information Collection Activities; Proposed Collection; Comment Request; Trade Secret...

    Science.gov (United States)

    2012-06-08

    ... Activities; Proposed Collection; Comment Request; Trade Secret Claims for Emergency Planning and Community... Planning and Community Right-to- Know Act (EPCRA). Title: Trade Secret Claims for Emergency Planning and...). Estimated total number of potential respondents: 481. Frequency of response: Trade secret claims are...

  12. Adipocyte activation of cancer stem cell signaling in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Benjamin; Wolfson; Gabriel; Eades; Qun; Zhou

    2015-01-01

    Signaling within the tumor microenvironment has a critical role in cancer initiation and progression. Adipocytes, one of the major components of the breast microenvironment,have been shown to provide pro-tumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo. Adipocyte secreted factors such as leptin and interleukin-6(IL-6) have a paracrine effect on breast cancer cells. In adipocyte-adjacent breast cancer cells, the leptin and IL-6 signaling pathways activate janus kinase 2/signal transducer and activatorof transcription 5, promoting the epithelial-mesenchymal transition, and upregulating stemness regulators such as Notch, Wnt and the Sex determining region Y-box 2/octamer binding transcription factor 4/Nanog signaling axis. In this review we will summarize the major signaling pathways that regulate cancer stem cells in breast cancer and describe the effects that adipocyte secreted IL-6 and leptin have on breast cancer stem cell signaling. Finally we will introduce a new potential treatment paradigm of inhibiting the adipocyte-breast cancer cell signaling via targeting the IL-6 or leptin pathways.

  13. The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger.

    Science.gov (United States)

    Xu, Yue; Wang, Yan-Hui; Liu, Tian-Qi; Zhang, Hui; Zhang, He; Li, Jie

    2018-03-31

    α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production. Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C. The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

  14. [ERK activation effects on GABA secretion inhibition induced by SDF-1 in hippocampal neurons of rats].

    Science.gov (United States)

    Zhang, Zi-juan; Guo, Mei-xia; Xing, Ying

    2015-09-01

    To investigate the effect of extracellular regulating kinase (ERK) signaling pathway on the secretion of gamma-aminobutyric acid (GABA) in cultured rat hippocampal neurons induced by stromal cell derived factor-1 (SDF-1). The hippocampal neurons of newborn SD rats were cultured and identified in vitro; the phosphorylation level of ERK1/2 was examined by Western blot; ELISA was used to detect the effect of PD98059, a ERK1/2 specific blocker on GABA secretion of cultured hippocampal neurons and Western blot were adopted to measure the protein expression levels of glutamate decarboxylase (GAD65/67) and gamma aminobutyric acid transporter (GAT); after blocking ERK1/2 signaling pathway with PD98059; RT-PCR was used to detect the mRNA expression levels of GAT-1 and GAD65 after treated with PD98059. The levels of ERKl/2 phosphorylation were increased significantly by SDF1 acting on hippocampal neurons, and CX-CR4 receptor blocker AMD3100, could inhibit SDF-1 induced ERK1/2 activation; SDF-1 could inhibit the secretion of GABA in cultured hippocampal neurons, and ERK1/2 specific inhibitor PD98059, could partly reverse the inhibition of GABA secretion by SDF-1. The effects of SDF-1 on cultured hippocampal neurons was to decrease the mRNA genesis of glutamic acid decarboxylase GAD65 and GABA transporter GAT-1, besides, ERK inhibitor PD98059 could effectively flip the effect of SDF-1. The results of Western blot showed that SDF-1 could inhibit the protein expression of GAT-1 and GAD65/67 in hippocampal neurons and the inhibition of GAT-1 and GAD65/67 protein expression could be partially restored by ERK1/2 blocker. SDF-1 acts on the CXCR4 of hippocampal neurons in vitro, and inhibits the expression of GAD by activating the ERK1/2 signaling pathway, and this may represent one possible pathway of GABA secretion inhibition.

  15. Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    João Vitor Dutra Molino

    Full Text Available Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

  16. Secretion and activation of the Serratia marcescens hemolysin by structurally defined ShlB mutants.

    Science.gov (United States)

    Pramanik, Avijit; Könninger, Ulrich; Selvam, Arun; Braun, Volkmar

    2014-05-01

    The ShlA hemolysin of Serratia marcescens is secreted across the outer membrane by the ShlB protein; ShlB belongs to the two-partner secretion system (type Vb), a subfamily of the Omp85 outer membrane protein assembly and secretion superfamily. During secretion, ShlA is converted from an inactive non-hemolytic form into an active hemolytic form. The structure of ShlB is predicted to consist of the N-terminal α-helix H1, followed by the two polypeptide-transport-associated domains POTRA P1 and P2, and the β-barrel of 16 β-strands. H1 is inserted into the pore of the β-barrel in the outer membrane; P1 and P2 are located in the periplasm. To obtain insights into the secretion and activation of ShlA by ShlB, we isolated ShlB mutants impaired in secretion and/or activation. The triple H1 P1 P2 mutant did not secrete ShlA. The P1 and P2 deletion derivatives secreted reduced amounts of ShlA, of which P1 showed some hemolysis, whereas P2 was inactive. Deletion of loop 6 (L6), which is conserved among exporters of the Omp85 family, compromised activation but retained low secretion. Secretion-negative mutants generated by random mutagenesis were located in loop 6. The inactive secreted ShlA derivatives were complemented in vitro to active ShlA by an N-terminal ShlA fragment (ShlA242) secreted by ShlB. Deletion of H1 did not impair secretion of hemolytic ShlA. The study defines domains of ShlB which are important for ShlA secretion and activation. Copyright © 2013 Elsevier GmbH. All rights reserved.

  17. Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC.

    Directory of Open Access Journals (Sweden)

    Lucia Gonzales

    Full Text Available Enterotoxigenic Escherichia coli (ETEC cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP on the regulation of production and secretion of heat labile (LT enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

  18. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

    Science.gov (United States)

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

  19. Adrenal androgen secretion and dopaminergic activity in anorexia nervosa.

    Science.gov (United States)

    Devesa, J; Pérez-Fernández, R; Bokser, L; Gaudiero, G J; Lima, L; Casanueva, F F

    1988-01-01

    The aim of the present study was to investigate if the postulated deficient adrenal androgen secretion in Anorexia Nervosa (AN), could be associated with a status of sustained dopaminergic hyperactivity. The adrenal responses to ACTH and PRL response to dopaminergic receptor blockade were studied in seven patients with Anorexia Nervosa and seven regularly menstruating women. AN patients showed lower baseline DHEA-sulphate (DHEA-S), androstenedione (Adione) and prolactin (PRL) levels than controls. The response to ACTH revealed evidences of significantly decreased 17-20 desmolase activity in AN, with apparent predominance of glucocorticoid over androgenic pathways relative to controls. Because dopaminergic receptor blockade with Domperidone (DOM) showed intense dopaminergic hyperactivity in AN, we postulate that the adrenal regression seen in the disease is the consequence of a reduced zona reticularis as a consequence of the lack of trophic support by PRL and/or intermediate lobe proopiomelanocortin (IL-POMC). This is consistent with our previous results in pre-adrenarchal dogs and rabbits.

  20. Antifungal activity of epithelial secretions from selected frog species ...

    African Journals Online (AJOL)

    Ezedom Theresa

    2013-11-06

    Nov 6, 2013 ... Chemical stimulation gave the best yield by mass, and secretions from A. fuscigula showed the best ..... electrical stimulation (Dourado et al., 2007; Kim et al.,. 2007 .... Hematopoietic Stem Cell Transplants: An International.

  1. Glucose decouples intracellular Ca2+ activity from glucagon secretion in mouse pancreatic islet alpha-cells.

    Directory of Open Access Journals (Sweden)

    Sylvain J Le Marchand

    Full Text Available The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca(2+](i and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca(2+](i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K(ATP channel activity but not on tetrodotoxin-sensitive Na(+ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca(2+](i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K(ATP channel activity or reduction in α-cell [Ca(2+](i. Our results demonstrate that glucose uncouples the positive relationship between [Ca(2+](i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.

  2. Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

    Directory of Open Access Journals (Sweden)

    Floriane Auderset

    Full Text Available The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+ T helper (Th 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1 and Notch2 (N2 are expressed on activated CD4(+ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

  3. Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation.

    Science.gov (United States)

    Wan, Xiaojuan; Wang, Songbo; Xu, Jingren; Zhuang, Lu; Xing, Kongping; Zhang, Mengyuan; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Sun, Jiajie; Zhang, Yongliang; Li, Tiejun; Shu, Gang; Jiang, Qingyan

    2017-01-01

    Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

  4. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

    International Nuclear Information System (INIS)

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

    2012-01-01

    Highlights: ► G protein coupled receptor TGR5 is expressed in mouse and human islets. ► TGR5 is coupled to activation of Gs and Ca 2+ release via cAMP/Epac/PLC-ε pathway. ► Activation of TGR5 by bile salts and selective ligands causes insulin secretion. ► TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gα s and caused an increase in intracellular cAMP and Ca 2+ . OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gα s inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G s /cAMP/Ca 2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  5. CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

    International Nuclear Information System (INIS)

    Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh; Nagre, Nagaraja N.; Inamdar, Shashikala R.; Swamy, Bale M.; Shastry, Padma

    2012-01-01

    Highlights: ► RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. ► RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. ► Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. ► RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL–induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.

  6. Luteolin decreases invasiveness, deactivates STAT3 signaling, and reverses interleukin-6 induced epithelial–mesenchymal transition and matrix metalloproteinase secretion of pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Huang XC

    2015-10-01

    Full Text Available Xince Huang,1 Shengjie Dai,1 Juji Dai,1 Yuwu Xiao,1 Yongyu Bai,1 Bicheng Chen,1,2 Mengtao Zhou1 1Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of China; 2Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery, Wenzhou, Zhejiang Province, People’s Republic of China Abstract: Luteolin, a flavone, has been shown to exhibit anticancer properties. Here, we investigated whether luteolin affects epithelial–mesenchymal transition (EMT and invasiveness of pancreatic cancer cell lines and their underlying mechanism. Pancreatic cancer cell lines PANC-1 and SW1990 were used in our study, and their EMT characters, matrix metalloproteinase (MMP expression level, invasiveness, and signal transducer and activator of transcription 3 (STAT3 activity were determined after luteolin treatment. We also treated pancreatic cancer cells with interleukin-6 (IL-6 to see whether IL-6-induced activation of STAT3, EMT, and MMP secretion was affected by luteolin. We found that luteolin inhibits EMT and MMP2, MMP7, and MMP9 expression in a dose-dependent manner, similar to STAT3 signaling. Through Transwell assay, we found that invasiveness of pancreatic cancer cells was inhibited by luteolin. EMT characters and MMP secretion increase with STAT3 activity after IL-6 treatment and these effects, caused by IL-6, were inhibited by luteolin. We concluded that luteolin inhibits invasiveness of pancreatic cancer cells, and we speculated that luteolin inhibits EMT and MMP secretion likely through deactivation of STAT3 signaling. Luteolin has potential antitumor effects and merits further investigation. Keywords: epithelial–mesenchymal transition, matrix metalloproteinase, luteolin, STAT3

  7. AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

    Science.gov (United States)

    Neuman, Rugina I; van Kalmthout, Juliette A M; Pfau, Daniel J; Menendez, Dhariyat M; Young, Lawrence H; Forrest, John N

    2018-04-01

    The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr 172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr 172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr 172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

  8. Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

    Science.gov (United States)

    Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

    2018-03-01

    The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

  9. The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.

    Science.gov (United States)

    Jacobsen, Annette V; Murphy, James M

    2017-06-15

    Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic diversity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Loss of a neural AMP-activated kinase mimics the effects of elevated serotonin on fat, movement, and hormonal secretions.

    Directory of Open Access Journals (Sweden)

    Katherine A Cunningham

    2014-06-01

    Full Text Available AMP-activated protein kinase (AMPK is an evolutionarily conserved master regulator of metabolism and a therapeutic target in type 2 diabetes. As an energy sensor, AMPK activity is responsive to both metabolic inputs, for instance the ratio of AMP to ATP, and numerous hormonal cues. As in mammals, each of two genes, aak-1 and aak-2, encode for the catalytic subunit of AMPK in C. elegans. Here we show that in C. elegans loss of aak-2 mimics the effects of elevated serotonin signaling on fat reduction, slowed movement, and promoting exit from dauer arrest. Reconstitution of aak-2 in only the nervous system restored wild type fat levels and movement rate to aak-2 mutants and reconstitution in only the ASI neurons was sufficient to significantly restore dauer maintenance to the mutant animals. As in elevated serotonin signaling, inactivation of AAK-2 in the ASI neurons caused enhanced secretion of dense core vesicles from these neurons. The ASI neurons are the site of production of the DAF-7 TGF-β ligand and the DAF-28 insulin, both of which are secreted by dense core vesicles and play critical roles in whether animals stay in dauer or undergo reproductive development. These findings show that elevated levels of serotonin promote enhanced secretions of systemic regulators of pro-growth and differentiation pathways through inactivation of AAK-2. As such, AMPK is not only a recipient of hormonal signals but can also be an upstream regulator. Our data suggest that some of the physiological phenotypes previously attributed to peripheral AAK-2 activity on metabolic targets may instead be due to the role of this kinase in neural serotonin signaling.

  11. Bile salt receptor complex activates a pathogenic type III secretion system

    Science.gov (United States)

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N; Salomon, Dor; Tomchick, Diana R; Grishin, Nick V; Orth, Kim

    2016-01-01

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment. DOI: http://dx.doi.org/10.7554/eLife.15718.001 PMID:27377244

  12. Bile salt receptor complex activates a pathogenic type III secretion system

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; Salomon, Dor; Tomchick, Diana R.; Grishin, Nick V.; Orth, Kim

    2016-07-05

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered thatVibrio parahaemolyticusVtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.

  13. Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L

    2005-01-01

    The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are la...

  14. Mathematical Modeling of Interacting Glucose-Sensing Mechanisms and Electrical Activity Underlying Glucagon-Like Peptide 1 Secretion.

    Directory of Open Access Journals (Sweden)

    Michela Riz

    2015-12-01

    Full Text Available Intestinal L-cells sense glucose and other nutrients, and in response release glucagon-like peptide 1 (GLP-1, peptide YY and other hormones with anti-diabetic and weight-reducing effects. The stimulus-secretion pathway in L-cells is still poorly understood, although it is known that GLP-1 secreting cells use sodium-glucose co-transporters (SGLT and ATP-sensitive K+-channels (K(ATP-channels to sense intestinal glucose levels. Electrical activity then transduces glucose sensing to Ca2+-stimulated exocytosis. This particular glucose-sensing arrangement with glucose triggering both a depolarizing SGLT current as well as leading to closure of the hyperpolarizing K(ATP current is of more general interest for our understanding of glucose-sensing cells. To dissect the interactions of these two glucose-sensing mechanisms, we build a mathematical model of electrical activity underlying GLP-1 secretion. Two sets of model parameters are presented: one set represents primary mouse colonic L-cells; the other set is based on data from the GLP-1 secreting GLUTag cell line. The model is then used to obtain insight into the differences in glucose-sensing between primary L-cells and GLUTag cells. Our results illuminate how the two glucose-sensing mechanisms interact, and suggest that the depolarizing effect of SGLT currents is modulated by K(ATP-channel activity. Based on our simulations, we propose that primary L-cells encode the glucose signal as changes in action potential amplitude, whereas GLUTag cells rely mainly on frequency modulation. The model should be useful for further basic, pharmacological and theoretical investigations of the cellular signals underlying endogenous GLP-1 and peptide YY release.

  15. Perlecan Domain V induces VEGf secretion in brain endothelial cells through integrin α5β1 and ERK-dependent signaling pathways.

    Directory of Open Access Journals (Sweden)

    Douglas N Clarke

    Full Text Available Perlecan Domain V (DV promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs following stroke. In this study, we define the specific mechanism of DV interaction with the α(5β(1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV's angio-modulatory activity outside of the brain, binds poorly to α(5β(1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV's DGR sequence as an important element for the interaction of DV with α(5β(1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV's induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV's mechanism of action on BECs, and further support its potential as a novel stroke therapy.

  16. Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

    Science.gov (United States)

    Clarke, Douglas N.; Al Ahmad, Abraham; Lee, Boyeon; Parham, Christi; Auckland, Lisa; Fertala, Andrezj; Kahle, Michael; Shaw, Courtney S.; Roberts, Jill; Bix, Gregory J.

    2012-01-01

    Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy. PMID:23028886

  17. Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

    Science.gov (United States)

    Dalmas, Elise; Lehmann, Frank M; Dror, Erez; Wueest, Stephan; Thienel, Constanze; Borsigova, Marcela; Stawiski, Marc; Traunecker, Emmanuel; Lucchini, Fabrizio C; Dapito, Dianne H; Kallert, Sandra M; Guigas, Bruno; Pattou, Francois; Kerr-Conte, Julie; Maechler, Pierre; Girard, Jean-Philippe; Konrad, Daniel; Wolfrum, Christian; Böni-Schnetzler, Marianne; Finke, Daniela; Donath, Marc Y

    2017-11-21

    Pancreatic-islet inflammation contributes to the failure of β cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1β, and palmitate). IL-33 promoted β cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the β cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute β cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Naidoo, N.; Pillay, B.; Bubb, M.; Kumar, A.; Chiliza, T.; Pillay, M.

    2017-07-01

    Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli. Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a

  19. Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

    International Nuclear Information System (INIS)

    Naidoo, N.; Pillay, B.; Bubb, M.; Kumar, A.; Chiliza, T.; Pillay, M.

    2017-01-01

    Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli. Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a

  20. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    International Nuclear Information System (INIS)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-01-01

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

  1. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  2. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

    Science.gov (United States)

    Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-05-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  4. Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-α.

    Directory of Open Access Journals (Sweden)

    Suneesh Kumar Pachathundikandi

    2011-05-01

    Full Text Available Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI encoding a type-IV secretion system (T4SS for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5 during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression

  5. Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

    Directory of Open Access Journals (Sweden)

    Gary W. Cline

    2011-10-01

    Full Text Available The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet β-cells.

  6. Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid secretion in airway epithelia

    Science.gov (United States)

    Turner, Mark J.; Saint‐Criq, Vinciane; Patel, Waseema; Ibrahim, Salam H.; Verdon, Bernard; Ward, Christopher; Garnett, James P.; Tarran, Robert; Cann, Martin J.

    2015-01-01

    Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid

  7. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: skarnam@vcu.edu [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: asanyal@mcvh-vcu.edu [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  8. Pancreatic β-Cell Electrical Activity and Insulin Secretion: of Mice and Men

    Science.gov (United States)

    Rorsman, Patrik; Ashcroft, Frances M

    2018-01-01

    The pancreatic β-cell plays a key role in glucose homeostasis by secreting insulin, the only hormone capable of lowering the blood glucose concentration. Impaired insulin secretion results in the chronic hyperglycaemia that characterizes type 2 diabetes (T2DM), which currently afflicts >450 million people worldwide. The healthy β-cell acts as a glucose sensor matching its output to the circulating glucose concentration. It does so via metabolically induced changes in electrical activity, which culminate in an increase in the cytoplasmic Ca2+ concentration and initiation of Ca2+-dependent exocytosis of insulin-containing secretory granules. Here, we review recent advances in our understanding of the β-cell transcriptome, electrical activity and insulin exocytosis. We highlight salient differences between mouse and human β-cells, provide models of how the different ion channels contribute to their electrical activity and insulin secretion, and conclude by discussing how these processes become perturbed in T2DM. PMID:29212789

  9. FTO Inhibits Insulin Secretion and Promotes NF-κB Activation through Positively Regulating ROS Production in Pancreatic β cells.

    Directory of Open Access Journals (Sweden)

    Hong-Qi Fan

    Full Text Available FTO (Fat mass and obesity-associated is associated with increased risk of obesity and type 2 diabetes incurrence. Pancreas islet β cells dysfunction and insulin resistance are major causes of type 2 diabetes. However, whether FTO plays an important functional role in pancreatic β cells as well as the related molecular mechanism is still unclear. In the present study, the tissue expression profile of FTO was firstly determined using quantitative PCR and western blot. FTO is widely expressed in various tissues and presented with relative high expression in pancreas tissue, especially in endocrine pancreas. FTO overexpression in MIN6 cells achieved by lentivirus delivery significantly inhibits insulin secretion in the presence of glucose stimulus as well as KCl. FTO silence has no effect on insulin secretion of MIN6 cells. However, FTO overexpression doesn't affect the transcription of insulin gene. Furthermore, reactive oxygen species (ROS production and NF-κB activation are significantly promoted by FTO overexpression. Inhibition of intracellular ROS production by N-acetyl-L-cysteine (NAC can alleviate NF-κB activation and restore the insulin secretion mediated by FTO overexpression. A whole transcript-microarray is employed to analyze the differential gene expression mediated by FTO overexpression. The genes which are modulated by FTO are involved in many important biological pathways such as G-protein coupled receptor signaling and NF-κB signaling. Therefore, our study indicates that FTO may contribute to pancreas islet β cells dysfunction and the inhibition of FTO activity is a potential target for the treatment of diabetes.

  10. Antibacterial activity of the parotid glands secretions of sudanese indigenous african toad (Bufo spp.)

    International Nuclear Information System (INIS)

    Abugabr, H. E.; Elhussein, S. A.; Mahmoud, Z. N.

    2009-01-01

    The study revealed a well-built first line innate immunity system in Bufo Spp., the skin extracts illustrated a very active antibiotic attitude which inhibited the growth of human pathogenic bacteria species, Escherichia coli (ATCC 19615), staphylococcus aureus(ATCC29213) and streptococcus pyogenes (ATCC25922). Heat treatment of secretion supported the fact that the antibacterial components possess an enzymatic attitude. Gel filtration chromatography accompanied with sensitivity tests against S.aureus showed the existence of four groups responsible for antibacterial activity in the parotoid glands secretions.(Author)

  11. Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells.

    Science.gov (United States)

    Ishihara, Seiichiro; Inman, David R; Li, Wan-Ju; Ponik, Suzanne M; Keely, Patricia J

    2017-11-15

    In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179-89. ©2017 AACR . ©2017 American Association for Cancer Research.

  12. A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, Garry W.; Niemann, George; Baker, Erin Shammel; Belov, Mikhail E.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; McDermott, Jason E.

    2010-11-08

    Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. While it has been determined that the first 20 - 30 N-terminal residues usually contain the ‘secretion signal’ that targets effector proteins for translocation, the molecular basis for recognition of this signal is not understood. Recent machine-learning approaches, such as SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1-30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal-CyaA fusion assay and their structures systematically probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry-mass spectrometry. The signal-CyaA fusion assay showed that the native and SseJ-H fusion constructs were secreted into J774 macrophage at similar levels via the SPI-2 secretion pathway while secretion of the SseJ-L fusion construct was substantially retarded, suggesting that the SseJ secretion signal was sequence order dependent. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with only a small predisposition to adopt nascent helical structure in the presence of the powerful structure stabilizing agent, 1

  13. Antibacterial, modulatory activity of antibiotics and toxicity from Rhinella jimi (Stevaux, 2002) (Anura: Bufonidae) glandular secretions.

    Science.gov (United States)

    Sales, Débora Lima; Morais-Braga, Maria Flaviana Bezerra; Santos, Antonia Thassya Lucas Dos; Machado, Antonio Judson Targino; Araujo Filho, João Antonio de; Dias, Diógenes de Queiroz; Cunha, Francisco Assis Bezerra da; Saraiva, Rogério de Aquino; Menezes, Irwin Rose Alencar de; Coutinho, Henrique Douglas Melo; Costa, José Galberto Martins; Ferreira, Felipe Silva; Alves, Rômulo Romeu da Nóbrega; Almeida, Waltécio de Oliveira

    2017-08-01

    The increase in microorganisms with resistance to medications has caused a strong preoccupation within the medical and scientific community. Animal toxins studies, such as parotoid glandular secretions from amphibians, possesses a great potential in the development of drugs, such as antimicrobials, as these possess bioactive compounds. It was evaluated Rhinella jimi (Stevaux, 2002) glandular secretions against standard and multi-resistant bacterial strains; the effect of secretions combined with drugs; and determined the toxicity using two biologic in vivo models, and a in vitro model with mice livers. Standard strains were used for the determination of the Minimum Inhibitory Concentration (MIC), while for the modulatory activity of antibiotics, the clinical isolates Escherichia coli 06, Pseudomonas aeruginosa 03 and Staphylococcus aureus 10 were used. Modulatory activity was evaluated by the broth microdilution method with aminoglycosides and β-lactams as target antibiotics. The secretions in association with the antibiotics have a significant reduction in MIC, both the aminoglycosides and β-lactams. The toxicity and cytotoxicity results were lower than the values used in the modulation. R. jimi glandular secretions demonstrated clinically relevant results regarding the modulation of the tested antimicrobials. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

    Science.gov (United States)

    Almario, R U; Karakas, S E

    2015-02-01

    Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (pPCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

    Science.gov (United States)

    Chang, Chih-Jung; Chen, Yi-Yuan M; Lu, Chia-Chen; Lin, Chuan-Sheng; Martel, Jan; Tsai, Sheng-Hui; Ko, Yun-Fei; Huang, Tsung-Teng; Ojcius, David M; Young, John D; Lai, Hsin-Chih

    2014-04-01

    Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

  16. Platelet-activating factor increases platelet-dependent glycoconjugate secretion from tracheal submucosal gland

    International Nuclear Information System (INIS)

    Sasaki, T.; Shimura, S.; Ikeda, K.; Sasaki, H.; Takishima, T.

    1989-01-01

    Using isolated glands from feline trachea, we examined the effect of platelet-activating factor (PAF) on radiolabeled glycoconjugate release and glandular contraction by measuring induced tension in the absence or presence of platelets. PAF alone did not produce any significant glandular contraction nor any significant change in glycoconjugate release from isolated glands. In the presence of purified platelets containing no plasma, PAF (10(-8) to 10(-5) M) produced significant glycoconjugate secretion in a dose-dependent fashion, but it produced no significant glandular contraction. PAF-evoked glycoconjugate secretion was time dependent, reaching a peak response of 277% of control 15-30 min after the exposure of isolated glands to 10(-5) M PAF in the presence of platelets and returning to 135% of controls at 2 h. Platelets alone did not produce any significant stimulation in glycoconjugate release. CV-3988, a known PAF antagonist, inhibited the secretory response to PAF. Methysergide, a known antagonist to receptors for 5-hydroxytryptamine, did not alter PAF-evoked glycoconjugate secretion. Both indomethacin and SQ 29,548, a thromboxane receptor antagonist, abolished the PAF-evoked glycoconjugate secretion from isolated submucosal glands. Epithiomethanothromboxane A2, a stable thromboxane A2 analogue, produced a significant increase in glycoconjugate secretion in a dose-dependent fashion. These findings indicate that PAF increases glycoconjugate release in the presence of platelets and that the increase is dependent on some aspect of platelet function, namely thromboxane generation

  17. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    Directory of Open Access Journals (Sweden)

    Xuefei Song

    2015-01-01

    Full Text Available Purpose. The purpose of this study was to determine the impact of cross-linking (CXL on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham’s F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2 during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA. Results. Following CXL, cell viability and proliferation decreased (P0.06. Five hours after CXL, FGFb secretion increased significantly (P=0.037; however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P>0.12. Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours, normalizing after 24 hours.

  18. Mining anaerobic digester consortia metagenomes for secreted carbohydrate active enzymes

    DEFF Research Database (Denmark)

    Wilkens, Casper; Busk, Peter Kamp; Pilgaard, Bo

    thermophilic and mesophilic ADs a wide variety of carbohydrate active enzyme functions were discovered in the metagenomic sequencing of the microbial consortia. The most dominating type of glycoside hydrolases were β-glucosidases (up to 27%), α-amylases (up to 10%), α-glucosidases (up to 8%), α......, and food wastes (Alvarado et al., 2014). The processes and the roles of the microorganisms that are involved in biomass conversion and methane production in ADs are still not fully understood. We are investigating thermophilic and mesophilic ADs that use wastewater surplus sludge for methane production...... was done with the Peptide Pattern Recognition (PPR) program (Busk and Lange, 2013), which is a novel non-alignment based approach that can predict function of e.g. CAZymes. PPR identifies a set of short conserved sequences, which can be used as a finger print when mining genomes for novel enzymes. In both...

  19. Exocrine Gland-Secreting Peptide 1 Is a Key Chemosensory Signal Responsible for the Bruce Effect in Mice.

    Science.gov (United States)

    Hattori, Tatsuya; Osakada, Takuya; Masaoka, Takuto; Ooyama, Rumi; Horio, Nao; Mogi, Kazutaka; Nagasawa, Miho; Haga-Yamanaka, Sachiko; Touhara, Kazushige; Kikusui, Takefumi

    2017-10-23

    The Bruce effect refers to pregnancy termination in recently pregnant female rodents upon exposure to unfamiliar males [1]. This event occurs in specific combinations of laboratory mouse strains via the vomeronasal system [2, 3]; however, the responsible chemosensory signals have not been fully identified. Here we demonstrate that the male pheromone exocrine gland-secreting peptide 1 (ESP1) is one of the key factors that causes pregnancy block. Female mice exhibited high pregnancy failure rates upon encountering males that secreted different levels of ESP1 compared to the mated male. The effect was not observed in mice that lacked the ESP1 receptor, V2Rp5, which is expressed in vomeronasal sensory neurons. Prolactin surges in the blood after mating, which are essential for maintaining luteal function, were suppressed by ESP1 exposure, suggesting that a neuroendocrine mechanism underlies ESP1-mediated pregnancy failure. The single peptide pheromone ESP1 conveys not only maleness to promote female receptivity but also the males' characteristics to facilitate memorization of the mating partner. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A novel gene's role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior-posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos.

    Science.gov (United States)

    Khadka, Anita; Martínez-Bartolomé, Marina; Burr, Stephanie D; Range, Ryan C

    2018-01-01

    The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior-posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8-Fzl5/8-JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8-Fzl5/8-JNK signaling pathway throughout ANE restriction, providing precise

  1. A novel gene’s role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior–posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos

    Directory of Open Access Journals (Sweden)

    Anita Khadka

    2018-01-01

    Full Text Available Abstract The anterior neuroectoderm (ANE in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates is progressively restricted along the anterior–posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5 are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1, during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling to antagonize Wnt1/Wnt8–Fzl5/8–JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8–Fzl5/8–JNK signaling pathway throughout ANE

  2. Insulin secretion and signaling in response to dietary restriction and subsequent re-alimentation in cattle.

    Science.gov (United States)

    Keogh, Kate; Kenny, David A; Kelly, Alan K; Waters, Sinéad M

    2015-08-01

    The objectives of this study were to examine systemic insulin response to a glucose tolerance test (GTT) and transcript abundance of genes of the insulin signaling pathway in skeletal muscle, during both dietary restriction and re-alimentation-induced compensatory growth. Holstein Friesian bulls were blocked to one of two groups: 1) restricted feed allowance for 125 days (period 1) (RES, n = 15) followed by ad libitum feeding for 55 days (period 2) or 2) ad libitum access to feed throughout (periods 1 and 2) (ADLIB, n = 15). On days 90 and 36 of periods 1 and 2, respectively, a GTT was performed. M. longissimus dorsi biopsies were harvested from all bulls on days 120 and 15 of periods 1 and 2, respectively, and RNA-Seq analysis was performed. RES displayed a lower growth rate during period 1 (RES: 0.6 kg/day, ADLIB: 1.9 kg/day; P alimentation (RES: 2.5 kg/day, ADLIB: 1.4 kg/day; P alimentation (P > 0.05). Genes differentially expressed in the insulin signaling pathway suggested a greater sensitivity to insulin in skeletal muscle, with pleiotropic effects of insulin signaling interrupted during dietary restriction. Collectively, these results indicate increased sensitivity to glucose clearance and skeletal muscle insulin signaling during dietary restriction; however, no overall role for insulin was apparent in expressing compensatory growth. Copyright © 2015 the American Physiological Society.

  3. AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells.

    Directory of Open Access Journals (Sweden)

    Elisabeth Panther

    Full Text Available The nucleotide adenosine-5'-monophosphate (AMP can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1 and A(2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC function. AMP increased intracellular Ca(2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+CD45RA(+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene diphosphate (APCP. Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

  4. The Ewing sarcoma secretome and its response to activation of Wnt/beta-catenin signaling.

    Science.gov (United States)

    Hawkins, Allegra G; Basrur, Venkatesha; da Veiga Leprevost, Felipe; Pedersen, Elisabeth; Sperring, Colin; Nesvizhskii, Alexey I; Lawlor, Elizabeth R

    2018-01-31

    Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells upregulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic

  5. Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase.

    Science.gov (United States)

    Andersson, Lotta E; Shcherbina, Liliya; Al-Majdoub, Mahmoud; Vishnu, Neelanjan; Arroyo, Claudia Balderas; Aste Carrara, Jonathan; Wollheim, Claes B; Fex, Malin; Mulder, Hindrik; Wierup, Nils; Spégel, Peter

    2018-03-01

    Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo. © 2017 by the American Diabetes Association.

  6. Antimicrobial activity of the pygidial gland secretion of three ground beetle species (Insecta: Coleoptera: Carabidae)

    Science.gov (United States)

    Nenadić, Marija; Soković, Marina; Glamočlija, Jasmina; Ćirić, Ana; Perić-Mataruga, Vesna; Ilijin, Larisa; Tešević, Vele; Vujisić, Ljubodrag; Todosijević, Marina; Vesović, Nikola; Ćurčić, Srećko

    2016-04-01

    The antimicrobial properties of the pygidial gland secretions released by the adults of the three ground beetle species, Carabus ullrichii, C. coriaceus, and Abax parallelepipedus, have been tested. Microdilution method was applied for detection of minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), and minimal fungicidal concentrations (MFCs). Additionally, morpho-histology of the pygidial glands is investigated. We have tested 16 laboratory and clinical strains of human pathogens—eight bacterial both gram-positive and gram-negative species and eight fungal species. The pygidial secretion samples of C. ullrichii have showed the strongest antimicrobial effect against all strains of treated bacteria and fungi. Staphylococcus aureus, Lysteria monocytogenes, and Salmonella typhimurium proved to be the most sensitive bacterial strains. Penicillium funiculosum proved to be the most sensitive micromycete, while P. ochrochloron and P. verrucosum var . cyclopium the most resistant micromycetes. The pygidial secretion of C. coriaceus has showed antibacterial potential solely against Pseudomonas aeruginosa and antifungal activity against Aspergillus fumigatus, A. versicolor, A. ochraceus, and P. ochrochloron. Antibacterial properties of pygidial gland secretion of A. parallelepipedus were achieved against P. aeruginosa, while antifungal activity was detected against five of the eight tested micromycetes (A. fumigatus, A. versicolor, A. ochraceus, Trichoderma viride, and P. verrucosum var . cyclopium). Commercial antibiotics Streptomycin and Ampicillin and mycotics Ketoconazole and Bifonazole, applied as the positive controls, showed higher antibacterial/antifungal properties for all bacterial and fungal strains. The results of this observation might have a significant impact on the environmental aspects and possible medical purpose in the future.

  7. Effect of Boron on Thymic Cytokine Expression, Hormone Secretion, Antioxidant Functions, Cell Proliferation, and Apoptosis Potential via the Extracellular Signal-Regulated Kinases 1 and 2 Signaling Pathway.

    Science.gov (United States)

    Jin, Erhui; Ren, Man; Liu, Wenwen; Liang, Shuang; Hu, Qianqian; Gu, Youfang; Li, Shenghe

    2017-12-27

    Boron is an essential trace element in animals. Appropriate boron supplementation can promote thymus development; however, a high dose of boron can lead to adverse effects and cause toxicity. The influencing mechanism of boron on the animal body remains unclear. In this study, we examined the effect of boron on cytokine expression, thymosin and thymopoietin secretion, antioxidant function, cell proliferation and apoptosis, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in the thymus of rats. We found that supplementation with 10 and 20 mg/L boron to the drinking water significantly elevated levels of interleukin 2 (IL-2), interferon γ (IFN-γ), interleukin 4 (IL-4), and thymosin α1 in the thymus of rats (p boron had no apparent effect on many of the above indicators. In contrast, supplementation with 480 and 640 mg/L boron had the opposite effect on the above indicators in rats and elevated levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) (p boron to the drinking water had a U-shaped dose-effect relationship with thymic cytokine expression, hormone secretion, antioxidant function, cell proliferation, and apoptosis. Specifically, supplementation with 10 and 20 mg/L boron promoted thymocyte proliferation and enhanced thymic functions. However, supplementation with 480 and 640 mg/L boron inhibited thymic functions and increased the number of apoptotic thymocytes, suggesting that the effects of boron on thymic functions may be caused via the ERK1/2 signaling pathway.

  8. Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism.

    Science.gov (United States)

    Campion, Katherine L; McCormick, Wanda D; Warwicker, Jim; Khayat, Mohd Ezuan Bin; Atkinson-Dell, Rebecca; Steward, Martin C; Delbridge, Leigh W; Mun, Hee-Chang; Conigrave, Arthur D; Ward, Donald T

    2015-09-01

    The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca(2+)i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca(2+)o). Similar pHo effects were observed for Ca(2+)o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca(2+)i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo. Copyright © 2015 by the American Society of

  9. Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines

    International Nuclear Information System (INIS)

    Razonable, Raymund R.; Henault, Martin; Paya, Carlos V.

    2006-01-01

    The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-κB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-κB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-κB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study

  10. Covert radar and signals interception the secret career of Eric Ackermann

    CERN Document Server

    Hayson, David

    2014-01-01

    Of German stock dating back to 1530 in Saxony, Eric George Ackermann GM was born on the Isle of Wight in 1919 and became a leading figure in the world of signals and electronic intelligence. As a Junior Scientific Officer at the Telecommunication Research Establishment, Boscombe Down, with an honorary commission in the Royal Air Force, he made numerous flights over occupied territory searching for, monitoring and destroying Germany's Wuerzburg, Knickebein and X Band radar systems. Much of his research was passed to the highest levels of wartime government, and was highly prized, ensuring that

  11. The Secret Life of NAD(+): An Old Metabolite Controlling New Metabolic Signaling Pathways

    NARCIS (Netherlands)

    Houtkooper, Riekelt H.; Cantó, Carles; Wanders, Ronald J.; Auwerx, Johan

    2010-01-01

    A century after the identification of a coenzymatic activity for NAD(+), NAD(+) metabolism has come into the spotlight again due to the potential therapeutic relevance of a set of enzymes whose activity is tightly regulated by the balance between the oxidized and reduced forms of this metabolite. In

  12. Activation of PPARd and RXRa stimulates fatty acid oxidatin and insulin secretion inpancreatic beta-cells

    DEFF Research Database (Denmark)

    Børgesen, Michael; Ravnskjær, Kim; Frigerio, Francesca

    as a central effector of unsaturated fatty acids in pancreatic ß-cells. Interestingly, activation of PPARd increases basal as well as glucose-stimulated insulin secretion of INS-1E cells. This increase is further potentiated by RXR agonists. This observation suggests that PPARd may mediate some of the positive......ACTIVATION OF PPARd AND RXRa STIMULATES FATTY ACID OXIDATION AND INSULIN SECRETION IN PANCREATIC b-CELLS Michael Boergesen1, Kim Ravnskjaer2, Francesca Frigerio3, Allan E. Karlsen4, Pierre Maechler3 and Susanne Mandrup1 1 Department of Biochemistry and Molecular Biology, University of Southern...... of genes as PPARd specific agonists and stimulates ß-oxidation. Importantly, oleate-induction of gene expression and ß-oxidation in INS-1E cells is abolished by knock-down of PPARd using adenoviral transfer of shRNA. Thus, PPARd appears to be a central regulator of fatty acid metabolism as well...

  13. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    OpenAIRE

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell...

  14. Sweet Taste Receptor Activation in the Gut Is of Limited Importance for Glucose-Stimulated GLP-1 and GIP Secretion

    DEFF Research Database (Denmark)

    Saltiel, Monika Yosifova; Kuhre, Rune Ehrenreich; Christiansen, Charlotte Bayer

    2017-01-01

    Glucose stimulates the secretion of the incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is debated whether the sweet taste receptor (STR) triggers this secretion. We investigated the role of STR activation for glucose-stimulated incretin...

  15. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    Science.gov (United States)

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

  16. Autophagy Inhibition Contributes to ROS-Producing NLRP3-Dependent Inflammasome Activation and Cytokine Secretion in High Glucose-Induced Macrophages.

    Science.gov (United States)

    Dai, Jiezhi; Zhang, Xiaotian; Li, Li; Chen, Hua; Chai, Yimin

    2017-01-01

    Type 2 diabetes is a persistent inflammatory response that impairs the healing process. We hypothesized that stimulation with high glucose following a pro-inflammatory signal would lead to autophagy inhibition, reactive oxygen species (ROS) production and eventually to the activation of the Nod-like receptor protein (NLRP) -3. Macrophages were isolated from human diabetic wound. We measured the expression of NLRP3, caspase1 and interleukin-1 beta (IL-1β) by western blot and real-time PCR, and the surface markers on cells by flow cytometry. THP-1-derived macrophages exposed to high glucose were applied to study the link between autophagy, ROS and NLRP3 activation. LC3-II, P62, NLRP3 inflammation and IL-1β expression were measured by western blot and real-time PCR. ROS production was measured with a Cellular Reactive Oxygen Species Detection Assay Kit. Macrophages isolated from diabetic wounds exhibited a pro-inflammatory phenotype, including sustained NLRP3 inflammasome activity associated with IL-1β secretion. Our data showed that high glucose inhibited autophagy, induced ROS production, and activated NLRP3 inflammasome and cytokine secretion in THP-1-derived macrophages. To study high glucose-induced NLRP3 inflammasome signalling, we performed studies using an autophagy inducer, a ROS inhibitor and a NLRP3 inhibitor and found that all reduced the NLRP3 inflammasome activation and cytokine secretion. Sustained NLRP3 inflammasome activity in wound-derived macrophages contributes to the hyper-inflammation in human diabetic wounds. Autophagy inhibition and ROS generation play an essential role in high glucose-induced NLRP3 inflammasome activation and cytokine secretion in macrophages. © 2017 The Author(s). Published by S. Karger AG, Basel.

  17. Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

    Science.gov (United States)

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2015-12-26

    To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced

  18. AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

    Science.gov (United States)

    Panther, Elisabeth; Dürk, Thorsten; Ferrari, Davide; Di Virgilio, Francesco; Grimm, Melanie; Sorichter, Stephan; Cicko, Sanja; Herouy, Yared; Norgauer, Johannes; Idzko, Marco; Müller, Tobias

    2012-01-01

    The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders. PMID:22624049

  19. Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; Bjelobaba, I.; Zemková, Hana

    2017-01-01

    Roč. 8, Jun 9 (2017), č. článku 126. ISSN 1664-2392 R&D Projects: GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA16-12695S Institutional support: RVO:67985823 Keywords : gonadotrophs * voltage-gated channels * ligand-gated channels * electrical activity Subject RIV: FH - Neurology OBOR OECD: Physiology (including cytology) Impact factor: 3.675, year: 2016

  20. Wise, a context-dependent activator and inhibitor of Wnt signalling.

    Science.gov (United States)

    Itasaki, Nobue; Jones, C Michael; Mercurio, Sara; Rowe, Alison; Domingos, Pedro M; Smith, James C; Krumlauf, Robb

    2003-09-01

    We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

  1. Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

    Directory of Open Access Journals (Sweden)

    Maarke J.E. Roelofs

    2014-01-01

    Full Text Available Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO, fluconazole (FLUC, flusilazole (FLUS, hexaconazole (HEXA, myconazole (MYC, penconazole (PEN, prochloraz (PRO, tebuconazole (TEBU, triadimefon (TRIA, and triticonazole (TRIT were examined using murine Leydig (MA-10 cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM or TEBU (IC50 = 2.4 μM in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM and effect potencies (REPs were calculated relative to the known AR antagonist flutamide (FLUT. FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61 and MYC the least potent (REP = 0.03 AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human risk assessment of this class of compounds.

  2. Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa

    OpenAIRE

    Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

    2017-01-01

    Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, bu...

  3. Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

    Directory of Open Access Journals (Sweden)

    Ailín C Rogers

    Full Text Available Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR, is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK, can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK. In order to substantiate our findings on the whole tissue level, short-circuit current (SCC was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

  4. Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa.

    Science.gov (United States)

    Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

    2017-06-30

    Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, but never with P-PIV. Since the activity of M-PIV was restored by adding the CS of QS-positive and PIV-deficient strain, we hypothesized that the propeptide binds to and inhibits PIV, and is degraded to activate PIV by a QS-dependent factor. In fact, the CS of the QS-positive and PIV-deficient strain was able to degrade the propeptide. Since the responsible factor should be a QS-dependently expressed extracellular protease, we tested QS-dependent proteases of P. aeruginosa and found that LasB (elastase) can degrade the propeptide and activate M-PIV. We purified the propeptide of PIV and confirmed that the propeptide can bind to and inhibit PIV. We suggest that PIV is post-secretionally activated through the extracellular degradation of the propeptide by LasB, a QS-dependent protease.

  5. A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

    Science.gov (United States)

    Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A; Schmid, Michael C; Schröder, Gunnar; Vergunst, Annette C; Carena, Ilaria; Dehio, Christoph

    2005-01-18

    Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

  6. Egg cell signaling by the secreted peptide ZmEAL1 controls antipodal cell fate.

    Science.gov (United States)

    Krohn, Nadia Graciele; Lausser, Andreas; Juranić, Martina; Dresselhaus, Thomas

    2012-07-17

    Unlike in animals, female gametes of flowering plants are not the direct products of meiosis but develop from a functional megaspore after three rounds of free mitotic divisions. After nuclei migration and positioning, the eight-nucleate syncytium differentiates into the embryo sac, which contains two female gametes as well as accessory cells at the micropylar and chalazal pole, respectively. We report that an egg-cell-specific gene, ZmEAL1, is activated at the micropylar pole of the eight-nucleate syncytium. ZmEAL1 translation is restricted to the egg cell, resulting in the generation of peptide-containing vesicles directed toward its chalazal pole. RNAi knockdown studies show that ZmEAL1 is required for robust expression of the proliferation-regulatory gene IG1 at the chalazal pole of the embryo sac in antipodal cells. We further show that ZmEAL1 is required to prevent antipodal cells from adopting central cell fate. These findings show how egg cells orchestrate differentiation of the embryo sac. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Signal detection by active, noisy hair bundles

    Science.gov (United States)

    O'Maoiléidigh, Dáibhid; Salvi, Joshua D.; Hudspeth, A. J.

    2018-05-01

    Vertebrate ears employ hair bundles to transduce mechanical movements into electrical signals, but their performance is limited by noise. Hair bundles are substantially more sensitive to periodic stimulation when they are mechanically active, however, than when they are passive. We developed a model of active hair-bundle mechanics that predicts the conditions under which a bundle is most sensitive to periodic stimulation. The model relies only on the existence of mechanotransduction channels and an active adaptation mechanism that recloses the channels. For a frequency-detuned stimulus, a noisy hair bundle's phase-locked response and degree of entrainment as well as its detection bandwidth are maximized when the bundle exhibits low-amplitude spontaneous oscillations. The phase-locked response and entrainment of a bundle are predicted to peak as functions of the noise level. We confirmed several of these predictions experimentally by periodically forcing hair bundles held near the onset of self-oscillation. A hair bundle's active process amplifies the stimulus preferentially over the noise, allowing the bundle to detect periodic forces less than 1 pN in amplitude. Moreover, the addition of noise can improve a bundle's ability to detect the stimulus. Although, mechanical activity has not yet been observed in mammalian hair bundles, a related model predicts that active but quiescent bundles can oscillate spontaneously when they are loaded by a sufficiently massive object such as the tectorial membrane. Overall, this work indicates that auditory systems rely on active elements, composed of hair cells and their mechanical environment, that operate on the brink of self-oscillation.

  8. The Effect of Bad Human Activities on Marine Life as Portrayed in Sammy's Adventure: the Secret Passage

    OpenAIRE

    LATHIFAH, ISNA NUR

    2015-01-01

    Keywords: ecocriticism, bad humans activities, marine life, Sammy's Adventure: the Secret Passage movie. The balance of marine life is often damaged by irresponsible humans who do not care about their environment. This problem has inspired some works to criticize humans' reckless behavior toward environment, especially ocean. Sammy's Adventure: the Secret Passage is one of the examples that have been created to criticize the bad human activities in the ocean. This research applies ecocritici...

  9. Plant flavones enhance antimicrobial activity of respiratory epithelial cell secretions against Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Benjamin M Hariri

    Full Text Available Flavones are a class of natural plant secondary metabolites that have anti-inflammatory and anti-bacterial effects. Some flavones also activate the T2R14 bitter taste receptor, which is expressed in motile cilia of the sinonasal epithelium and activates innate immune nitric oxide (NO production. Flavones may thus be potential therapeutics for respiratory infections. Our objective was to examine the anti-microbial effects of flavones on the common sinonasal pathogens Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, evaluating both planktonic and biofilm growth. Flavones had only very low-level antibacterial activity alone. They did not reduce biofilm formation, but did reduce production of the important P. aeruginosa inflammatory mediator and ciliotoxin pyocyanin. However, flavones exhibited synergy against P. aeruginosa in the presence of antibiotics or recombinant human lysozyme. They also enhanced the efficacy of antimicrobials secreted by cultured and primary human airway cells grown at air-liquid interface. This suggests that flavones may have anti-gram-negative potential as topical therapeutics when combined with antibiotics or in the context of innate antimicrobials secreted by the respiratory or other epithelia. This may have an additive effect when combined with T2R14-activated NO production. Additional studies are necessary to understand which flavone compounds or mixtures are the most efficacious.

  10. Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

    Science.gov (United States)

    Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

    2018-04-01

    Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

  11. Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Benjamin Mielich-Süss

    2017-11-01

    Full Text Available Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM, a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.

  12. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

    Science.gov (United States)

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  13. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway

    DEFF Research Database (Denmark)

    Abdallah, Basem; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-01-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchyma...

  14. Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1

    DEFF Research Database (Denmark)

    Frödin, M; Sekine, N; Roche, E

    1995-01-01

    The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways...... converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues......-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation...

  15. Hoopoes color their eggs with antimicrobial uropygial secretions

    Science.gov (United States)

    Soler, Juan J.; Martín-Vivaldi, M.; Peralta-Sánchez, J. M.; Arco, L.; Juárez-García-Pelayo, N.

    2014-09-01

    Uropygial gland secretions are used as cosmetics by some species of birds to color and enhance properties of feathers and teguments, which may signal individual quality. Uropygial secretions also reach eggshells during incubation and, therefore, may influence the coloration of birds' eggs, a trait that has attracted the attention of evolutionary biologists for more than one century. The color of hoopoe eggs typically changes along incubation, from bluish-gray to greenish-brown. Here, we test experimentally the hypothesis that dark uropygial secretion of females is responsible for such drastic color change. Moreover, since uropygial secretion of hoopoes has antimicrobial properties, we also explore the association between color and antimicrobial activity of the uropygial secretion of females. We found that eggs stayed bluish-gray in nests where female access to the uropygial secretion was experimentally blocked. Furthermore, experimental eggs that were maintained in incubators and manually smeared with uropygial secretion experienced similar color changes that naturally incubated eggs did, while control eggs that were not in contact with the secretions did not experience such color changes. All these results strongly support the hypothesis that female hoopoes use their uropygial gland secretion to color the eggs. Moreover, saturation of the uropygial secretion was associated with antimicrobial activity against Bacillus licheniformis. Given the known antimicrobial potential of uropygial secretions of birds, this finding opens the possibility that in scenarios of sexual selection, hoopoes in particular and birds in general signal antimicrobial properties of their uropygial secretion by mean of changes in egg coloration along incubation.

  16. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif.

    Directory of Open Access Journals (Sweden)

    Mei Ying Ng

    Full Text Available Cycle inhibiting factors (Cifs are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

  17. GnRH-induced Ca2+ signaling patterns and gonadotropin secretion in pituitary gonadotrophs. Functional adaptations to both ordinary and extraordinary physiological demands

    Directory of Open Access Journals (Sweden)

    María Luisa eDurán-Pastén

    2013-09-01

    Full Text Available Pituitary gonadotrophs are a small fraction of the anterior pituitary population, yet they synthesize gonadotropins: luteinizing (LH and follicle stimulating (FSH, essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca2+ rises produced do not influence secretion, which is mainly driven by Gonadotropin Releasing Hormone (GnRH, a decapeptide synthesized in the hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation. GnRH binding to G protein coupled receptors triggers Ca2+ mobilization from InsP3-sensitive intracellular pools, generating the global Ca2+ elevations necessary for secretion. Ca2+ signaling responses to increasing [GnRH] vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory, to biphasic (spike-oscillatory or spike-plateau. This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca2+ signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases and Ca2+ signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary

  18. Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

    Science.gov (United States)

    Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

    2009-07-01

    The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

  19. Active voltammetric microsensors with neural signal processing.

    Energy Technology Data Exchange (ETDEWEB)

    Vogt, M. C.

    1998-12-11

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical ''signatures'' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration, the calibration, sensing, and processing methods of these active voltammetric microsensors can

  20. Active voltammetric microsensors with neural signal processing

    Science.gov (United States)

    Vogt, Michael C.; Skubal, Laura R.

    1999-02-01

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical 'signatures' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration; the calibration, sensing, and processing methods of these active voltammetric microsensors can detect, recognize, and

  1. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  2. An Evolutionarily Conserved Network Mediates Development of the zona limitans intrathalamica, a Sonic Hedgehog-Secreting Caudal Forebrain Signaling Center

    Directory of Open Access Journals (Sweden)

    Elena Sena

    2016-10-01

    Full Text Available Recent studies revealed new insights into the development of a unique caudal forebrain-signaling center: the zona limitans intrathalamica (zli. The zli is the last brain signaling center to form and the first forebrain compartment to be established. It is the only part of the dorsal neural tube expressing the morphogen Sonic Hedgehog (Shh whose activity participates in the survival, growth and patterning of neuronal progenitor subpopulations within the thalamic complex. Here, we review the gene regulatory network of transcription factors and cis-regulatory elements that underlies formation of a shh-expressing delimitated domain in the anterior brain. We discuss evidence that this network predates the origin of chordates. We highlight the contribution of Shh, Wnt and Notch signaling to zli development and discuss implications for the fact that the morphogen Shh relies on primary cilia for signal transduction. The network that underlies zli development also contributes to thalamus induction, and to its patterning once the zli has been set up. We present an overview of the brain malformations possibly associated with developmental defects in this gene regulatory network (GRN.

  3. Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection.

    Directory of Open Access Journals (Sweden)

    Jorge Rubén Cabrera

    2015-01-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG from HSV-2, known to modulate immune mediators (chemokines, also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2 increases nerve growth factor (NGF-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (TrkA-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems.

  4. Activity of Genital Tract Secretions and Synthetic Antimicrobial Peptides against Group B Streptococcus.

    Science.gov (United States)

    Agarwal, Nidhi; Buckley, Niall; Nakra, Natasha; Gialanella, Philip; Yuan, Weirong; Ghartey, Jeny P

    2015-12-01

    Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. None of the CVL samples inhibited GBS. The human neutrophil peptide-1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 μg/mL and ≥50 μg/mL, respectively, while human beta-defensin 2 and LL-37 did not inhibit at highest concentration tested (100 μg/mL). In contrast, all AMP inhibited E. coli. Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Osteoclast TGF-β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

    Science.gov (United States)

    Weivoda, Megan M; Ruan, Ming; Pederson, Larry; Hachfeld, Christine; Davey, Rachel A; Zajac, Jeffrey D; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. PMID:26108893

  6. PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Hyo-Sup Kim

    Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

  7. High levels of DegU-P activate an Esat-6-like secretion system in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Catarina Baptista

    Full Text Available The recently discovered Type VII/Esat-6 secretion systems seem to be widespread among bacteria of the phyla Actinobacteria and Firmicutes. In some species they play an important role in pathogenic interactions with eukaryotic hosts. Several studies have predicted that the locus yukEDCByueBC of the non-pathogenic, Gram-positive bacterium Bacillus subtilis would encode an Esat-6-like secretion system (Ess. We provide here for the first time evidences for the functioning of this secretion pathway in an undomesticated B. subtilis strain. We show that YukE, a small protein with the typical features of the secretion substrates from the WXG100 superfamily is actively secreted to culture media. YukE secretion depends on intact yukDCByueBC genes, whose products share sequence or structural homology with known components of the S. aureus Ess. Biochemical characterization of YukE indicates that it exists as a dimer both in vitro and in vivo. We also show that the B. subtilis Ess essentially operates in late stationary growth phase in absolute dependence of phosphorylated DegU, the response regulator of the two-component system DegS-DegU. We present possible reasons that eventually have precluded the study of this secretion system in the B. subtilis laboratory strain 168.

  8. Distinct licensing of IL-18 and IL-1β secretion in response to NLRP3 inflammasome activation.

    Directory of Open Access Journals (Sweden)

    Rebecca L Schmidt

    Full Text Available Inflammasome activation permits processing of interleukins (IL-1β and 18 and elicits cell death (pyroptosis. Whether these responses are independently licensed or are "hard-wired" consequences of caspase-1 (casp1 activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a "non-canonical" stimulus, the secreted Listeria monocytogenes (Lm p60 protein. Primed murine dendritic cells (DCs responded to p60 stimulation with reactive oxygen species (ROS production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.

  9. Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Hadil F Al-Jallad

    2011-01-01

    Full Text Available Transglutaminase activity, arising potentially from transglutaminase 2 (TG2 and Factor XIIIA (FXIIIA, has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

  10. Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine

    DEFF Research Database (Denmark)

    Christensen, Louise Wulff; Kuhre, Rune Ehrenreich; Janus, Charlotte

    2015-01-01

    -protein-cou- pled receptor, FFAR1 (previously GPR40), expressed on L cells and activated by long-chain fatty acids (LCFAs) is a potential target. A link between FFAR1 activation and GLP-1 secretion has been demonstrated in cellular models and small-molecule FFAR1 agonists have been developed. In this study, we exam......- ined the effect of FFAR1 activation on GLP-1 secretion using isolated, per- fused small intestines from rats, a physiologically relevant model allowing distinction between direct and indirect effects of FFAR1 activation. The endogenous FFAR1 ligand, linoleic acid (LA), and four synthetic FFAR1 ago...

  11. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Science.gov (United States)

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-09-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  12. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  13. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  14. Lactobacillus proteins are associated with the bactericidal activity against E. coli of female genital tract secretions.

    Directory of Open Access Journals (Sweden)

    Sabah Kalyoussef

    Full Text Available Female genital tract secretions are bactericidal for Escherichia (E. coli ex vivo. However, the intersubject variability and molecules that contribute to this activity have not been defined.The bactericidal activity and concentration of immune mediators in cervicovaginal lavage (CVL collected from 99 healthy women were determined.CVL reduced the number of E. coli colonies by 68% [-26, 100] (median [range]. CVL were active against laboratory and clinical isolates of E. coli, but were inactive against Lactobacillus species. Bactericidal activity correlated with the concentration of protein recovered (p90% inhibitory activity (active and two with<30% activity were subjected to MS/MS proteomic analysis. 215 proteins were identified and six were found exclusively in active samples. Four of these corresponded to Lactobacillus crispatus or jensenii proteins. Moreover, culture supernatants from Lactobacillus jensenii were bactericidal for E. coli.Both host and commensal microbiota proteins contribute to mucosal defense. Identification of these proteins will facilitate the development of strategies to maintain a healthy vaginal microbiome and prevent colonization with pathogenic bacteria such as E. coli that increase the risk for urinary tract infections, preterm labor and perinatal infection.

  15. A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH-stimulated luteinizing hormone (LH secretion

    Directory of Open Access Journals (Sweden)

    Musgrove Lois C

    2001-11-01

    Full Text Available Abstract Background Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs. These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. Results A truncated version of RGS3 (RGS3T = RGS3 314–519 inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein. Conclusions RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino

  16. GLP-1 secretion is stimulated by 1,10-phenanthroline via colocalized T2R5 signal transduction in human enteroendocrine L cell

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jiyoung; Kim, Ki-Suk; Kim, Kang-Hoon; Lee, In-Seung; Jeong, Hyeon-soo; Kim, Yumi; Jang, Hyeung-Jin, E-mail: hjjang@khu.ac.kr

    2015-12-04

    Glucagon-like peptide-1 (GLP-1) hormone is known to regulate blood glucose by an insulinotropic effect and increases proliferation as and also prevents apoptosis of pancreatic β cells. We know that GLP-1 is secreted by nutrients such as fatty acids and sweet compounds but also bitter compounds via stimulation of G-protein coupled receptors (GPCRs) in the gut. Among these, bitter compounds are multiply-contained in phytochemicals or artificial materials and perceived as ligands of various bitter taste receptors. We hypothesized that GLP-1 hormone is secreted through stimulation of a single bitter taste receptor by 1,10-phenanthroline which is known agonist of taste receptor type 2 member 5 (T2R5). To prove this hypothesis, we used the representatively well-known 1,10-phenanthroline as ligand of single receptor and evaluated the existence of T2R5 by double-labeling immunofluorescence and then 1,10-phenanthroline is able to secrete GLP-1 hormone through stimulation of T2R5 in human enteroendocrine cells. Consequently, we verify that GLP-1 hormone is colocalized with T2R5 in the human duodenum and ileum tissue and is secreted by 1,10-phenanthroline via T2R5 signal transduction in differentiated human enteroendocrine L cells. - Highlights: • Taste receptor type 2 member 5 (T2R5) is colocalized with GLP-1 hormone in human enteroendocrine cells. • GLP-1 secretion is stimulated by 1,10-phenanthroline via stimulation of T2R5. • Inhibition of the bitter taste pathway reduce GLP-1 secretion.

  17. GLP-1 secretion is stimulated by 1,10-phenanthroline via colocalized T2R5 signal transduction in human enteroendocrine L cell

    International Nuclear Information System (INIS)

    Park, Jiyoung; Kim, Ki-Suk; Kim, Kang-Hoon; Lee, In-Seung; Jeong, Hyeon-soo; Kim, Yumi; Jang, Hyeung-Jin

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) hormone is known to regulate blood glucose by an insulinotropic effect and increases proliferation as and also prevents apoptosis of pancreatic β cells. We know that GLP-1 is secreted by nutrients such as fatty acids and sweet compounds but also bitter compounds via stimulation of G-protein coupled receptors (GPCRs) in the gut. Among these, bitter compounds are multiply-contained in phytochemicals or artificial materials and perceived as ligands of various bitter taste receptors. We hypothesized that GLP-1 hormone is secreted through stimulation of a single bitter taste receptor by 1,10-phenanthroline which is known agonist of taste receptor type 2 member 5 (T2R5). To prove this hypothesis, we used the representatively well-known 1,10-phenanthroline as ligand of single receptor and evaluated the existence of T2R5 by double-labeling immunofluorescence and then 1,10-phenanthroline is able to secrete GLP-1 hormone through stimulation of T2R5 in human enteroendocrine cells. Consequently, we verify that GLP-1 hormone is colocalized with T2R5 in the human duodenum and ileum tissue and is secreted by 1,10-phenanthroline via T2R5 signal transduction in differentiated human enteroendocrine L cells. - Highlights: • Taste receptor type 2 member 5 (T2R5) is colocalized with GLP-1 hormone in human enteroendocrine cells. • GLP-1 secretion is stimulated by 1,10-phenanthroline via stimulation of T2R5. • Inhibition of the bitter taste pathway reduce GLP-1 secretion.

  18. Promises and Challenges in Continuous Tracking Utilizing Amino Acids in Skin Secretions for Active Multi-Factor Biometric Authentication for Cybersecurity.

    Science.gov (United States)

    Agudelo, Juliana; Privman, Vladimir; Halámek, Jan

    2017-07-05

    We consider a new concept of biometric-based cybersecurity systems for active authentication by continuous tracking, which utilizes biochemical processing of metabolites present in skin secretions. Skin secretions contain a large number of metabolites and small molecules that can be targeted for analysis. Here we argue that amino acids found in sweat can be exploited for the establishment of an amino acid profile capable of identifying an individual user of a mobile or wearable device. Individual and combinations of amino acids processed by biocatalytic cascades yield physical (optical or electronic) signals, providing a time-series of several outputs that, in their entirety, should suffice to authenticate a specific user based on standard statistical criteria. Initial results, motivated by biometrics, indicate that single amino acid levels can provide analog signals that vary according to the individual donor, albeit with limited resolution versus noise. However, some such assays offer digital separation (into well-defined ranges of values) according to groups such as age, biological sex, race, and physiological state of the individual. Multi-input biocatalytic cascades that handle several amino acid signals to yield a single digital-type output, as well as continuous-tracking time-series data rather than a single-instance sample, should enable active authentication at the level of an individual. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences

    DEFF Research Database (Denmark)

    Jabobs, Myra F; Andersen, Jens Bo; Kontinen, Vesa P.

    1993-01-01

    In prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including alpha-amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted...

  20. Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, P.C.; Rossi, N.F.; Churchill, M.C.; Ellis, V.R. (Wayne State Univ. School of Medicine, Detroit, MI (USA))

    1990-01-01

    There is evidence that three inhibitors of Na,K-ATPase activity-ouabain, K-free extracellular fluid, and vanadate--inhibit renin secretion by increasing Ca{sup 2+} concentration in juxtaglomerular cells, but in the case of vanadate, it is uncertain whether the increase in Ca{sup 2+} is due to a decrease in Ca{sup 2+} efflux or to an increase in Ca{sup 2+} influx through potential operated Ca channels. In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free fluid, and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, A Ca channel blocker. Basal renin secretory rate averaged 7.7 {plus minus} 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by K-free fluid, by 0.5 mM vanadate, and by K-depolarization. Although 0.5 {mu}M methoxyverapamil completely blocked the inhibitory effect of K-depolarization, it failed to antagonize the inhibitory effects of ouabain, of K-free fluid, and of vanadate.

  1. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    Science.gov (United States)

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  2. Innate immunity in the vagina (Part II): Anti-HIV activity and antiviral content of human vaginal secretions.

    Science.gov (United States)

    Patel, Mickey V; Ghosh, Mimi; Fahey, John V; Ochsenbauer, Christina; Rossoll, Richard M; Wira, Charles R

    2014-07-01

    Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions change across the menstrual cycle is unknown. Using a menstrual cup, vaginal secretions from pre-menopausal women were recovered at the proliferative (d6-8), mid-cycle (d13-15), and secretory (d21-23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. CCL20, RANTES, elafin, HBD2, SDF-1α, and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women and in consecutive cycles from the same woman. The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and interindividual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life. © 2014 John Wiley & Sons Ltd.

  3. Immunity in the Vagina (Part II): Anti-HIV Activity and Antiviral Content of Human Vaginal Secretions

    Science.gov (United States)

    Patel, Mickey V.; Ghosh, Mimi; Fahey, John V.; Ochsenbauer, Christina; Rossoll, Richard M.; Wira, Charles R.

    2015-01-01

    Problem Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions changes across the menstrual cycle is unknown. Method of Study Using a menstrual cup, vaginal secretions from premenopausal women were recovered at the proliferative (d6–8), mid-cycle (d13–15) and secretory (d21–23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. Results CCL20, RANTES, elafin, HBD2, SDF-1α and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women, and in consecutive cycles from the same woman. Conclusion The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and inter-individual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life. PMID:24806967

  4. Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors.

    Science.gov (United States)

    Haugen, Martin; Dammen, Rikard; Svejda, Bernhard; Gustafsson, Bjorn I; Pfragner, Roswitha; Modlin, Irvin; Kidd, Mark

    2012-11-15

    The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (∼75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.

  5. Arsenite evokes IL-6 secretion, autocrine regulation of STAT3 signaling, and miR-21 expression, processes involved in the EMT and malignant transformation of human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Luo, Fei; Xu, Yuan; Ling, Min; Zhao, Yue; Xu, Wenchao; Liang, Xiao; Jiang, Rongrong; Wang, Bairu; Bian, Qian; Liu, Qizhan

    2013-01-01

    Arsenite is an established human carcinogen, and arsenite-induced inflammation contributes to malignant transformation of cells, but the molecular mechanisms by which cancers are produced remain to be established. The present results showed that, evoked by arsenite, secretion of interleukin-6 (IL-6), a pro-inflammatory cytokine, led to the activation of STAT3, a transcription activator, and to increased levels of a microRNA, miR-21. Blocking IL-6 with anti-IL-6 antibody and inhibiting STAT3 activation reduced miR-21 expression. For human bronchial epithelial cells, cultured in the presence of anti-IL-6 antibody for 3 days, the arsenite-induced EMT and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates miR-21in an autocrine manner, contributes to the EMT induced by arsenite. These data define a link from inflammation to EMT in the arsenite-induced malignant transformation of HBE cells. This link, mediated through miRNAs, establishes a mechanism for arsenite-induced lung carcinogenesis. - Highlights: • Arsenite evokes IL-6 secretion. • IL-6 autocrine mediates STAT3 signaling and up-regulates miR-21expression. • Inflammation is involved in arsenite-induced EMT

  6. Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

    Science.gov (United States)

    Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

    2017-01-01

    Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed. PMID:27845895

  7. Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

    Science.gov (United States)

    Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

    2017-03-21

    Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

  8. Glomerular parietal epithelial cell activation induces collagen secretion and thickening of Bowman's capsule in diabetes.

    Science.gov (United States)

    Holderied, Alexander; Romoli, Simone; Eberhard, Jonathan; Konrad, Lukas A; Devarapu, Satish K; Marschner, Julian A; Müller, Susanna; Anders, Hans-Joachim

    2015-03-01

    The metabolic and hemodynamic alterations in diabetes activate podocytes to increase extracellular matrix (ECM) production, leading to thickening of the glomerular basement membrane (GBM). We hypothesized that diabetes would activate parietal epithelial cells (PECs) in a similar manner and cause thickening of Bowman's capsules. Periodic acid Schiff staining of human kidney biopsies of 30 patients with diabetic nephropathy (DN) revealed a significantly thicker Bowman's capsule as compared with 20 non-diabetic controls. The average thickness was 4.55±0.21 μm in the group of patients with DN compared with 2.92±0.21 μm in the group of non-diabetic controls (PBowman's capsule showed strong association with CD44-positive PECs. In summary, metabolic alterations in diabetes activate PECs to increase the expression and secretion of Bowman's capsule proteins. This process may contribute to the thickening of the Bowman's capsule, similar to the thickening of the GBM that is driven by activated podocytes. These data may also imply that activated PECs contribute to ECM production once they migrate to the glomerular tuft, a process resulting in glomerular scaring, for example, in diabetic glomerulosclerosis.

  9. The Central Metabolism Regulator EIIAGlc Switches Salmonella from Growth Arrest to Acute Virulence through Activation of Virulence Factor Secretion

    Directory of Open Access Journals (Sweden)

    Alain Mazé

    2014-06-01

    Full Text Available The ability of Salmonella to cause disease depends on metabolic activities and virulence factors. Here, we show that a key metabolic protein, EIIAGlc, is absolutely essential for acute infection, but not for Salmonella survival, in a mouse typhoid fever model. Surprisingly, phosphorylation-dependent EIIAGlc functions, including carbohydrate transport and activation of adenylate cyclase for global regulation, do not explain this virulence phenotype. Instead, biochemical studies, in vitro secretion and translocation assays, and in vivo genetic epistasis experiments suggest that EIIAGlc binds to the type three secretion system 2 (TTSS-2 involved in systemic virulence, stabilizes its cytoplasmic part including the crucial TTSS-2 ATPase, and activates virulence factor secretion. This unexpected role of EIIAGlc reveals a striking direct link between central Salmonella metabolism and a crucial virulence mechanism.

  10. Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

    Science.gov (United States)

    Stubbs, M; Aiston, S; Agius, L

    2000-12-01

    We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

  11. Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

    Science.gov (United States)

    Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

    2016-07-01

    Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  12. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

    KAUST Repository

    Abdallah, Abdallah

    2011-09-28

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

  13. Spontaneous NF-κB activation by autocrine TNFα signaling: a computational analysis.

    Directory of Open Access Journals (Sweden)

    Jakub Pękalski

    Full Text Available NF-κB is a key transcription factor that regulates innate immune response. Its activity is tightly controlled by numerous feedback loops, including two negative loops mediated by NF-κB inducible inhibitors, IκBα and A20, which assure oscillatory responses, and by positive feedback loops arising due to the paracrine and autocrine regulation via TNFα, IL-1 and other cytokines. We study the NF-κB system of interlinked negative and positive feedback loops, combining bifurcation analysis of the deterministic approximation with stochastic numerical modeling. Positive feedback assures the existence of limit cycle oscillations in unstimulated wild-type cells and introduces bistability in A20-deficient cells. We demonstrated that cells of significant autocrine potential, i.e., cells characterized by high secretion of TNFα and its receptor TNFR1, may exhibit sustained cytoplasmic-nuclear NF-κB oscillations which start spontaneously due to stochastic fluctuations. In A20-deficient cells even a small TNFα expression rate qualitatively influences system kinetics, leading to long-lasting NF-κB activation in response to a short-pulsed TNFα stimulation. As a consequence, cells with impaired A20 expression or increased TNFα secretion rate are expected to have elevated NF-κB activity even in the absence of stimulation. This may lead to chronic inflammation and promote cancer due to the persistent activation of antiapoptotic genes induced by NF-κB. There is growing evidence that A20 mutations correlate with several types of lymphomas and elevated TNFα secretion is characteristic of many cancers. Interestingly, A20 loss or dysfunction also leaves the organism vulnerable to septic shock and massive apoptosis triggered by the uncontrolled TNFα secretion, which at high levels overcomes the antiapoptotic action of NF-κB. It is thus tempting to speculate that some cancers of deregulated NF-κB signaling may be prone to the pathogen-induced apoptosis.

  14. [Detection of surface EMG signal using active electrode].

    Science.gov (United States)

    He, Qinghua; Peng, Chenglin; Wu, Baoming; Wang, He

    2003-09-01

    Research of surface electromyogram(EMG) signal is important in rehabilitation medicine, sport medicine and clinical diagnosis, accurate detection of signal is the base of quantitative analysis of surface EMG signal. In this article were discussed how to reduce possible noise in the detection of surface EMG. Considerations on the design of electrode unit were presented. Instrumentation amplifier AD620 was employed to design a bipolar active electrode for use in surface EMG detection. The experiments showed that active electrode could be used to improve signal/noise ratio, reduce noise and detect surface EMG signal effectively.

  15. Corydalis edulis Maxim. Promotes Insulin Secretion via the Activation of Protein Kinase Cs (PKCs) in Mice and Pancreatic β Cells.

    Science.gov (United States)

    Zheng, Jiao; Zhao, Yunfang; Lun, Qixing; Song, Yuelin; Shi, Shepo; Gu, Xiaopan; Pan, Bo; Qu, Changhai; Li, Jun; Tu, Pengfei

    2017-01-16

    Corydalis edulis Maxim., a widely grown plant in China, had been proposed for the treatment for type 2 diabetes mellitus. In this study, we found that C. edulis extract (CE) is protective against diabetes in mice. The treatment of hyperglycemic and hyperlipidemic apolipoprotein E (ApoE)-/- mice with a high dose of CE reduced serum glucose by 28.84% and serum total cholesterol by 17.34% and increased insulin release. We also found that CE significantly enhanced insulin secretion in a glucose-independent manner in hamster pancreatic β cell (HIT-T15). Further investigation revealed that CE stimulated insulin exocytosis by a protein kinase C (PKC)-dependent signaling pathway and that CE selectively activated novel protein kinase Cs (nPKCs) and atypical PKCs (aPKCs) but not conventional PKCs (cPKCs) in HIT-T15 cells. To the best of our knowledge, our study is the first to identify the PKC pathway as a direct target and one of the major mechanisms underlying the antidiabetic effect of CE. Given the good insulinotropic effect of this herbal medicine, CE is a promising agent for the development of new drugs for treating diabetes.

  16. An ABC-transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

    Science.gov (United States)

    Casabona, Maria G.; Silverman, Julie M.; Sall, Khady M.; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D.; Elsen, Sylvie; Attree, Ina

    2012-01-01

    Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SS). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Thr kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and export of Hcp1 and Tse1. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signaling pathway that promotes H1-T6SS activity under optimal environmental conditions. PMID:22765374

  17. An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

    Science.gov (United States)

    Casabona, Maria G; Silverman, Julie M; Sall, Khady M; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D; Elsen, Sylvie; Attree, Ina

    2013-02-01

    Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  18. Novel strategies in drug discovery of the calcium-sensing receptor based on biased signaling

    DEFF Research Database (Denmark)

    Thomsen, Alex Rojas Bie; Smajilovic, Sanela; Bräuner-Osborne, Hans

    2012-01-01

    SR activating drug that selectively activates signaling pathways that inhibit PTH secretion while having no effect on signaling pathways involved in calcitonin secretion. Such a drug would have the same therapeutic value as cinacalcet in lowering PTH secretion while eliminating the side effect of hypocalcemia...

  19. Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities.

    Science.gov (United States)

    Ray, Ann; Schwartz, Nika; de Souza Santos, Marcela; Zhang, Junmei; Orth, Kim; Salomon, Dor

    2017-11-01

    Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

    Science.gov (United States)

    Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

    2013-09-01

    Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

  1. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    Science.gov (United States)

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Temperature oscillations drive cycles in the activity of MMP-2,9 secreted by a human trabecular meshwork cell line.

    Science.gov (United States)

    Li, Stanley Ka-Lok; Banerjee, Juni; Jang, Christopher; Sehgal, Amita; Stone, Richard A; Civan, Mortimer M

    2015-02-05

    Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

  3. A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis

    DEFF Research Database (Denmark)

    van der Plas, Mariena J A; Andersen, Anders S; Nazir, Sheresma

    2014-01-01

    Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasmin...

  4. Insulin secretion enhancing activity of roselle calyx extract in normal and streptozotocin-induced diabetic rats

    Science.gov (United States)

    Wisetmuen, Eamruthai; Pannangpetch, Patchareewan; Kongyingyoes, Bunkerd; Kukongviriyapan, Upa; Yutanawiboonchai, Wiboonchai; Itharat, Arunporn

    2013-01-01

    Background and Objective: Our recent study revealed the antihyperglycemic activity of an ethanolic extract of roselle calyxes (Hibiscus sabdariffa) in diabetic rats. The present study had, therefore, an objective to investigate the mechanism underlying this activity. Materials and Methods: Male Sprague Dawley rats were induced to be diabetes by intraperitoneal injection of 45 mg/kg streptozotocin (STZ). Normal rats as well as diabetic rats were administered with the ethanolic extract of H. sabdariffa calyxes (HS-EE) at 0.1 and 1.0 g/kg/day, respectively, for 6 weeks. Then, blood glucose and insulin levels, at basal and glucose-stimulated secretions, were measured. The pancreas was dissected to examine histologically. Results: HS-EE 1.0 g/kg/day significantly decreased the blood glucose level by 38 ± 12% in diabetic rats but not in normal rats. In normal rats, treatment with 1.0 g/kg HS-EE increased the basal insulin level significantly as compared with control normal rats (1.28 ± 0.25 and 0.55 ± 0.05 ng/ml, respectively). Interestingly, diabetic rats treated with 1.0 g/kg HS-EE also showed a significant increase in basal insulin level as compared with the control diabetic rats (0.30 ± 0.05 and 0.15 ± 0.01 ng/ml, respectively). Concerning microscopic histological examination, HS-EE 1.0 g/kg significantly increased the number of islets of Langerhans in both normal rats (1.2 ± 0.1 and 2.0 ± 0.1 islet number/10 low-power fields (LPF) for control and HS-EE treated group, respectively) and diabetic rats (1.0 ± 0.3 and 3.9 ± 0.6 islet number/10 LPF for control and HS-EE treated group, respectively). Conclusion: The antidiabetic activity of HS-EE may be partially mediated via the stimulating effect on insulin secretion. PMID:23798879

  5. Irradiation Effect on Secreting Function, Amylase Activity and Nucleic Acid Contents of Rat Parotid Gland

    International Nuclear Information System (INIS)

    Cho, Young Jin; Park, Tae Won

    1990-01-01

    This experiment was performed to clarify the effects of 60 Co gamma irradiation on secretory function, amylase activity and contents of nucleic acids of parotid gland in rat. Experimental animals were divided into 6th hours, 3rd, 7th, 14th and 28th days after irradiation and control. The experimental animals are singly irradiated with 20 Gy (2,000 rad) through protective lead block. Secretory function of parotid gland was evaluated by uptake and clearance of 99m TcO 4 . 99m TcO 4 , 0.2μ ci/gm, was injected into peritonium in uptake groups. Rats were sacrificed with cervical dislocation after 30 minutes and gland was excised. In the clearance group, pilocarpine nitrate (8 mg/kg) was intraperitoneally injected at 30 minutes after 99m TcO 4 injection and rats were sacrificed 30 minutes after pilocarpine infection. Radioactivity of excised parotid gland was measured by using of gamma counter and stimulation-secretion coefficient, uptake radioactivity divided by clearance radioactivity, was calculated. Amylase activity and contents of DNA and RNA were determined by spectrophotometry. The results obtained were as follows: 1. In the uptake test, the radioactivity of 99m TcO 4 per unit weight increase in experimental group except 6th hours group, compared with control groups and showed a peak at 3rd days after irradiation. 2. In the clearance test, the radioactivity of 99m TcO 4 per unit weight rose to a peak at 3rd days after irradiation and gradually recovered thereafter. 3. Stimulation-secretion coefficient of parotid gland decreased at 6th hours, 3rd and 7th days after irradiation, and gradually increased. 4. Amylase activity of parotid gland decreased in 3rd and 7th days group, and especially lowest in 3rd days after irradiation. 5. The contents of DNA showed no definite difference in all the experimental groups, but RNA was seemed to decrease with time after irradiation.

  6. Neuronal Functions of Activators of G Protein Signaling

    Directory of Open Access Journals (Sweden)

    Man K. Tse

    2012-05-01

    Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

  7. Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

    Science.gov (United States)

    Witzke, Kathrin E; Rosowski, Kristin; Müller, Christian; Ahrens, Maike; Eisenacher, Martin; Megger, Dominik A; Knobloch, Jürgen; Koch, Andrea; Bracht, Thilo; Sitek, Barbara

    2017-01-06

    Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

  8. Effect of certain active components from traditional Chinese medicinal herbs on Aβ secretion rate with L-[35S]-Methionine

    International Nuclear Information System (INIS)

    Hu Yaer; Zhang Naizheng; Li Aimin; Xia Zongqin

    2006-01-01

    To observe the effect of certain active components from traditional Chinese medicinal herbs on Aβ secretion rates with L-[ 35 S]-Methionine, β-amyloid peptide (Aβ) in SK-N-SH cell lines stably transfected with APP695 was metabolically labeled with L-[ 35 S]-Methionine. the supernatant from culture medium was immunoprecipitated with monoclonal antibody against Aβ 22-35 , Western blot was carried out, and the gray density of Aβ band in the autoradiograph was measured by an image analysis system. The active components from certain traditional Chinese medicinal herbs (ZMS from Zhimu and AST and HT from Huangqi) were added to the culture medium at a final concentration of 10 -5 mol/L. An Aβ band in the autoradiograph was clearly viewed in the culture medium after 24 h incorporation of [ -35 S]-Methionine which represent the secretion rate of Aβ by the cells. One of the 3 tested components (AST) could significantly reduce the Aβ secretion rate while the other two showed no effect. The preliminary result showed that certain active component from traditional Chinese medicines could decrease the Aβ secretion rate but other active components could not. Combined use of the AST and ZMS was more effective than single AST. (authors)

  9. Propionic acid secreted from propionibacteria induces NKG2D ligand expression on human-activated T lymphocytes and cancer cells

    DEFF Research Database (Denmark)

    Andresen, Lars; Hansen, Karen Aagaard; Jensen, Helle

    2009-01-01

    We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone cou...

  10. In-vitro secretion of inhibin-like activity by Sertoli cells from normal and prenatally irradiated immature rats

    International Nuclear Information System (INIS)

    Ultee-van Gessel, A.M.; Leemborg, F.G.; Jong, F.H. de; Molen, H.J. van der

    1986-01-01

    The influence of in-vitro conditions on the production of inhibin by Sertoli cells from 21-day-old normal and prenatally irradiated rat testes was studied by measuring inhibin activity in culture media, using the suppression of the release of FSH from cultured rat pituitary cells. Sertoli cells secreted inhibin-like activity during at least 21 days of culture, and cells cultured at 37 0 C produced significantly more inhibin than those cultured at 32 0 C. The presence of fetal calf serum had no significant effect on inhibin production at 32 0 C, while at 37 0 C the production was decreased. The presence of ovine FSH stimulated inhibin secretion, while inhibin concentrations in Sertoli cell culture media were decreased after the addition of testosterone. Testosterone, added together with ovine FSH, suppressed inhibin secretion when compared with the levels found in the presence of FSH alone. The presence of spermatogenic cells decreased the release of inhibin. From these results it was concluded that both Sertoli cells isolated from normal immature rat testes and those from testes without spermatogenic cells can secrete inhibin-like activity in culture. A number of discrepancies with in-vivo observations was observed. (author)

  11. Effects of Steaming Time and Frequency for Manufactured Red Liriope platyphylla on the Insulin Secretion Ability and Insulin Receptor Signaling Pathway.

    Science.gov (United States)

    Choi, Sun Il; Lee, Hye Ryun; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Young Ju; Prak, So Hae; Lee, Hee Seob; Lee, Jong Sup; Jang, In Surk; Son, Hong Ju; Hwang, Dae Youn

    2011-06-01

    In oriental medicine, Liriope platyphylla (LP) has long been regarded as a curative herb useful for the treatment of diabetes, asthma, and neurodegenerative disorders. The principal objective of this study was to assess the effects of steaming time and frequency for manufactured Red LP (RLP) on insulin secretion ability and insulin receptor signaling pathway. To achieve our goal, several types of LPs manufactured under different conditions were applied to INS cells and streptozotocin (STZ)-induced diabetic ICR mice, after which alterations in insulin concentrations were detected in the culture supernatants and sera. The optimal concentration for the investigation of insulin secretion ability was found to be 50 ug/mL of LP. At this concentration, maximum insulin secretion was observed in the INS cells treated with LP extract steamed for 3 h (3-SLP) with two repeated steps (3 h steaming and 24 h air-dried) carried out 9 times (9-SALP); no significant changes in viability were detected in any of the treated cells. Additionally, the expression and phosphorylation levels of most components in the insulin receptor signaling pathway were increased significantly in the majority of cells treated with steaming-processed LP as compared to the cells treated with LP prepared without steaming. With regard to glucose transporter (GLUT) expression, alterations of steaming time induced similar responses on the expression levels of GLUT-2 and GLUT-3. However, differences in steaming frequency were also shown to induce dose-dependent responses in the expression level of GLUT-2 only; no significant differences in GLUT-3 expression were detected under these conditions. Furthermore, these responses observed in vitro were similarly detected in STZ-induced diabetic mice. 24-SLP and 9-SALP treatment applied for 14 days induced the down-regulation of glucose concentration and upregulation of insulin concentration. Therefore, these results indicated that the steaming processed LP may

  12. Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

    DEFF Research Database (Denmark)

    Hautmann, Matthias; Friis, Ulla G; Desch, Michael

    2007-01-01

    Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because...

  13. Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

    Science.gov (United States)

    Denys, A; Allain, F; Masy, E; Dessaint, J P; Spik, G

    1998-04-27

    Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

  14. Open Secrets

    OpenAIRE

    Madison, Michael

    2017-01-01

    The law of trade secrets is often conceptualized in bilateral terms, as creating and enforcing rights between trade secret owners, on the one hand, and misappropriators on the other hand. This paper, a chapter in a forthcoming collection on the law of trade secrets, argues that trade secrets and the law that guards them can serve structural and insitutional roles as well. Somewhat surprisingly, given the law’s focus on secrecy, among the institutional products of trade secrets law are commons...

  15. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-01

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

  16. Recently activated naive CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2-type cytokines.

    Science.gov (United States)

    Croft, M; Swain, S L

    1995-05-01

    Development of T cells during primary responses was investigated using pigeon cytochrome C-specific naive Th from TCR transgenic mice. Naive CD4 cells did not activate and help resting B cells. This failure was found to be primarily because the resting B cells were incapable of stimulating the naive Th. Provision of a costimulatory signal such as anti-CD28, or addition of APCs that express costimulatory molecules, such as dendritic cells, activated B cells, and B7+ and B7+ICAM(+)-expressing fibroblasts, induced naive Th activation and promoted T cell-dependent help for IgM secretion. T cell activation for as little as 24 h promoted helper activity, and Ig secretion required production of small amounts of IL-4 by the activated naive Th. On initial stimulation, naive Th secrete only IL-2. By mRNA analysis, activated naive Th were also shown to produce IL-4, however induction of IL-4 message only occurred 24 h after initial activation and required additional stimulation with Ag. A single exposure of naive CD4 to Ag/APC followed by 4 to 12 days in culture led to generation of effector Th which secreted IL-2 and some IFN-gamma, and no detectable IL-4 or IL-5, and which could only help B cells to IgM secretion. In contrast, similar cultures that received Ag/APC one or more times during this period generated effector cells capable of secreting easily detectable titers of IL-4 and IL-5, as well as IL-2 and IFN-gamma, and able to now promote IgG1 and IgE responses. Generation of these Th0-like effectors was accompanied by increasing amounts of IL-4 secreted during the culture period after each restimulation, and addition of anti-IL-4 in culture inhibited development of the capacity to produce Th2 cytokines. These studies reinforce the notion that naive CD4 must interact with a costimulatory professional APC, rather than a resting B cell, for initiation of the primary response, but show that such an interaction can result in rapid development of the ability to interact with

  17. NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals.

    Science.gov (United States)

    Bakker, A B; Wu, J; Phillips, J H; Lanier, L L

    2000-01-01

    A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.

  18. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Marina Pasca di Magliano

    2007-11-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDA is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

  19. Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)*

    Science.gov (United States)

    Hershko, Klilah; Simhadri, Vijaya L.; Blaisdell, Adam; Hunt, Ryan C.; Newell, Jordan; Tseng, Sandra C.; Hershko, Alon Y.; Choi, Jae Won; Sauna, Zuben E.; Wu, Andrew; Bram, Richard J.; Komar, Anton A.; Kimchi-Sarfaty, Chava

    2012-01-01

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients. PMID:23144461

  20. Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

    Science.gov (United States)

    Hershko, Klilah; Simhadri, Vijaya L; Blaisdell, Adam; Hunt, Ryan C; Newell, Jordan; Tseng, Sandra C; Hershko, Alon Y; Choi, Jae Won; Sauna, Zuben E; Wu, Andrew; Bram, Richard J; Komar, Anton A; Kimchi-Sarfaty, Chava

    2012-12-28

    The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

  1. Essential roles of aspartate aminotransferase 1 and vesicular glutamate transporters in β-cell glutamate signaling for incretin-induced insulin secretion.

    Directory of Open Access Journals (Sweden)

    Naoya Murao

    Full Text Available Incretins (GLP-1 and GIP potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin secretion (IIIS that requires two critical processes: 1 generation of cytosolic glutamate through the malate-aspartate (MA shuttle in glucose metabolism and 2 glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol Got1, a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol Slc17a7, Slc17a6, and Slc17a8, respectively, which participate in glutamate transport into secretory vesicles. Got1 knockout (KO β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.

  2. Acidity enhances the effectiveness of active chemical defensive secretions of sea hares, Aplysia californica, against spiny lobsters, Panulirus interruptus.

    Science.gov (United States)

    Shabani, Shkelzen; Yaldiz, Seymanur; Vu, Luan; Derby, Charles D

    2007-12-01

    Sea hares such as Aplysia californica, gastropod molluscs lacking a protective shell, can release a purple cloud of chemicals when vigorously attacked by predators. This active chemical defense is composed of two glandular secretions, ink and opaline, both of which contain an array of compounds. This secretion defends sea hares against predators such as California spiny lobsters Panulirus interruptus via multiple mechanisms, one of which is phagomimicry, in which secretions containing feeding chemicals attract and distract predators toward the secretion and away from the sea hare. We show here that ink and opaline are highly acidic, both having a pH of approximately 5. We examined if the acidity of ink and opaline affects their phagomimetic properties. We tested behavioral and electrophysiological responses of chemoreceptor neurons in the olfactory and gustatory organs of P. interruptus, to ink and opaline of A. californica within their natural range of pH values, from approximately 5 to 8. Both behavioral and electrophysiological responses to ink and opaline were enhanced at low pH, and low pH alone accounted for most of this effect. Our data suggest that acidity enhances the phagomimetic chemical defense of sea hares.

  3. Reducing the activity and secretion of microbial antioxidants enhances the immunogenicity of BCG.

    Directory of Open Access Journals (Sweden)

    Shanmugalakshmi Sadagopal

    Full Text Available In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB. Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production.To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli.We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.

  4. SECRETLY ECCENTRIC: THE GIANT PLANET AND ACTIVITY CYCLE OF GJ 328

    International Nuclear Information System (INIS)

    Robertson, Paul; Endl, Michael; Cochran, William D.; MacQueen, Phillip J.; Boss, Alan P.

    2013-01-01

    We announce the discovery of a ∼2 Jupiter-mass planet in an eccentric 11 yr orbit around the K7/M0 dwarf GJ 328. Our result is based on 10 years of radial velocity (RV) data from the Hobby-Eberly and Harlan J. Smith telescopes at McDonald Observatory, and from the Keck Telescope at Mauna Kea. Our analysis of GJ 328's magnetic activity via the Na I D features reveals a long-period stellar activity cycle, which creates an additional signal in the star's RV curve with amplitude 6-10 m s –1 . After correcting for this stellar RV contribution, we see that the orbit of the planet is more eccentric than suggested by the raw RV data. GJ 328b is currently the most massive, longest-period planet discovered around a low-mass dwarf

  5. Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2

    DEFF Research Database (Denmark)

    Owens, T

    1991-01-01

    23.1+ TH1 clone E9.D4 in F23.1 (anti-T cell receptor V-beta 8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did......The mechanism whereby noncognate contact with activated IL-2-producing Type 1 helper T cells (TH1) induces B cell activation was examined. Small resting B cells from C57B1/6 mice were cultured, in the absence of any ligand for surface Ig, with irradiated cells of the hapten-specific, CBA-derived, F...... not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-gamma mAb partially inhibited Ig secretion, without affecting entry into cycle. LPS...

  6. Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

    Science.gov (United States)

    Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

    2016-05-01

    Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

  7. Cell Secretion: Current Structural and Biochemical Insights

    Directory of Open Access Journals (Sweden)

    Saurabh Trikha

    2010-01-01

    Full Text Available Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

  8. Defocused low-energy shock wave activates adipose tissue-derived stem cells in vitro via multiple signaling pathways.

    Science.gov (United States)

    Xu, Lina; Zhao, Yong; Wang, Muwen; Song, Wei; Li, Bo; Liu, Wei; Jin, Xunbo; Zhang, Haiyang

    2016-12-01

    We found defocused low-energy shock wave (DLSW) could be applied in regenerative medicine by activating mesenchymal stromal cells. However, the possible signaling pathways that participated in this process remain unknown. In the present study, DLSW was applied in cultured rat adipose tissue-derived stem cells (ADSCs) to explore its effect on ADSCs and the activated signaling pathways. After treating with DLSW, the cellular morphology and cytoskeleton of ADSCs were observed. The secretions of ADSCs were detected. The expressions of ADSC surface antigens were analyzed using flow cytometry. The expressions of proliferating cell nuclear antigen and Ki67 were analyzed using western blot. The expression of CXCR2 and the migrations of ADSCs in vitro and in vivo were detected. The phosphorylation of selected signaling pathways with or without inhibitors was also detected. DLSW did not change the morphology and phenotype of ADSCs, and could promote the secretion, proliferation and migration of ADSCs. The phosphorylation levels were significantly higher in mitogen-activated protein kinases (MAPK) pathway, phosphoinositide 3-kinase (PI-3K)/AKT pathway and nuclear factor-kappa B (NF-κB) signaling pathway but not in Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Furthermore, ADSCs were not activated by DLSW after adding the inhibitors of these pathways simultaneously. Our results demonstrated for the first time that DLSW could activate ADSCs through MAPK, PI-3K/AKT and NF-κB signaling pathways. Combination of DLSW and agonists targeting these pathways might improve the efficacy of ADSCs in regenerative medicine in the future. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Roč. 20, č. 12 (2011), s. 2004-2012 ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  10. CTNNB1 signaling in sertoli cells downregulates spermatogonial stem cell activity via WNT4.

    Directory of Open Access Journals (Sweden)

    Alexandre Boyer

    Full Text Available Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin in the Sertoli cells of the Ctnnb1(tm1Mmt/+;Amhr2(tm3(creBhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC activity. Reciprocal SSC transplants between Ctnnb1(tm1Mmt/+;Amhr2(tm3(creBhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1(tm1Mmt/+;Amhr2(tm3(creBhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1(tm1Mmt/+;Amhr2(tm3(creBhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1(tm1Mmt/+;Amhr2(tm3(creBhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development.

  11. Dental Calculus Stimulates Interleukin-1β Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes.

    Directory of Open Access Journals (Sweden)

    Jorge Luis Montenegro Raudales

    Full Text Available Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs, and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs and peripheral blood mononuclear cells (PBMCs with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a

  12. Dental Calculus Stimulates Interleukin-1β Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes.

    Science.gov (United States)

    Montenegro Raudales, Jorge Luis; Yoshimura, Atsutoshi; Sm, Ziauddin; Kaneko, Takashi; Ozaki, Yukio; Ukai, Takashi; Miyazaki, Toshihiro; Latz, Eicke; Hara, Yoshitaka

    2016-01-01

    Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in

  13. Dental Calculus Stimulates Interleukin-1β Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes

    Science.gov (United States)

    Montenegro Raudales, Jorge Luis; Yoshimura, Atsutoshi; SM, Ziauddin; Kaneko, Takashi; Ozaki, Yukio; Ukai, Takashi; Miyazaki, Toshihiro; Latz, Eicke; Hara, Yoshitaka

    2016-01-01

    Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in

  14. Coco is a dual activity modulator of TGFβ signaling

    Science.gov (United States)

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  15. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

    DEFF Research Database (Denmark)

    Sasaki, S; Watanabe, J; Ohtaki, H

    2017-01-01

    BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is requir...

  16. Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator

    International Nuclear Information System (INIS)

    Vlaicu, Philip; Mertins, Philipp; Mayr, Thomas; Widschwendter, Peter; Ataseven, Beyhan; Högel, Bernhard; Eiermann, Wolfgang; Knyazev, Pjotr; Ullrich, Axel

    2013-01-01

    Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors. Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients. Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease. Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy

  17. Modulation of β-catenin signaling by glucagon receptor activation.

    Directory of Open Access Journals (Sweden)

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  18. Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

    Science.gov (United States)

    Perera Córdova, Wilmer H; Leitão, Suzana Guimarães; Cunha-Filho, Geraldino; Bosch, Roberto Alonso; Alonso, Isel Pascual; Pereda-Miranda, Rogelio; Gervou, Rodrigo; Touza, Natália Araújo; Quintas, Luis Eduardo M; Noël, François

    2016-02-01

    Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase. Published by Elsevier Ltd.

  19. Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

    Science.gov (United States)

    Branham-O'Connor, Melissa; Robichaux, William G; Zhang, Xian-Kui; Cho, Hyeseon; Kehrl, John H; Lanier, Stephen M; Blumer, Joe B

    2014-04-11

    Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

  20. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  1. BIOLOGICAL ACTIVITY OF SKIN SECRETIONS IN THE PERCIDAE FAMILY OF FISH WITH REGARD TO ERYSIPELOTHRIX RHUSIOPATHIAE BACTERIA

    Directory of Open Access Journals (Sweden)

    А. Нulaі

    2015-09-01

    Full Text Available Purpose. To determine the effect of skin secretions in Percidae family of fish (Sander lucioperca, Perca fluviatilis on the cultures of pathogenic E. rhusiopathiae bacteria. Methodology. Filter paper was placed on the skin of live fish; after 1 minute it was removed, and the extraction of water-soluble components in the rate of 0.1 cm3 of water per 1 cm2 area of the paper was carried out. The resulting solutions of the secretions of fish skin glands were sterilized by vacuum filtration through the filters with a pore diameter The test samples contained E. rhusiopathiae cultures and fish skin secretions in 1:10, 1:100, 1:1000 and 1:10000 dilutions. The control samples contained a similar ratio of sterile water and cultures of the experimental bacteria type. The content of E. rhusiopathiae cells in the experimental and control samples was determined after 48 hours, the samples were stored at a temperature of +18... +20 °C. Findings. The presence of water-soluble extracts of skin secretions obtained from Percidae family of freshwater fish in the environment causes an increase in the density of E. rhusiopathiae bacteria cultures. The degree of the detected stimulating effect of E. rhusiopathiae directly depend on the concentration of the secretions of fish skin glands in the experimental samples. Under the conditions of freshwater ecosystems, such fish as S. lucioperca and P. fluviatilis and E. rhusiopathiae bacteria may form biocenotical relations of the trophic, topical and phoric types. Originality. The quantitative data proving the biological activity of skin secretions in Percidae family of fish with regard to E. rhusiopathiae pathogenic bacteria have been obtained for the first time. Practical Value. One of the factors contributing to E. rhusiopathiae bacteria staying in freshwater conditions for a long time can be their biocenotical relations with the components of freshwater biocenoses, including such fish as S. lucioperca and P

  2. Enhanced secretion of natto phytase by Bacillus subtilis.

    Science.gov (United States)

    Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

    2015-01-01

    Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

  3. Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

    Science.gov (United States)

    Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

    2005-01-01

    We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

  4. Inhibition of the JAK2/STAT3/SOSC1 Signaling Pathway Improves Secretion Function of Vascular Endothelial Cells in a Rat Model of Pregnancy-Induced Hypertension

    Directory of Open Access Journals (Sweden)

    Jian-Ying Luo

    2016-11-01

    Full Text Available Background/Aims: The present study aimed to investigate the effects of the JAK2/STAT3/SOSC1 signaling pathway on the secretion function of vascular endothelial cells (VECs in a rat model of pregnancy-induced hypertension (PIH. Methods: A PIH rat model was established. Forty-eight pregnant Sprague-Dawley female rats were selected and assigned into four groups: the normal group (normal non-pregnant rats, the non-PIH group (pregnant rats without PIH, the PIH group (pregnant rats with PIH and the AG490 group (pregnant rats with PIH treated with AG490. Systolic blood pressure (SBP and urinary protein (UP were measured. The expressions of JAK2/STAT3/SOSC1 signaling pathway-related proteins in placenta tissues were detect by Western blotting. Radioimmunoassay was applied to detect serum levels of nitric oxide (NO, super oxide dismutase (SOD, placental growth factor (PGF, thromboxane B2 (TXB2 and endothelin (ET. Enzyme-linked immunosorbent assay (ELISA was used to determine serum levels of interleukin-6 (IL-6, interleukin-10 (IL-10 and tumor necrosis factor-α (TNF-α. Results: Compared with the normal and non-PIH groups, the PIH and AG490 groups had higher SBP and UP levels at 17th and 25th day of pregnancy. The expressions of p/t-JAK2, p/t-STAT3 and SOSC1 in the PIH and AG490 groups were higher than those in the non-PIH group, while the expressions of p/t-JAK2, p/t-STAT3 and SOSC1 in the AG490 group were lower than those in the PIH group. Compared with the non-PIH group, serum levels of ET, TXB2, IL-6 and TNF-α were increased in the PIH and AG490 groups, while serum levels of NO, SOD, 6-keto-PGF1a and IL-10 levels were reduced. Furthermore, the AG490 had lower serum levels of ET, TXB2, IL-6 and TNF-α and higher serum levels of NO, SOD, 6-keto-PGF1a and IL-10 than those in the PIH group. Conclusion: Our study provides evidence that inhibition of the JAK2/STAT3/SOSC1 signaling pathway could improve the secretion function of VECs in PIH rats.

  5. A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis.

    Science.gov (United States)

    Sun, Hui; Kamanova, Jana; Lara-Tejero, Maria; Galán, Jorge E

    2016-03-01

    Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen's virulence.

  6. Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

    Science.gov (United States)

    Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

    2017-11-02

    The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

  7. Antifungal activity of the pygidial gland secretion of Laemostenus punctatus (Coleoptera: Carabidae) against cave-dwelling micromycetes

    Science.gov (United States)

    Nenadić, Marija; Ljaljević-Grbić, Milica; Stupar, Miloš; Vukojević, Jelena; Ćirić, Ana; Tešević, Vele; Vujisić, Ljubodrag; Todosijević, Marina; Vesović, Nikola; Živković, Nemanja; Ćurčić, Srećko

    2017-06-01

    The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.

  8. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

    Science.gov (United States)

    Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

    2018-01-01

    Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  9. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Zheng Lu

    2018-05-01

    Full Text Available Arctigenin (ARG, one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21 and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α and transforming growth factor beta1 (TGF-β1 in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126. Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  10. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway.

    Science.gov (United States)

    Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

    2018-01-01

    Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L. , has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  11. Epithelial cells detect functional type III secretion system of enteropathogenic Escherichia coli through a novel NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Yael Litvak

    2017-07-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC, a common cause of infant diarrhea, is associated with high risk of mortality in developing countries. The primary niche of infecting EPEC is the apical surface of intestinal epithelial cells. EPEC employs a type three secretion system (TTSS to inject the host cells with dozens of effector proteins, which facilitate attachment to these cells and successful colonization. Here we show that EPEC elicit strong NF-κB activation in infected host cells. Furthermore, the data indicate that active, pore-forming TTSS per se is necessary and sufficient for this NF-κB activation, regardless of any specific effector or protein translocation. Importantly, upon infection with wild type EPEC this NF-κB activation is antagonized by anti-NF-κB effectors, including NleB, NleC and NleE. Accordingly, this NF-κB activation is evident only in cells infected with EPEC mutants deleted of nleB, nleC, and nleE. The TTSS-dependent NF-κB activation involves a unique pathway, which is independent of TLRs and Nod1/2 and converges with other pathways at the level of TAK1 activation. Taken together, our results imply that epithelial cells have the capacity to sense the EPEC TTSS and activate NF-κB in response. Notably, EPEC antagonizes this capacity by delivering anti-NF-κB effectors into the infected cells.

  12. Lactobacillus crispatus dominant vaginal microbiome is associated with inhibitory activity of female genital tract secretions against Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Jeny P Ghartey

    Full Text Available Female genital tract secretions inhibit E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity.Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition or low (<20% inhibition anti-E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity.Sixteen samples (10 with high and 6 with low activity yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each. However, there were significant differences between women with high compared to low E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007 and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus.Microbiome communities with abundant L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.

  13. Adrenergic receptors and gastric secretion in dogs. Is a "tonic balance" relationship between vagal and beta 2-adrenergic activity a possibility?

    DEFF Research Database (Denmark)

    Gottrup, F; Hovendal, C; Bech, K

    1984-01-01

    The relative influence of adrenergic receptors on gastric acid secretion in the dog stomach with different vagal activity or "tone" is almost unknown. beta-adrenoceptors seem to be most important for the direct effect of adrenergic stimulation on acid secretion. In this study the effects...... acid secretion was not influenced significantly by beta-blockade. Similar dose-response curves were found for non-vagotomized dogs with high beta 2-adrenergic tone and dogs with low vagal tone (vagotomy) after pentagastrin and histamine stimulated acid secretion. This study indicates...... that a counterbalance between beta 2-adrenergic and cholinergic vagal tone exists. A "tonic balance theory" is suggested and is probably involved in the resulting acid secretion after vagotomy....

  14. The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System.

    Science.gov (United States)

    Walker, Kimberly A; Griggs, Lauren A; Obrist, Markus; Bode, Addys; Summers, R Patrick; Miller, Virginia L

    2016-06-15

    The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes

  15. Metabolic signals and innate immune activation in obesity and exercise.

    Science.gov (United States)

    Ringseis, Robert; Eder, Klaus; Mooren, Frank C; Krüger, Karsten

    2015-01-01

    The combination of a sedentary lifestyle and excess energy intake has led to an increased prevalence of obesity which constitutes a major risk factor for several co-morbidities including type 2 diabetes and cardiovascular diseases. Intensive research during the last two decades has revealed that a characteristic feature of obesity linking it to insulin resistance is the presence of chronic low-grade inflammation being indicative of activation of the innate immune system. Recent evidence suggests that activation of the innate immune system in the course of obesity is mediated by metabolic signals, such as free fatty acids (FFAs), being elevated in many obese subjects, through activation of pattern recognition receptors thereby leading to stimulation of critical inflammatory signaling cascades, like IκBα kinase/nuclear factor-κB (IKK/NF- κB), endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and NOD-like receptor P3 (NLRP3) inflammasome pathway, that interfere with insulin signaling. Exercise is one of the main prescribed interventions in obesity management improving insulin sensitivity and reducing obesity- induced chronic inflammation. This review summarizes current knowledge of the cellular recognition mechanisms for FFAs, the inflammatory signaling pathways triggered by excess FFAs in obesity and the counteractive effects of both acute and chronic exercise on obesity-induced activation of inflammatory signaling pathways. A deeper understanding of the effects of exercise on inflammatory signaling pathways in obesity is useful to optimize preventive and therapeutic strategies to combat the increasing incidence of obesity and its comorbidities. Copyright © 2015 International Society of Exercise and Immunology. All rights reserved.

  16. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  17. Cholinergic signaling mediates the effects of xenin-25 on secretion of pancreatic polypeptide but not insulin or glucagon in humans with impaired glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Songyan Wang

    Full Text Available We previously demonstrated that infusion of an intestinal peptide called xenin-25 (Xen amplifies the effects of glucose-dependent insulinotropic polypeptide (GIP on insulin secretion rates (ISRs and plasma glucagon levels in humans. However, these effects of Xen, but not GIP, were blunted in humans with type 2 diabetes. Thus, Xen rather than GIP signaling to islets fails early during development of type 2 diabetes. The current crossover study determines if cholinergic signaling relays the effects of Xen on insulin and glucagon release in humans as in mice. Fasted subjects with impaired glucose tolerance were studied. On eight separate occasions, each person underwent a single graded glucose infusion- two each with infusion of albumin, Xen, GIP, and GIP plus Xen. Each infusate was administered ± atropine. Heart rate and plasma glucose, insulin, C-peptide, glucagon, and pancreatic polypeptide (PP levels were measured. ISRs were calculated from C-peptide levels. All peptides profoundly increased PP responses. From 0 to 40 min, peptide(s infusions had little effect on plasma glucose concentrations. However, GIP, but not Xen, rapidly and transiently increased ISRs and glucagon levels. Both responses were further amplified when Xen was co-administered with GIP. From 40 to 240 min, glucose levels and ISRs continually increased while glucagon concentrations declined, regardless of infusate. Atropine increased resting heart rate and blocked all PP responses but did not affect ISRs or plasma glucagon levels during any of the peptide infusions. Thus, cholinergic signaling mediates the effects of Xen on insulin and glucagon release in mice but not humans.

  18. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiang-Zhu Xie

    2016-01-01

    Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

  19. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion.

    Science.gov (United States)

    de Mingo Pulido, Álvaro; de Gregorio, Estefanía; Chandra, Shilpi; Colell, Anna; Morales, Albert; Kronenberg, Mitchell; Marí, Montserrat

    2018-01-01

    Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs), probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro .

  20. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    Directory of Open Access Journals (Sweden)

    Álvaro de Mingo Pulido

    2018-02-01

    Full Text Available Natural killer T (NKT cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ, and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB and cathepsin S (CTSS, regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs, probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro.

  1. Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells.

    Science.gov (United States)

    Manning Fox, Jocelyn E; Karaman, Gunce; Wheeler, Michael B

    2006-11-17

    Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.

  2. Survey of activated FLT3 signaling in leukemia.

    Directory of Open Access Journals (Sweden)

    Ting-lei Gu

    Full Text Available Activating mutations of FMS-like tyrosine kinase-3 (FLT3 are found in approximately 30% of patients with acute myeloid leukemia (AML. FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3 and B cell acute lymphoblastic leukemia (normal and amplification of FLT3 cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC, we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.

  3. Epstein Barr virus Latent Membrane Protein-1 enhances dendritic cell therapy lymph node migration, activation, and IL-12 secretion.

    Directory of Open Access Journals (Sweden)

    James M Termini

    Full Text Available Dendritic cells (DC are a promising cell type for cancer vaccines due to their high immunostimulatory capacity. However, improper maturation of DC prior to treatment may account for the limited efficacy of DC vaccine clinical trials. Latent Membrane Protein-1 (LMP1 of Epstein-Barr virus was examined for its ability to mature and activate DC as a gene-based molecular adjuvant for DC vaccines. DC were transduced with an adenovirus 5 vector (Ad5 expressing LMP1 under the control of a Tet-inducible promoter. Ad5-LMP1 was found to mature and activate both human and mouse DC. LMP1 enhanced in vitro migration of DC toward CCL19, as well as in vivo migration of DC to the inguinal lymph nodes of mice following intradermal injection. LMP1-transduced DC increased T cell proliferation in a Pmel-1 adoptive transfer model and enhanced survival in B16-F10 melanoma models. LMP1-DC also enhanced protection in a vaccinia-Gag viral challenge assay. LMP1 induced high levels of IL-12p70 secretion in mouse DC when compared to standard maturation protocols. Importantly, LMP1-transduced human DC retained the capacity to secrete IL-12p70 and TNF in response to DC restimulation. In contrast, DC matured with Monocyte Conditioned Media-Mimic cocktail (Mimic were impaired in IL-12p70 secretion following restimulation. Overall, LMP1 matured and activated DC, induced migration to the lymph node, and generated high levels of IL-12p70 in a murine model. We propose LMP1 as a promising molecular adjuvant for DC vaccines.

  4. Systematic high-yield production of human secreted proteins in Escherichia coli

    International Nuclear Information System (INIS)

    Dai Xueyu; Chen Qiang; Lian Min; Zhou Yanfeng; Zhou Mo; Lu Shanyun; Chen Yunjia; Luo Jingchu; Gu Xiaocheng; Jiang Ying; Luo Ming; Zheng Xiaofeng

    2005-01-01

    Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies

  5. ROS detoxification and proinflammatory cytokines are linked by p38 MAPK signaling in a model of mature astrocyte activation.

    Directory of Open Access Journals (Sweden)

    Adrian Nahirnyj

    Full Text Available Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL and optic nerve head (ONH, and perform essential roles in maintaining retinal ganglion cell (RGC detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1, with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.

  6. Effect of dietary roughage level on chewing activity, ruminal pH, and saliva secretion in lactating Holstein cows.

    Science.gov (United States)

    Jiang, F G; Lin, X Y; Yan, Z G; Hu, Z Y; Liu, G M; Sun, Y D; Liu, X W; Wang, Z H

    2017-04-01

    Increasing dietary roughage level is a commonly used strategy to prevent subacute ruminal acidosis. We hypothesized that high-roughage diets could promote chewing activity, saliva secretion, and hence more alkaline to buffer rumen pH. To verify the hypothesis, 12 multiparous Holstein cows in mid lactation were randomly allocated to 4 treatments in a triplicated 4 × 4 Latin square experiment with one cow in each treatment surgically fitted with a ruminal cannula. Treatments were diets containing 40, 50, 60, or 70% of roughage on a DM basis. Increasing dietary roughage level decreased DM, CP, OM, starch, and NE L intake, increased ADF intake, and decreased milk yield linearly. Intake of NDF was quite stable across treatments and ranged from 7.8 to 8.1 kg/d per cow. Daily eating time increased linearly with increased roughage level. The increase in eating time was due to increased eating time per meal but not number of meals per day, which was stable and ranged from 8.3 to 8.5 meals per day across treatments. Increasing dietary roughage level had no effect on ruminating time (min/d), the number of ruminating periods (rumination periods per d), and chewing time per ruminating period (min/ruminating period). Ruminating time per kilogram of NDF intake and total chewing time per kilogram of ADF intake were similar across treatments (57.4 and 183.8 min/kg, respectively). Increasing dietary roughage level linearly increased daily total chewing time; linearly elevated the mean, maximum, and minimum ruminal pH; and linearly decreased total VFA concentration and molar proportion of propionate in ruminal fluid. Saliva secretion during eating was increased, the secretion during rumination was unaffected, but the secretion during resting tended to decrease with increased dietary roughage level. As a result, total saliva secretion was not affected by treatments. In conclusion, the results of the present study did not support the concept that high-roughage diets elevated ruminal p

  7. Peroxisome proliferator-activated receptor α agonists modulate Th1 and Th2 chemokine secretion in normal thyrocytes and Graves' disease

    International Nuclear Information System (INIS)

    Antonelli, Alessandro; Ferrari, Silvia Martina; Frascerra, Silvia; Corrado, Alda; Pupilli, Cinzia; Bernini, Giampaolo; Benvenga, Salvatore; Ferrannini, Ele; Fallahi, Poupak

    2011-01-01

    Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR)α activation on the prototype Th1 [chemokine (C-X-C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C-C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells. The role of PPARα and PPARγ activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN)γ and tumor necrosis factor (TNF)α. IFNγ stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNFα alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFNγ and TNFα had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPARα activators inhibited the secretion of both chemokines (stimulated with IFNγ and TNFα) at a level higher (for CXCL10, about 60-72%) than PPARγ agonists (about 25-35%), which were confirmed to inhibit CXCL10, but not CCL2. Our data show that CCL2 is modulated by IFNγ and TNFα in GD and normal thyrocytes. Furthermore we first show that PPARα activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPARα may be involved in the modulation of the immune response in the thyroid.

  8. The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways.

    Science.gov (United States)

    De Nisco, Nicole J; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-05-16

    Bacterial effectors potently manipulate host signaling pathways. The marine bacterium Vibrio parahaemolyticus ( V. para ) delivers effectors into host cells through two type 3 secretion systems (T3SSs). T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate nonapoptotic cell death. To understand how the concerted action of T3SS1 effectors globally affects host cell signaling, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1 + ) to those in cells infected with V. para lacking T3SS1 (T3SS1 - ). Overall, the host transcriptional response to both T3SS1 + and T3SS1 - V. para was rapid, robust, and temporally dynamic. T3SS1 rewired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors targeted host cells at the posttranslational level to cause cytotoxicity, V. para T3SS1 also precipitated a host transcriptional response that initially activated cell survival and repressed cell death networks. The increased expression of several key prosurvival transcripts mediated by T3SS1 depended on a host signaling pathway that is silenced posttranslationally later in infection. Together, our analysis reveals a complex interplay between the roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. Copyright © 2017, American Association for the Advancement of Science.

  9. Reciprocal signals between microglia and neurons regulate alpha-synuclein secretion by exophagy through a neuronal cJU-N-Nterminal kinase-signaling axis

    DEFF Research Database (Denmark)

    Christensen, Dan Ploug; Ejlerskov, Patrick; Rasmussen, Izabela

    2016-01-01

    implicate stress kinases of the JNK family in the regulation of exophagy and release of alpha-SNC following endogenous or exogenous stimulation. In a wider scope, our results imply that microglia not only inflict bystander damage to neurons in late phases of inflammatory brain disease but may also be active...

  10. Methylglyoxal and carboxyethyllysine reduce glutamate uptake and S100B secretion in the hippocampus independently of RAGE activation.

    Science.gov (United States)

    Hansen, Fernanda; Battú, Cíntia Eickhoff; Dutra, Márcio Ferreira; Galland, Fabiana; Lirio, Franciane; Broetto, Núbia; Nardin, Patrícia; Gonçalves, Carlos-Alberto

    2016-02-01

    Diabetes is a metabolic disease characterized by high fasting-glucose levels. Diabetic complications have been associated with hyperglycemia and high levels of reactive compounds, such as methylglyoxal (MG) and advanced glycation endproducts (AGEs) formation derived from glucose. Diabetic patients have a higher risk of developing neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. Herein, we examined the effect of high glucose, MG and carboxyethyllysine (CEL), a MG-derived AGE of lysine, on oxidative, metabolic and astrocyte-specific parameters in acute hippocampal slices, and investigated some of the mechanisms that could mediate these effects. Glucose, MG and CEL did not alter reactive oxygen species (ROS) formation, glucose uptake or glutamine synthetase activity. However, glutamate uptake and S100B secretion were decreased after MG and CEL exposure. RAGE activation and glycation reactions, examined by aminoguanidine and L-lysine co-incubation, did not mediate these changes. Acute MG and CEL exposure, but not glucose, were able to induce similar effects on hippocampal slices, suggesting that conditions of high glucose concentrations are primarily toxic by elevating the rates of these glycation compounds, such as MG, and by generation of protein cross-links. Alterations in the secretion of S100B and the glutamatergic activity mediated by MG and AGEs can contribute to the brain dysfunction observed in diabetic patients.

  11. M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity.

    Directory of Open Access Journals (Sweden)

    Linda Cambier

    Full Text Available Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.

  12. Corticosteroid-Induced MKP-1 Represses Pro-Inflammatory Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM Cells.

    Science.gov (United States)

    Prabhala, Pavan; Bunge, Kristin; Ge, Qi; Ammit, Alaina J

    2016-10-01

    Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti-inflammatory pharmacotherapies, including corticosteroids, are front-line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid-inducible gene, mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP-1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein-tristetraprolin (TTP). But whether corticosteroid-induced MKP-1 acts via p38 MAPK-mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK-dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro-asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL-6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP-1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone-induced MKP-1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro-inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153-2158, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Mark W Robinson

    Full Text Available BACKGROUND: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3, liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains. Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. CONCLUSIONS/SIGNIFICANCE: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.

  14. Nonreciprocal signal routing in an active quantum network

    Science.gov (United States)

    Metelmann, A.; Türeci, H. E.

    2018-04-01

    As superconductor quantum technologies are moving towards large-scale integrated circuits, a robust and flexible approach to routing photons at the quantum level becomes a critical problem. Active circuits, which contain parametrically driven elements selectively embedded in the circuit, offer a viable solution. Here, we present a general strategy for routing nonreciprocally quantum signals between two sites of a given lattice of oscillators, implementable with existing superconducting circuit components. Our approach makes use of a dual lattice of overdamped oscillators linking the nodes of the main lattice. Solutions for spatially selective driving of the lattice elements can be found, which optimally balance coherent and dissipative hopping of microwave photons to nonreciprocally route signals between two given nodes. In certain lattices these optimal solutions are obtained at the exceptional point of the dynamical matrix of the network. We also demonstrate that signal and noise transmission characteristics can be separately optimized.

  15. Non-Cationic Proteins Are Associated with HIV Neutralizing Activity in Genital Secretions of Female Sex Workers.

    Science.gov (United States)

    Birse, Kenzie D M; Cole, Amy L; Hirbod, Taha; McKinnon, Lyle; Ball, Terry B; Westmacott, Garrett R; Kimani, Joshua; Plummer, Frank; Cole, Alexander M; Burgener, Adam; Broliden, Kristina

    2015-01-01

    Cationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection. CVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics. HIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3 yr, HESNHIV-positive groups, respectively. Gene ontology placed these proteins into functional categories including proteolysis, oxidation-reduction, and epidermal development. The proteins identified in this study include proteins previously associated with the HESN phenotype in other cohorts as well as novel proteins not yet associated with anti-HIV activities. While cationic proteins appear to contribute to the majority of the intrinsic HIV neutralizing activity in the CVS of low-risk women, a broader range of non-cationic proteins were associated with HIV neutralizing activity in HESN and HIV-positive female sex workers. These results indicate that novel protein factors found in CVS of women with high-risk sexual practices may have inherent antiviral activity, or are involved in other aspects of anti-HIV host defense, and warrant further exploration into their mode of action.

  16. The impact of pregnancy on anti-HIV activity of cervicovaginal secretions.

    Science.gov (United States)

    Hughes, Brenna L; Dutt, Riana; Raker, Christina; Barthelemy, Melody; Rossoll, Richard M; Ramratnam, Bharat; Wira, Charles R; Cu-Uvin, Susan

    2016-12-01

    Mucosal immunity of the female genital tract plays a critical role in defense against sexually transmitted infections like HIV. Pregnancy is associated with both structural and immunologic alterations in the genital mucosa, but the impact of these changes on its ability to suppress HIV infection is unknown. Current epidemiologic data are conflicting as to whether pregnancy increases the risk of HIV acquisition. The purpose of this study was to define the association between antimicrobial peptides and chemokines in cervicovaginal secretions and in vitro HIV infectivity among pregnant and nonpregnant women. Forty pregnant and 37 nonpregnant women were enrolled in a prospective longitudinal cohort study at a single tertiary care women's hospital in Providence, RI. Cervicovaginal lavage was performed at each study visit. For pregnant women, study visits occurred once per trimester, and there was an optional postpartum visit. For nonpregnant women, study visits occurred across a single cycle that was timed to occur in the proliferative, ovulatory, and secretory phases based on the presumption of a regular menstrual cycle. The impact of cervicovaginal lavage on HIV infectivity was evaluated using a TZM-bl assay and compared between pregnant and nonpregnant women for each visit. The previously validated TZM-bl assay, which uses a luciferase reporting gene to indicate HIV infection of TZM-bl cells, was measured with a luminometer with higher relative light units that indicate greater levels of in vitro HIV infection. Immune mediators were measured with a multiplex bead assay. HIV infectivity and median concentration of each mediator were compared between pregnant and nonpregnant groups with the Wilcoxon rank sum test. Cervicovaginal fluid from pregnant and nonpregnant women significantly decreased HIV infectivity in both groups compared with positive control (virus only; PHIV infectivity in both groups across all visits, except at the postpartum visit in the pregnant

  17. Chemokines: a new dendritic cell signal for T cell activation

    Directory of Open Access Journals (Sweden)

    Christoph A Thaiss

    2011-08-01

    Full Text Available Dendritic cells (DCs are the main inducers and regulators of cytotoxic T lymphocyte (CTL responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and licensed by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (classical licensing or by NKT cells (alternative licensing. Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

  18. Robust Indoor Human Activity Recognition Using Wireless Signals.

    Science.gov (United States)

    Wang, Yi; Jiang, Xinli; Cao, Rongyu; Wang, Xiyang

    2015-07-15

    Wireless signals-based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI) of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP) and access points (AP). First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions' CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO) signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM) based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds.

  19. Structural basis of arrestin-3 activation and signaling

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qiuyan; Perry, Nicole A.; Vishnivetskiy, Sergey A.; Berndt, Sandra; Gilbert, Nathaniel C.; Zhuo, Ya; Singh, Prashant K.; Tholen, Jonas; Ohi, Melanie D.; Gurevich, Eugenia V.; Brautigam, Chad A.; Klug, Candice S.; Gurevich, Vsevolod V.; Iverson, T.M. (UTSMC); (MCW); (Vanderbilt); (UASANS)

    2017-11-10

    A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP6) is a non-receptor activator of arrestin-3 and report the structure of IP6-activated arrestin-3 at 2.4-Å resolution. IP6-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP6 binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP6. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.

  20. Artifact suppression and analysis of brain activities with electroencephalography signals.

    Science.gov (United States)

    Rashed-Al-Mahfuz, Md; Islam, Md Rabiul; Hirose, Keikichi; Molla, Md Khademul Islam

    2013-06-05

    Brain-computer interface is a communication system that connects the brain with computer (or other devices) but is not dependent on the normal output of the brain (i.e., peripheral nerve and muscle). Electro-oculogram is a dominant artifact which has a significant negative influence on further analysis of real electroencephalography data. This paper presented a data adaptive technique for artifact suppression and brain wave extraction from electroencephalography signals to detect regional brain activities. Empirical mode decomposition based adaptive thresholding approach was employed here to suppress the electro-oculogram artifact. Fractional Gaussian noise was used to determine the threshold level derived from the analysis data without any training. The purified electroencephalography signal was composed of the brain waves also called rhythmic components which represent the brain activities. The rhythmic components were extracted from each electroencephalography channel using adaptive wiener filter with the original scale. The regional brain activities were mapped on the basis of the spatial distribution of rhythmic components, and the results showed that different regions of the brain are activated in response to different stimuli. This research analyzed the activities of a single rhythmic component, alpha with respect to different motor imaginations. The experimental results showed that the proposed method is very efficient in artifact suppression and identifying individual motor imagery based on the activities of alpha component.

  1. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  2. Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants

    NARCIS (Netherlands)

    Dirnberger, D.; Steinkellner, H.; Abdennebi, L.; Remy, J.J.; Wiel, van de D.

    2001-01-01

    We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal

  3. Activation of the sigma-1 receptor by haloperidol metabolites facilitates brain-derived neurotrophic factor secretion from human astroglia.

    Science.gov (United States)

    Dalwadi, Dhwanil A; Kim, Seongcheol; Schetz, John A

    2017-05-01

    Glial cells play a critical role in neuronal support which includes the production and release of the neurotrophin brain-derived neurotrophic factor (BDNF). Activation of the sigma-1 receptor (S1R) has been shown to attenuate inflammatory stress-mediated brain injuries, and there is emerging evidence that this may involve a BDNF-dependent mechanism. In this report we studied S1R-mediated BDNF release from human astrocytic glial cells. Astrocytes express the S1R, which mediates BDNF release when stimulated with the prototypical S1R agonists 4-PPBP and (+)-SKF10047. This effect could be antagonized by a selective concentration of the S1R antagonist BD1063. Haloperidol is known to have high affinity interactions with the S1R, yet it was unable to facilitate BDNF release. Remarkably, however, two metabolites of haloperidol, haloperidol I and haloperidol II (reduced haloperidol), were discovered to facilitate BDNF secretion and this effect was antagonized by BD1063. Neither 4-PPBP, nor either of the haloperidol metabolites affected the level of BDNF mRNA as assessed by qPCR. These results demonstrate for the first time that haloperidol metabolites I and II facilitate the secretion of BDNF from astrocytes by acting as functionally selective S1R agonists. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

    Directory of Open Access Journals (Sweden)

    Scott W. Messenger

    2015-11-01

    Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

  5. Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

    Science.gov (United States)

    Srivastava, Ankit; Ståhle, Mona; Pivarcsi, Andor; Sonkoly, Enikö

    2018-05-08

    Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

  6. Disentangling stellar activity from exoplanetary signals with interferometry

    Directory of Open Access Journals (Sweden)

    Ligi Roxanne

    2015-01-01

    Full Text Available Stellar activity can express as many forms at stellar surfaces: dark spots, convective cells, bright plages. Particularly, dark spots and bright plages add noise on photometric data or radial velocity measurements used to detect exoplanets, and thus lead to false detection or disrupt their derived parameters. Since interferometry provides a very high angular resolution, it may constitute an interesting solution to distinguish the signal of a transiting exoplanet and that of stellar activity. It has also been shown that granulation adds bias in visibility and closure phase measurements, affecting in turn the derived stellar parameters. We analyze the noises generated by dark spots on interferometric observables and compare them to exoplanet signals. We investigate the current interferometric instruments able to measure and disentangle these signals, and show that there is a lack in spatial resolution. We thus give a prospective of the improvements to be brought on future interferometers, which would also significantly extend the number of available targets.

  7. Dapper1 attenuates hepatic gluconeogenesis and lipogenesis by activating PI3K/Akt signaling.

    Science.gov (United States)

    Kuang, Jian-Ren; Zhang, Zhi-Hui; Leng, Wei-Ling; Lei, Xiao-Tian; Liang, Zi-Wen

    2017-05-15

    Studies have shown that hepatic insulin resistance, a disorder of glucose and lipid metabolism, plays a vital role in type 2 diabetes (T2D). To clarify the function of Dapper1 in glucose and lipid metabolism in the liver, we investigated the relationships between Dapper1 and adenosine triphosphate (ATP)- and Ca 2+ -mediated activation of PI3K/Akt. We observed a reduction in hepatic Dapper1 in db/db (mice that are homozygous for a spontaneous diabetes mutation) and HFD-induced diabetic mice with T2D. Hepatic overexpression of Dapper1 improved hyperglycemia, insulin resistance, and fatty liver. It also increased Akt (pAkt) signaling and repressed both gluconeogenesis and lipogenesis. Conversely, Ad-shDapper1-induced knockdown of hepatic Dapper1 promoted gluconeogenesis and lipogenesis. Furthermore, Dapper1 activated PI3K p110α/Akt in an insulin-independent manner by inducing ATP production and secretion in vitro. Blockade of P2 ATP receptors, the downstream phospholipase C (PLC), or the inositol triphosphate receptor (IP3R all reduced the Dapper1-induced increase in cytosolic free calcium and Dapper1-mediated PI3K/Akt activation, as did removal of calcium in the medium. In conclusion, Dapper1 attenuates hepatic gluconeogenesis and lipogenesis in T2D. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Robust Indoor Human Activity Recognition Using Wireless Signals

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2015-07-01

    Full Text Available Wireless signals–based activity detection and recognition technology may be complementary to the existing vision-based methods, especially under the circumstance of occlusions, viewpoint change, complex background, lighting condition change, and so on. This paper explores the properties of the channel state information (CSI of Wi-Fi signals, and presents a robust indoor daily human activity recognition framework with only one pair of transmission points (TP and access points (AP. First of all, some indoor human actions are selected as primitive actions forming a training set. Then, an online filtering method is designed to make actions’ CSI curves smooth and allow them to contain enough pattern information. Each primitive action pattern can be segmented from the outliers of its multi-input multi-output (MIMO signals by a proposed segmentation method. Lastly, in online activities recognition, by selecting proper features and Support Vector Machine (SVM based multi-classification, activities constituted by primitive actions can be recognized insensitive to the locations, orientations, and speeds.

  9. The microbe-secreted isopeptide poly-γ-glutamic acid induces stress tolerance in Brassica napus L. seedlings by activating crosstalk between H2O2 and Ca2+

    Science.gov (United States)

    Lei, Peng; Pang, Xiao; Feng, Xiaohai; Li, Sha; Chi, Bo; Wang, Rui; Xu, Zongqi; Xu, Hong

    2017-01-01

    Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide that has been shown to promote growth and enhance stress tolerance in crops. However, its site of action and downstream signaling pathways are still unknown. In this study, we investigated γ-PGA-induced tolerance to salt and cold stresses in Brassica napus L. seedlings. Fluorescent labeling of γ-PGA was used to locate the site of its activity in root protoplasts. The relationship between γ-PGA-induced stress tolerance and two signal molecules, H2O2 and Ca2+, as well as the γ-PGA-elicited signaling pathway at the whole plant level, were explored. Fluorescent labeling showed that γ-PGA did not enter the cytoplasm but instead attached to the surface of root protoplasm. Here, it triggered a burst of H2O2 in roots by enhancing the transcription of RbohD and RbohF, and the elicited H2O2 further activated an influx of Ca2+ into root cells. Ca2+ signaling was transmitted via the stem from roots to leaves, where it elicited a fresh burst of H2O2, thus promoting plant growth and enhancing stress tolerance. On the basis of these observation, we propose that γ-PGA mediates stress tolerance in Brassica napus seedlings by activating an H2O2 burst and subsequent crosstalk between H2O2 and Ca2+ signaling. PMID:28198821

  10. GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production

    International Nuclear Information System (INIS)

    Wu, Yong-hong; Li, Quan; Li, Ping; Liu, Bei

    2016-01-01

    LPS stimulation in macrophages/monocytes induces TNFα production. We here tested the potential effect of GSK621, a novel AMP-activated protein kinase (AMPK) activator, against the process. In RAW264.7 macrophages, murine bone marrow-derived macrophages (BMDMs), and chronic obstructive pulmonary disease (COPD) patients' monocytes, GSK621 significantly inhibited LPS-induced TNFα protein secretion and mRNA synthesis. Inhibition of AMPK, through AMPKα shRNA knockdown or dominant negative mutation (T172A), almost abolished GSK621's suppression on TNFα in RAW264.7 cells. Reversely, forced-expression of a constitutively-active AMPKα (T172D) mimicked GSK621 actions and reduced LPS-induced TNFα production. Molecularly, GSK621 suppressed LPS-induced reactive oxygen species (ROS) production and nuclear factor kappa B (NFκB) activation. In vivo, GSK621 oral administration inhibited LPS-induced TNFα production and endotoxin shock in mice. In summary, GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production in macrophages/monocytes. - Highlights: • GSK621 inhibits LPS-induced TNFα production/expression in RAW264.7 cells and BMDMs. • GSK621 inhibits LPS-induced TNFα production/expression in COPD patients' PBMCs. • GSK621's inhibition on TNFα production by LPS requires AMPK activation. • GSK621 inhibits LPS-induced ROS production and NFκB activation, dependent on AMPK. • GSK621 oral administration inhibits LPS-induced TNFα production and endotoxin shock in mice.

  11. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from Lebanese cow’s milk

    Directory of Open Access Journals (Sweden)

    Imad AL Kassaa

    2016-12-01

    Full Text Available AL Kassaa, I., Safourim, N., Mostafa, N. and Hamze, M. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from lebanese cow’s milk. 2016. Lebanese Science Journal, 17(2: 166-176. Lactic acid bacteria (LAB are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are used as probiotics and preservatives against pathogens and spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow’s milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showed an antimicrobial activity against Listeria monocytogenes ATCC® 19115™. One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtii by pyrosequencing method. The active substance was extracted using solvent method. This Bacteriocin like Inhibitory Substances “BLIS” can support a high temperature (121 ˚C for a long time and resist pH variation. The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeria monocytogenes and Penicillium spp. BLIS BL4 can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food products.

  12. Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-κB and MAPK

    International Nuclear Information System (INIS)

    Ci Xinxin; Song Yu; Zeng Fanqin; Zhang Xuemei; Li Hongyu; Wang Xinrui; Cui Junqing; Deng Xuming

    2008-01-01

    Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH 2 -terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-κB translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells

  13. Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

    Science.gov (United States)

    Kimura, Yukihiro; Chihara, Kazuyasu; Honjoh, Chisato; Takeuchi, Kenji; Yamauchi, Shota; Yoshiki, Hatsumi; Fujieda, Shigeharu; Sada, Kiyonao

    2014-11-07

    Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. MMTV-Wnt1 and -DeltaN89beta-catenin induce canonical signaling in distinct progenitors and differentially activate Hedgehog signaling within mammary tumors.

    Directory of Open Access Journals (Sweden)

    Brigitte Teissedre

    Full Text Available Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+ER(-PR(-CD24(highCD49f(low profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14(+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+/p63(+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.

  15. DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

    Science.gov (United States)

    Pettigrew, Christopher A; Clerkin, John S; Cotter, Thomas G

    2012-12-01

    Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

  16. Apoptosis of bone marrow mesenchymal stem cells caused by homocysteine via activating JNK signal.

    Directory of Open Access Journals (Sweden)

    Benzhi Cai

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs are capable of homing to and repair damaged myocardial tissues. Apoptosis of BMSCs in response to various pathological stimuli leads to the attenuation of healing ability of BMSCs. Plenty of evidence has shown that elevated homocysteine level is a novel independent risk factor of cardiovascular diseases. The present study was aimed to investigate whether homocysteine may induce apoptosis of BMSCs and its underlying mechanisms. Here we uncovered that homocysteine significantly inhibited the cellular viability of BMSCs. Furthermore, TUNEL, AO/EB, Hoechst 333342 and Live/Death staining demonstrated the apoptotic morphological appearance of BMSCs after homocysteine treatment. A distinct increase of ROS level was also observed in homocysteine-treated BMSCs. The blockage of ROS by DMTU and NAC prevented the apoptosis of BMSCs induced by homocysteine, indicating ROS was involved in the apoptosis of BMSCs. Moreover, homocysteine also caused the depolarization of mitochondrial membrane potential of BMSCs. Furthermore, apoptotic appearance and mitochondrial membrane potential depolarization in homocysteine-treated BMSCs was significantly reversed by JNK inhibitor but not p38 MAPK and ERK inhibitors. Western blot also confirmed that p-JNK was significantly activated after exposing BMSCs to homocysteine. Homocysteine treatment caused a significant reduction of BMSCs-secreted VEGF and IGF-1 in the culture medium. Collectively, elevated homocysteine induced the apoptosis of BMSCs via ROS-induced the activation of JNK signal, which provides more insight into the molecular mechanisms of hyperhomocysteinemia-related cardiovascular diseases.

  17. Inflammation activates the interferon signaling pathways in taste bud cells.

    Science.gov (United States)

    Wang, Hong; Zhou, Minliang; Brand, Joseph; Huang, Liquan

    2007-10-03

    Patients with viral and bacterial infections or other inflammatory illnesses often experience taste dysfunctions. The agents responsible for these taste disorders are thought to be related to infection-induced inflammation, but the mechanisms are not known. As a first step in characterizing the possible role of inflammation in taste disorders, we report here evidence for the presence of interferon (IFN)-mediated signaling pathways in taste bud cells. IFN receptors, particularly the IFN-gamma receptor IFNGR1, are coexpressed with the taste cell-type markers neuronal cell adhesion molecule and alpha-gustducin, suggesting that both the taste receptor cells and synapse-forming cells in the taste bud can be stimulated by IFN. Incubation of taste bud-containing lingual epithelia with recombinant IFN-alpha and IFN-gamma triggered the IFN-mediated signaling cascades, resulting in the phosphorylation of the downstream STAT1 (signal transducer and activator of transcription protein 1) transcription factor. Intraperitoneal injection of lipopolysaccharide or polyinosinic:polycytidylic acid into mice, mimicking bacterial and viral infections, respectively, altered gene expression patterns in taste bud cells. Furthermore, the systemic administration of either IFN-alpha or IFN-gamma significantly increased the number of taste bud cells undergoing programmed cell death. These findings suggest that bacterial and viral infection-induced IFNs can act directly on taste bud cells, affecting their cellular function in taste transduction, and that IFN-induced apoptosis in taste buds may cause abnormal cell turnover and skew the representation of different taste bud cell types, leading to the development of taste disorders. To our knowledge, this is the first study providing direct evidence that inflammation can affect taste buds through cytokine signaling pathways.

  18. Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes.

    Science.gov (United States)

    Jiang, X C; Inouchi, J; Wang, D; Halpern, M

    1990-05-25

    Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal

  19. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    Directory of Open Access Journals (Sweden)

    Taslima T. Lina

    2016-07-01

    Full Text Available Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40% were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2 and p38 mitogen-activated protein kinase (MAPK pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4 expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

  20. Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

    Science.gov (United States)

    Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

    2016-09-15

    We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. Copyright © 2016. Published by Elsevier B.V.

  1. Peroxisome proliferator-activated receptor {alpha} agonists modulate Th1 and Th2 chemokine secretion in normal thyrocytes and Graves' disease

    Energy Technology Data Exchange (ETDEWEB)

    Antonelli, Alessandro, E-mail: a.antonelli@med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Ferrari, Silvia Martina, E-mail: sm.ferrari@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Frascerra, Silvia, E-mail: lafrasce@gmail.com [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Corrado, Alda, E-mail: dala_res@hotmail.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Pupilli, Cinzia, E-mail: c.pupilli@dfc.unifi.it [Endocrinology Unit, Azienda Ospedaliera Careggi and University of Florence, Viale Morgagni 85, I-50134, Florence (Italy); Bernini, Giampaolo, E-mail: g.bernini@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Benvenga, Salvatore, E-mail: s.benvenga@me.nettuno.it [Department of Clinical and Experimental Medicine, Section of Endocrinology, University of Messina, Piazza Pugliatti 1, I-98122, Messina (Italy); Ferrannini, Ele, E-mail: eferrannini@med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy); Fallahi, Poupak, E-mail: poupak@int.med.unipi.it [Department of Internal Medicine, University of Pisa-School of Medicine, Via Roma 67, I-56100, Pisa (Italy)

    2011-07-01

    Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR){alpha} activation on the prototype Th1 [chemokine (C-X-C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C-C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells. The role of PPAR{alpha} and PPAR{gamma} activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN){gamma} and tumor necrosis factor (TNF){alpha}. IFN{gamma} stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNF{alpha} alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFN{gamma} and TNF{alpha} had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPAR{alpha} activators inhibited the secretion of both chemokines (stimulated with IFN{gamma} and TNF{alpha}) at a level higher (for CXCL10, about 60-72%) than PPAR{gamma} agonists (about 25-35%), which were confirmed to inhibit CXCL10, but not CCL2. Our data show that CCL2 is modulated by IFN{gamma} and TNF{alpha} in GD and normal thyrocytes. Furthermore we first show that PPAR{alpha} activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPAR{alpha} may be involved in the modulation of the immune response in the thyroid.

  2. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    Science.gov (United States)

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  3. Signaling lymphocytic activation molecules Slam and cancers: friends or foes?

    Science.gov (United States)

    Fouquet, Gregory; Marcq, Ingrid; Debuysscher, Véronique; Bayry, Jagadeesh; Rabbind Singh, Amrathlal; Bengrine, Abderrahmane; Nguyen-Khac, Eric; Naassila, Mickael; Bouhlal, Hicham

    2018-03-23

    Signaling Lymphocytic Activation Molecules (SLAM) family receptors are initially described in immune cells. These receptors recruit both activating and inhibitory SH2 domain containing proteins through their Immunoreceptor Tyrosine based Switch Motifs (ITSMs). Accumulating evidence suggest that the members of this family are intimately involved in different physiological and pathophysiological events such as regulation of immune responses and entry pathways of certain viruses. Recently, other functions of SLAM, principally in the pathophysiology of neoplastic transformations have also been deciphered. These new findings may prompt SLAM to be considered as new tumor markers, diagnostic tools or potential therapeutic targets for controlling the tumor progression. In this review, we summarize the major observations describing the implications and features of SLAM in oncology and discuss the therapeutic potential attributed to these molecules.

  4. Herpes simplex virus triggers activation of calcium-signaling pathways

    Science.gov (United States)

    Cheshenko, Natalia; Del Rosario, Brian; Woda, Craig; Marcellino, Daniel; Satlin, Lisa M.; Herold, Betsy C.

    2003-01-01

    The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy. PMID:14568989

  5. High Fat Diet Attenuates the Anticontractile Activity of Aortic PVAT via a Mechanism Involving AMPK and Reduced Adiponectin Secretion

    Directory of Open Access Journals (Sweden)

    Tarek A. M. Almabrouk

    2018-02-01

    Full Text Available Background and aim: Perivascular adipose tissue (PVAT positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD on aortic PVAT and whether any changes involved AMPK.Methods: Wild type Sv129 (WT and AMPKα1 knockout (KO mice aged 8 weeks were fed normal diet (ND or HFD (42% kcal fat for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later.Key results: Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markers Nos2 and Il1b and the M2 marker Chil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice.Conclusions: High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to

  6. Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

    Science.gov (United States)

    Gysler, Stefan M; Mulla, Melissa J; Guerra, Marta; Brosens, Jan J; Salmon, Jane E; Chamley, Lawrence W; Abrahams, Vikki M

    2016-07-01

    What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was

  7. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  8. Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA, an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f. var. glaucocalyx (Maxim. Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml significantly inhibited platelet aggregation induced by collagen (P<0.001 and CRP (P<0.01, a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

  9. Andrographolide Inhibits Inflammatory Cytokines Secretion in LPS-Stimulated RAW264.7 Cells through Suppression of NF-κB/MAPK Signaling Pathway.

    Science.gov (United States)

    Li, Yu; He, Shengnan; Tang, Jishun; Ding, Nana; Chu, Xiaoyan; Cheng, Lianping; Ding, Xuedong; Liang, Ting; Feng, Shibin; Rahman, Sajid Ur; Wang, Xichun; Wu, Jinjie

    2017-01-01

    Andrographolide, the main active component extracted from Andrographis paniculata (Burm.f.) Wall. ex Nees, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the molecular mechanisms of Andrographolide in modifying lipopolysaccharide- (LPS-) induced signaling pathway in RAW264.7 cells. An in vitro model of inflammation was induced by LPS in mouse RAW264.7 cells in the presence of Andrographolide. The concentration and expression levels of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The nuclear level of NF- κ B was measured by an electrophoretic mobility shift assay (EMSA). The expression levels of NF- κ B, p38, ERK, and JNK were determined by western blot. Andrographolide dose-dependently inhibited the release and mRNA expression of TNF- α , IL-6, and IL-1 β in LPS-stimulated RAW264.7 cells. The nuclear level of p65 protein was decreased in Andrographolide treatment group. Western blot analysis showed that Andrographolide suppressed LPS-induced NF- κ B activation and the phosphorylation of IkBa, ERK1/2, JNK, and p38. These results suggest that Andrographolide exerts an anti-inflammatory effect by inhibiting the activation of NF- κ B/MAPK signaling pathway and the induction of proinflammatory cytokines.

  10. Andrographolide Inhibits Inflammatory Cytokines Secretion in LPS-Stimulated RAW264.7 Cells through Suppression of NF-κB/MAPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yu Li

    2017-01-01

    Full Text Available Andrographolide, the main active component extracted from Andrographis paniculata (Burm.f. Wall. ex Nees, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the molecular mechanisms of Andrographolide in modifying lipopolysaccharide- (LPS- induced signaling pathway in RAW264.7 cells. An in vitro model of inflammation was induced by LPS in mouse RAW264.7 cells in the presence of Andrographolide. The concentration and expression levels of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay (ELISA and quantitative real-time polymerase chain reaction (qRT-PCR, respectively. The nuclear level of NF-κB was measured by an electrophoretic mobility shift assay (EMSA. The expression levels of NF-κB, p38, ERK, and JNK were determined by western blot. Andrographolide dose-dependently inhibited the release and mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 cells. The nuclear level of p65 protein was decreased in Andrographolide treatment group. Western blot analysis showed that Andrographolide suppressed LPS-induced NF-κB activation and the phosphorylation of IkBa, ERK1/2, JNK, and p38. These results suggest that Andrographolide exerts an anti-inflammatory effect by inhibiting the activation of NF-κB/MAPK signaling pathway and the induction of proinflammatory cytokines.

  11. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  12. Differential effects of the steaming time and frequency for manufactured red Liriope platyphylla on nerve growth factor secretion ability, nerve growth factor receptor signaling pathway and regulation of calcium concentration.

    Science.gov (United States)

    Choi, Sun Il; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Hye Ryun; Lee, Young Ju; Son, Hong Joo; Lee, Hee Seob; Lee, Jong Sup; Kim, Hak Jin; Hwang, Dae Youn

    2012-11-01

    The herb Liriope platyphylla (LP) has been considered to have curative properties for diabetes, asthma and neurodegenerative disorders. To examine the effects of steaming time and frequency of manufactured red LP (RLP) on the nerve growth factor (NGF) secretion ability and NGF receptor signaling pathway, the NGF concentration, cell differentiation, NGF signaling pathway and calcium concentration were analyzed in neuronal cells treated with several types of LPs manufactured under different conditions. The maximum NGF secretion was observed in B35 cells treated with 50 µg/ml LP extract steamed for 9 h (9-SLP) and with two repeated steps (3 h steaming and 24 h air-dried) carried out 7 times (7-SALP). No significant changes in viability were detected in any of the cells treated with the various LPs, with the exception of 0-SLP and 0-SALP. In addition, PC12 cell differentiation was induced by treatment with the NGF-containing conditional medium (CM) collected from the RLP-treated cells. The levels of TrkA and extracellular signal-regulated kinase (ERK) phosphorylation in the high affinity NGF receptor signaling pathway were significantly higher in the cells treated with 3-SLP or 1-SALP/3-SALP CM compared with those treated with the vehicle CM. In the low affinity NGF receptor pathway, the expression levels of most components were higher in the 9-, 15- and 24-SALP CM-treated cells compared with the vehicle CM-treated cells. However, this level was significantly altered in cells treated with 3-SALP CM. Furthermore, an examination of the RLP function on calcium regulation revealed that only the LP- or RLP-treated cells exhibited changes in intracellular and extracellular calcium levels. RLP induced a significant decrease in the intracellular calcium levels and an increase in the extracellular calcium levels. These results suggest the possibility that steaming-processed LP may aid in the relief of neurodegenerative diseases through the NGF secretion ability and NGF

  13. Secret Places.

    Science.gov (United States)

    Ridolfi, Kerry

    1997-01-01

    Argues that children are as deep as the ocean, with secret places inside of them waiting to be opened. Notes that it is powerful for students to learn they can make sense of the world through words, and describes inviting them into poetry as they read poetry, create poetry packets, and write and revise poems. (SR)

  14. Transfer function between EEG and BOLD signals of epileptic activity

    Directory of Open Access Journals (Sweden)

    Marco eLeite

    2013-01-01

    Full Text Available Simultaneous EEG-fMRI recordings have seen growing application in the evaluation of epilepsy, namely in the characterization of brain networks related to epileptic activity. In EEG-correlated fMRI studies, epileptic events are usually described as boxcar signals based on the timing information retrieved from the EEG, and subsequently convolved with a heamodynamic response function to model the associated BOLD changes. Although more flexible approaches may allow a higher degree of complexity for the haemodynamics, the issue of how to model these dynamics based on the EEG remains an open question. In this work, a new methodology for the integration of simultaneous EEG-fMRI data in epilepsy is proposed, which incorporates a transfer function from the EEG to the BOLD signal. Independent component analysis (ICA of the EEG is performed, and a number of metrics expressing different models of the EEG-BOLD transfer function are extracted from the resulting time courses. These metrics are then used to predict the fMRI data and to identify brain areas associated with the EEG epileptic activity. The methodology was tested on both ictal and interictal EEG-fMRI recordings from one patient with a hypothalamic hamartoma. When compared to the conventional analysis approach, plausible, consistent and more significant activations were obtained. Importantly, frequency-weighted EEG metrics yielded superior results than those weighted solely on the EEG power, which comes in agreement with previous literature. Reproducibility, specificity and sensitivity should be addressed in an extended group of patients in order to further validate the proposed methodology and generalize the presented proof of concept.

  15. Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

    Science.gov (United States)

    Lu, D; Yang, H; Raizada, M K

    1996-12-01

    Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

  16. DMPD: Signaling pathways activated by microorganisms. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17303405 Signaling pathways activated by microorganisms. Takeuchi O, Akira S. Curr ...Opin Cell Biol. 2007 Apr;19(2):185-91. Epub 2007 Feb 15. (.png) (.svg) (.html) (.csml) Show Signaling pathways activated by microorg...anisms. PubmedID 17303405 Title Signaling pathways activated by microorganisms. Auth

  17. DMPD: Macrophage activation by endogenous danger signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18161744 Macrophage activation by endogenous danger signals. Zhang X, Mosser DM. J ...Pathol. 2008 Jan;214(2):161-78. (.png) (.svg) (.html) (.csml) Show Macrophage activation by endogenous dange...r signals. PubmedID 18161744 Title Macrophage activation by endogenous danger signals. Authors Zhang X, Moss

  18. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    Science.gov (United States)

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  19. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xue-Feng [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Respiration, Qinghai Provincial People' s Hospital, Xining (China); Wang, Hua; Xiao, Feng-Jun [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Yin, Yue [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Hematology, Peking University First Hospital, Beijing (China); Xu, Qin-Qin [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Ge, Ri-Li, E-mail: geriligao@hotmail.com [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Wang, Li-Sheng, E-mail: wangls@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2016-02-12

    MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

  20. Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation

    International Nuclear Information System (INIS)

    Stunz, L.L.; Feldbush, T.L.

    1986-01-01

    A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in this laboratory for its ability to act as a mitogen for rat lymphocytes. This preparation (STM) has been found to be a potent simulator of B lymphocyte proliferation, as measured both by 3 H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle. Previous investigations by others have shown that at least two sets of signals are required for B cell differentiation; (a) proliferation signals that may consist of both a stimulator of B cell conversion from G 0 to G 1 and growth factors, and (b) differentiation signals that probably include at least two B cell differentiation factors (BCDF). When STM was tested in a differentiation system it did not drive purified B cells to differentiate to PFC, either alone or when supplemented with a supernatant from concanavalin A-stimulated spleen cells (CAS). However, when both CAS and dextran sulfate (DXS) were supplied to the STM-stimulated cells, a large number of PFC resulted. DXT does not act by stimulating an additional, CAS-responsive B cell subset, since it has only a marginal effect upon 3 H-TdR uptake and does not increase the number of B cells in cycle when used together with STM. The authors that the two agents may be acting sequentially: STM stimulates the B cells to proliferate, and DXS drives the proliferating cells to become responsive to CAS. This suggests that the signals for B cell differentiation must consist of at least three activities: a trigger to stimulate the cells to proliferate, a factor to drive the cells to a BCDF-responsive state, and a BCDF that can drive the cells to secrete antibody

  1. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  2. Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways.

    Science.gov (United States)

    Xie, Song-Zi; Hao, Ran; Zha, Xue-Qiang; Pan, Li-Hua; Liu, Jian; Luo, Jian-Ping

    2016-08-01

    The present work aimed at investigating the pattern recognition receptor (PRR) and immunostimulatory mechanism of a purified Dendrobium huoshanense polysaccharide (DHP). We found that DHP could bind to the surface of macrophages and stimulate macrophages to secrete NO, TNF-α and IL-1β. To unravel the mechanism for the binding of DHP to macrophages, flow cytometry, confocal laser-scanning microscopy, affinity electrophoresis, SDS-PAGE and western blotting were employed to verify the type of PRR responsible for the recognition of DHP by RAW264.7 macrophages and peritoneal macrophages of C3H/HeN and C3H/HeJ macrophages. Results showed that toll-like receptor 4 (TLR4) was an essential receptor for macrophages to directly bind DHP. Further, the phosphorylation of ERK, JNK, Akt and p38 were observed to be time-dependently promoted by DHP, as well as the nuclear translocation of NF-κB p65. These results suggest that DHP activates macrophages via its direct binding to TLR4 to trigger TLR4 signaling pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Shared Genetic Control of Brain Activity During Sleep and Insulin Secretion: A Laboratory-Based Family Study.

    Science.gov (United States)

    Morselli, Lisa L; Gamazon, Eric R; Tasali, Esra; Cox, Nancy J; Van Cauter, Eve; Davis, Lea K

    2018-01-01

    Over the past 20 years, a large body of experimental and epidemiologic evidence has linked sleep duration and quality to glucose homeostasis, although the mechanistic pathways remain unclear. The aim of the current study was to determine whether genetic variation influencing both sleep and glucose regulation could underlie their functional relationship. We hypothesized that the genetic regulation of electroencephalographic (EEG) activity during non-rapid eye movement sleep, a highly heritable trait with fingerprint reproducibility, is correlated with the genetic control of metabolic traits including insulin sensitivity and β-cell function. We tested our hypotheses through univariate and bivariate heritability analyses in a three-generation pedigree with in-depth phenotyping of both sleep EEG and metabolic traits in 48 family members. Our analyses accounted for age, sex, adiposity, and the use of psychoactive medications. In univariate analyses, we found significant heritability for measures of fasting insulin sensitivity and β-cell function, for time spent in slow-wave sleep, and for EEG spectral power in the delta, theta, and sigma ranges. Bivariate heritability analyses provided the first evidence for a shared genetic control of brain activity during deep sleep and fasting insulin secretion rate. © 2017 by the American Diabetes Association.

  4. Wnt/β-catenin signaling activates nephronectin expression in osteoblasts

    International Nuclear Information System (INIS)

    Ikehata, Mikiko; Yamada, Atsushi; Morimura, Naoko; Itose, Masakatsu; Suzawa, Tetsuo; Shirota, Tatsuo; Chikazu, Daichi; Kamijo, Ryutaro

    2017-01-01

    Nephronectin (Npnt), an extracellular matrix protein, is considered to play critical roles as an adhesion molecule in the development and functions of various organs and tissues, such as the kidneys and bone. In the present study, we found that Wnt3a strongly enhanced Npnt mRNA expression in osteoblast-like MC3T3-E1 cells, while it also induced an increase in Npnt gene expression in both time- and dose-dependent manners via the Wnt/β-catenin signaling pathway. These results suggest novel mechanisms for Wnt3a-induced osteoblast proliferation and cell survival via Npnt gene expression. - Highlights: • Wnt3a enhances nephronectin gene expression. • Nephronectin gene induction by Wnt3a is occurred by time- and dose-dependent manner. • Expression of nephronectin is regulated via β-catenin activation.

  5. RSA-Based Secret Handshakes

    OpenAIRE

    Vergnaud , Damien

    2006-01-01

    A secret handshake mechanism allows two entities, members of a same group, to authenticate each other secretly. This primitive was introduced recently by Balfanz, Durfee, Shankar, Smetters, Staddon and Wong and, so far, all the schemes proposed are based on discrete log systems. This paper proposes three new secret handshake protocols secure against active impersonator and detector adversaries. Inspired by two RSA-based key agreement protocols introduced by Okamoto and Tanaka in 1989 and Gira...

  6. Unraveling the Wnt secretion pathway

    NARCIS (Netherlands)

    Harterink, M.

    2011-01-01

    The Wnt family of signaling proteins has essential functions in development and adult tissue homeostasis throughout the animal kingdom. Although signaling cascades triggered by Wnt proteins have been extensively studied, much remains to be learned about how Wnts are produced and secreted and how

  7. Studies quantifying modulatory effects of inhaled NO2 and SO2 on tracheal mucus secretion, proliferative activity of airway epithelium and architecture of lung parenchyma

    International Nuclear Information System (INIS)

    Wagner, U.; Barth, P.J.; Bredenbroeker, D.; Haase, H.; Locher, A.; Janssen, P.; Yu, F.; Wichert, P. von

    1995-10-01

    The following studies were designed to quantify changes in tracheal mucus secretion and epithelial proliferation of peripheral airways induced by inhaled NO 2 and SO 2 . Groups of male Sprague-Dawley rats were exposed alternatively to 1, 5, 10 and 20 ppm NO 2 and SO 2 the exposure-time being 3 or 25 days (d) respectively. Studies of tracheal mucus secretion radiolabelling mucins with 35 S clearly demonstrated a concentration dependant modulation of mucus secretion. We were able to demonstrate for the first time a significant increase of mucus secretion due to submucosal application of the peptide hormone GLP-1(7-36)amide. We were able to demonstrate amylin to be a potent secretagogue, dose-dependently stimulating mucus secretion. Our morphologic data reveal the effects caused by concentrations between 4-5 ppm NO 2 to be so small, that they are hardly detectable at light microscopic level. The assessment of proliferative activity, however, clearly demonstrates an increased proliferation due to even lower concentrations indicating, that even 1 ppm is able to cause epithelial impairment with consecutive regeneration. Double-labelling techniques of proliferation markers and the 10 kD Clara cell specific antigen reveal the Clara cell to be the only source for epithelial regeneration in peripheral airways under the reported experimental conditions of this study. (orig.) [de

  8. Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters.

    Science.gov (United States)

    Zhang, Xuejun C; Liu, Min; Lu, Guangyuan; Heng, Jie

    2018-03-01

    Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR. Based on our membrane potential-driving hypothesis, we propose a general energy-coupling mechanism for secondary-active antiporters. This putative mechanism provides a common framework for understanding poly-specificity of most-if not all-MDR transporters. © 2017 The Protein Society.

  9. Profiling of Cytokines Secreted by Conventional Aqueous Outflow Pathway Endothelial Cells Activated In Vitro and Ex Vivo With Laser Irradiation.

    Science.gov (United States)

    Alvarado, Jorge A; Chau, Phuonglan; Wu, Jianfeng; Juster, Richard; Shifera, Amde Selassie; Geske, Michael

    2015-11-01

    To profile which cytokine genes are differentially expressed (DE) as up- or downregulated by cultured human trabecular meshwork (TMEs) and Schlemm's canal endothelial cells (SCEs) after three experimental treatments consisting of selective laser trabeculoplasty (SLT) irradiation, exposure to media conditioned either by SLT-irradiated TMEs (TME-cm) or by SCEs (SCE-cm). Also, to profile which cytokines are upregulated ex vivo in SLT-irradiated human conventional aqueous outflow pathway (CAOP) tissues. After each treatment, Affymetrix microarray assays were used to detect upregulated and downregulated genes for cytokines and their receptors in TMEs and SCEs. ELISA and protein antibody arrays were used to detect upregulated cytokines secreted in SLT-irradiated CAOP tissues ex vivo. The SLT irradiation upregulated numerous cytokine genes in TMEs, but only a few in SCEs. Exposure to TME- and SCE-cm induced SCEs to upregulate many more cytokine genes than TMEs. Selective laser trabeculoplasty irradiation and exposure to TME-cm downregulated several cytokine genes in TMEs but none in SCEs. Selective laser trabeculoplasty irradiation induced one upregulated and three downregulated cytokine-receptor genes in TMEs but none in SCEs. Exposure to TME-cm induced upregulation of one and downregulation of another receptor gene in TMEs, whereas two unique cytokine-receptor genes were upregulated in SCEs. Cytokine protein expression analysis showed that at least eight cytokines were upregulated in SLT-irradiated human CAOP tissues in situ/ex vivo. This study has helped us identify a cytokine signaling pathway and to consider newly identified mechanisms regulating aqueous outflow that may lay the foundation for the future development of cytokine-based glaucoma therapies.

  10. Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells*

    Science.gov (United States)

    Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

    2012-01-01

    The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. PMID:22669945

  11. Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells.

    Science.gov (United States)

    Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

    2012-07-27

    The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

  12. Pretreatment with quercetin prevents changes in lymphocytes E-NTPDase/E-ADA activities and cytokines secretion in hyperlipidemic rats.

    Science.gov (United States)

    Braun, Josiane B S; Ruchel, Jader B; Manzoni, Alessandra G; Abdalla, Fátima H; Casalli, Emerson A; Castilhos, Lívia G; Passos, Daniela F; Leal, Daniela B R

    2017-11-29

    Hyperlipidemia (HL) is a condition associated with endothelial dysfunction and inflammatory disorders. Purinergic system ectoenzymes play an important role in modulating the inflammatory and immune response. This study investigated whether the preventive treatment with quercetin is able to prevent changes caused by hyperlipidemia in the purinergic system, through the activities of E-NTPDase and E-ADA in lymphocytes, and quantify the nucleotides and nucleoside, and the secretion of anti- and proinflammatory cytokines. Animals were divided into saline/control, saline/quercetin 5 mg/kg, saline/quercetin 25 mg/kg, saline/quercetin 50 mg/kg, saline/simvastatin (0.04 mg/kg), hyperlipidemia, hyperlipidemia/quercetin 5 mg/kg, hyperlipidemia/quercetin 25 mg/kg, hyperlipidemia/quercetin 50 mg/kg, and hyperlipidemia/simvastatin. Animals were pretreated with quercetin for 30 days and hyperlipidemia was subsequently induced by intraperitoneal administration of 500 mg/kg of poloxamer-407. Simvastatin was administered after the induction of hyperlipidemia. Lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Serum was separated for the cytokines and nucleotide/nucleoside quantification. E-NTPDase and E-ADA activities were increased in lymphocytes from hyperlipidemic rats and pretreatment with quercetin was able to prevent the increase in the activities of these enzymes caused by hyperlipidemia. Hyperlipidemic rats when receiving pretreatment with quercetin and treatment with simvastatin showed decreased levels of ATP and ADP when compared to the untreated hyperlipidemic group. The IFN-γ and IL-4 cytokines were increased in the hyperlipidemic group when compared with control group, and decreased when hyperlipidemic rats received the pretreatment with quercetin. However, pretreatment with quercetin was able to prevent the alterations caused by hyperlipidemia probably by regulating the inflammatory process. We can suggest that the quercetin is a

  13. Egg cell-secreted EC1 triggers sperm cell activation during double fertilization.

    Science.gov (United States)

    Sprunck, Stefanie; Rademacher, Svenja; Vogler, Frank; Gheyselinck, Jacqueline; Grossniklaus, Ueli; Dresselhaus, Thomas

    2012-11-23

    Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

  14. Cdk5 inhibitory peptide (CIP inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

    Directory of Open Access Journals (Sweden)

    Ya-Li Zheng

    Full Text Available Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs, however was detectable in the long exposure in HG cells (24 hrs and 48 hrs. Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5 with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.

  15. BLM promotes the activation of Fanconi Anemia signaling pathway.

    Science.gov (United States)

    Panneerselvam, Jayabal; Wang, Hong; Zhang, Jun; Che, Raymond; Yu, Herbert; Fei, Peiwen

    2016-05-31

    Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.

  16. Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qiang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); The First Affiliated Hospital of Xiamen University, Xiamen (China); Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); Yu, Chundong, E-mail: cdyu@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China)

    2011-06-17

    Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

  17. Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

    International Nuclear Information System (INIS)

    Chen, Qiang; Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang; Yu, Chundong

    2011-01-01

    Highlights: → Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. → FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. → FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. → FGFR4-ECD reduced tetracycline-induced fatty liver in mice. → FGFR4-ECD partially restored tetracycline-repressed PPARα expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor α (PPARα), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

  18. Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2−/− Lung Fibroblasts

    Directory of Open Access Journals (Sweden)

    Mandar Dave

    2017-12-01

    Full Text Available The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF in lung fibroblasts derived from COX-2−/− but not wild-type (WT or COX-1−/− mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2−/− fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2−/− was “prostaglandin-independent.” GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2−/− cells. Furthermore, COX-2−/− fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

  19. The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

    2012-10-01

    Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

  20. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    International Nuclear Information System (INIS)

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-01-01

    Highlights: → Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. → Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca 2+ permeant SACs. → The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. → Stretch-induced ET-1 production depends on a calcium influx. → SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch ( 2+ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  1. Corticosteroid-Induced MKP-1 Represses Pro-Inflammatory Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM Cells

    NARCIS (Netherlands)

    Prabhala, Pavan; Ammit, Alaina; Bunge, Kristin; Ge, Qi

    2016-01-01

    Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti-inflammatory pharmacotherapies, including corticosteroids, are front-line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their

  2. Effects of oral stimulation with capsaicin on salivary secretion and neural activities in the autonomic system and the brain

    Directory of Open Access Journals (Sweden)

    Yoko Kono

    2018-06-01

    Conclusion: These results suggest that oral stimulation with capsaicin may be effective in improving oral conditions by increasing salivary flow and SIgA secretion, and in enhancing physical and mental conditions as indicated by sympathetic nerve and EEG changes.

  3. Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

    Science.gov (United States)

    Bhattacharyya, S; Ghosh, S; Jhonson, P L; Bhattacharya, S K; Majumdar, S

    2001-03-01

    Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

  4. Salivary Gland Secretion.

    Science.gov (United States)

    Dorman, H. L.; And Others

    1981-01-01

    Describes materials and procedures for an experiment utilizing a live dog to demonstrate: (1) physiology of the salivary gland; (2) parasympathetic control of the salivary gland; (3) influence of varying salivary flow rates on sodium and potassium ions, osmolarity and pH; and (4) salivary secretion as an active process. (DS)

  5. The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity

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    Dieter Wicher

    2007-12-01

    Full Text Available The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR, we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH: PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage.

  6. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

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    Brian Tomkowicz

    Full Text Available TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3 is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion. Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK, the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i NF-kB/NFAT activation, ii CD69 expression, and iii suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  7. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

    Directory of Open Access Journals (Sweden)

    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  8. Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

    International Nuclear Information System (INIS)

    Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

    2017-01-01

    We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.

  9. Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina

    Directory of Open Access Journals (Sweden)

    Yi Hyun

    2007-12-01

    Full Text Available Abstract Background The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1 and Dickkopf 2 (Dkk2 are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3 protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death. Results Dkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis. Conclusion These data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

  10. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

    Directory of Open Access Journals (Sweden)

    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  11. Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor.

    Science.gov (United States)

    Flores-Pliego, Arturo; Espejel-Nuñez, Aurora; Castillo-Castrejon, Marisol; Meraz-Cruz, Noemi; Beltran-Montoya, Jorge; Zaga-Clavellina, Veronica; Nava-Salazar, Sonia; Sanchez-Martinez, Maribel; Vadillo-Ortega, Felipe; Estrada-Gutierrez, Guadalupe

    2015-01-01

    The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. In this work we confirm that placental leukocytes from human term

  12. Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT.

    Directory of Open Access Journals (Sweden)

    Ashish Misra

    Full Text Available Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial-Mesenchymal Transition (EMT. EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor. Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.

  13. Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2.

    Directory of Open Access Journals (Sweden)

    Dor Salomon

    Full Text Available Vibrio parahaemolyticus is a marine bacterium that thrives in warm climates. It is a leading cause of gastroenteritis resulting from consumption of contaminated uncooked shellfish. This bacterium harbors two putative type VI secretion systems (T6SS. T6SSs are widespread protein secretion systems found in many Gram-negative bacteria, and are often tightly regulated. For many T6SSs studied to date, the conditions and cues, as well as the regulatory mechanisms that control T6SS activity are unknown. In this study, we characterized the environmental conditions and cues that activate both V. parahaemolyticus T6SSs, and identified regulatory mechanisms that control T6SS gene expression and activity. We monitored the expression and secretion of the signature T6SS secreted proteins Hcp1 and Hcp2, and found that both T6SSs are differentially regulated by quorum sensing and surface sensing. We also showed that T6SS1 and T6SS2 require different temperature and salinity conditions to be active. Interestingly, T6SS1, which is found predominantly in clinical isolates, was most active under warm marine-like conditions. Moreover, we found that T6SS1 has anti-bacterial activity under these conditions. In addition, we identified two transcription regulators in the T6SS1 gene cluster that regulate Hcp1 expression, but are not required for immunity against self-intoxication. Further examination of environmental isolates revealed a correlation between the presence of T6SS1 and virulence of V. parahaemolyticus against other bacteria, and we also showed that different V. parahaemolyticus isolates can outcompete each other. We propose that T6SS1 and T6SS2 play different roles in the V. parahaemolyticus lifestyles, and suggest a role for T6SS1 in enhancing environmental fitness of V. parahaemolyticus in marine environments when competing for a niche in the presence of other bacterial populations.

  14. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

    Directory of Open Access Journals (Sweden)

    Sachin Gupta

    Full Text Available Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG, including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1. Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5 expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.

  15. Salmonella-secreted Virulence Factors

    Energy Technology Data Exchange (ETDEWEB)

    Heffron, Fred; Niemann, George; Yoon, Hyunjin; Kidwai, Afshan S.; Brown, Roslyn N.; McDermott, Jason E.; Smith, Richard D.; Adkins, Joshua N.

    2011-05-01

    In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellent reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.

  16. Peroxisome proliferator-activated receptor δ modulates MMP-2 secretion and elastin expression in human dermal fibroblasts exposed to ultraviolet B radiation.

    Science.gov (United States)

    Ham, Sun Ah; Yoo, Taesik; Hwang, Jung Seok; Kang, Eun Sil; Paek, Kyung Shin; Park, Chankyu; Kim, Jin-Hoi; Do, Jeong Tae; Seo, Han Geuk

    2014-10-01

    Changes in skin connective tissues mediated by ultraviolet (UV) radiation have been suggested to cause the skin wrinkling normally associated with premature aging of the skin. Recent investigations have shown that peroxisome proliferator-activated receptor (PPAR) δ plays multiple biological roles in skin homeostasis. We attempted to investigate whether PPARδ modulates elastin protein levels and secretion of matrix metalloproteinase (MMP)-2 in UVB-irradiated human dermal fibroblasts (HDFs) and mouse skin. These studies were undertaken in primary HDFs or HR-1 hairless mice using Western blot analyses, small interfering (si)RNA-mediated gene silencing, and Fluorescence microscopy. In HDFs, UVB irradiation induced increased secretion of MMP-2 and reduced levels of elastin. Activation of PPARδ by GW501516, a ligand specific for PPARδ, markedly attenuated UVB-induced MMP-2 secretion with a concomitant increase in the level of elastin. These effects were reduced by the presence of siRNAs against PPARδ or treatment with GSK0660, a specific inhibitor of PPARδ. Furthermore, GW501516 elicited a dose- and time-dependent increase in the expression of elastin. Modulation of MMP-2 secretion and elastin levels by GW501516 was associated with a reduction in reactive oxygen species (ROS) production in HDFs exposed to UVB. Finally, in HR-1 hairless mice, administration of GW501516 significantly reduced UVB-induced MMP-2 expression with a concomitant increase in elastin levels, and these effects were significantly reduced by the presence of GSK0660. Our results suggest that PPARδ-mediated modulation of MMP-2 secretion and elastin expression may contribute to the maintenance of skin integrity by inhibiting ROS generation. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  17. Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

    Science.gov (United States)

    Huang, F-M; Chen, Y-J; Chou, M-Y; Chang, Y-C

    2005-12-01

    To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P endodontalis stimulated t-PA production respectively (P endodontalis stimulated t-PA production (P > 0.05). Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

  18. The activity of 11β-hydroxysteroid dehydrogenase type 2 enzyme and cortisol secretion in patients with adrenal incidentalomas.

    Science.gov (United States)

    Morelli, Valentina; Polledri, Elisa; Mercadante, Rosa; Zhukouskaya, Volha; Palmieri, Serena; Beck-Peccoz, Paolo; Spada, Anna; Fustinoni, Silvia; Chiodini, Iacopo

    2016-09-01

    In adrenal incidentaloma (AI) patients, beside the cortisol secretion, a different 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity, measurable by 24-h urinary cortisol/cortisone ratio (R-UFF/UFE) (the higher R-UFF/UFE the lower HSD11B2 activity), could influence the occurrence of the subclinical hypercortisolism (SH)-related complications (hypertension, type 2 diabetes, obesity). We evaluated whether in AI patients, UFF levels are associated to UFE levels, and the HSD11B2 activity to the complications presence. In 156 AI patients (93F, age 65.2 ± 9.5 years), the following were measured: serum cortisol after 1 mg-dexamethasone test (1 mg-DST), ACTH, UFF, UFE levels, and R-UFF/UFE (by liquid chromatography-tandem mass spectrometry), the latter was also evaluated in 63 matched-controls. We diagnosed SH (n = 22) in the presence of ≥2 among ACTH levels, and 1 mg-DST >83 nmol/L. Patients showed higher UFF levels and R-UFF/UFE than controls (75.9 ± 43.1 vs 54.4 ± 22.9 nmol/24 h and 0.26 ± 0.12 vs 0.20 ± 0.07, p levels (291 ± 91.1 vs 268 ± 61.5, p = 0.069). The R-UFF/UFE was higher in patients with high (h-UFF, n = 28, 0.41 ± 0.20) than in those with normal (n-UFF, 0.22 ± 0.10, p levels and in patients with SH than in those without SH (0.30 ± 0.12 vs 0.25 ± 0.12, p = 0.04). UFF levels were associated with R-UFF/UFE (r = 0.849, p levels in n-UFF patients but not in h-UFF patients, and it is not associated with the SH complications.

  19. Visfatin Is Actively Secreted In Vitro From U-937 Macrophages, but Only Passively Released From 3T3-L1 Adipocytes and HepG2 Hepatocytes

    Czech Academy of Sciences Publication Activity Database

    Svoboda, Petr; Křížová, E.; Čeňková, K.; Vápenková, K.; Zídková, J.; Zídek, Václav; Škop, V.

    2017-01-01

    Roč. 66, č. 4 (2017), s. 709-714 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA16-04859S; GA ČR(CZ) GA13-04580S Institutional support: RVO:67985823 Keywords : Nampt * Visfatin * active secretion * adipocytes * hepatocytes * macrophages Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Endocrinology and metabolism (including diabetes, hormones) Impact factor: 1.461, year: 2016

  20. The immunomodulatory effect of Zingiber cassumunar ethanolic extract on phagocytic activity, nitrit oxide and reaxtive oxygen intermediate secretions of macrophage in mice

    Science.gov (United States)

    Nurkhasanah; Santoso, R. D.; Fauziah, R.

    2017-11-01

    Immunomodulators could protect the body from a variety of infectious agents and boost immunity. Zingiber cassumunar rhizome or bangle potentially showed as an immunomodulator through increasing of macrophage activity in vitro. The objective of the study was to determine the effect of Z. cassumunar rhizome ethanolic extract on phagocytic activity, nitrite oxide (NO) and reactive oxygen intermediate (ROI) secretions in macrophages in vivo. A total of 200 g of Z. cassumunar rhizome was powdered, macerated in 96% ethanol and evaporated to get concentrated extract. Mice were divided into 5 groups as follow: the normal group was given by water only, the negative control group was given by a 0.94% CMC-Na suspension, the treatment groups were given by 250, 500 and 1000 mg/kgBW, respectively, of Z. cassumunar ethanolic extract. The extract was administered orally for 7 days. On the 8th day the mice were injected intraperitoneally 0.7 mg/kg BW of lipopolysaccharide. Four hours later macrophage was isolated. Furthermore, the determination of the phagocytic activity, NO and ROI secretions levels of macrophage were performed. The treatments of 250, 500 and 1000 mg/kg BW of Z. cassumunar ethanolic extract significantly increase the ROI and NO secretions levels (p0.05) of macrophage. Z. cassumunar ethanolic extract have immunomodulatory effect in vivo.

  1. In Vivo Characterization of Intracellular Signaling Pathways Activated by the Nerve Agent Sarin

    National Research Council Canada - National Science Library

    Shih, Tsung-Ming A; Snyder, Gretchen L; Hendrick, Joseph P; Fienberg, Allen A; McDonough, John H

    2004-01-01

    ..., an excessive stimulation of nicotinic and muscarinic receptors. Preliminary evidence using diverse OPs indicates that the DARPP-32/PP-1 signaling pathway is activated by nicotinic receptor stimulation...

  2. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

    NARCIS (Netherlands)

    Claes, I.J.; Schoofs, G.; Regulski, K.; Vos, de W.M.

    2012-01-01

    Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with

  3. Venom allergen-like proteins in secretions of plant-parasitic nematodes activate and suppress extracellular plant immune receptors

    NARCIS (Netherlands)

    Lozano Torres, J.L.

    2014-01-01

    Parasitic worms threaten human, animal and plant health by infecting people, livestock and crops worldwide. Animals and plants share an anciently evolved innate immune system. Parasites modulate this immune system by secreting proteins to maintain their parasitic lifestyle. This thesis

  4. Suppression or activation of immune responses by predicted secreted proteins of the soybean rust pathogen Phakopsora pachyrhizi

    Science.gov (United States)

    Rust fungi, such as Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are intimately associated with plant cells. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characte...

  5. Pancreatic bicarbonate secretion involves two proton pumps

    DEFF Research Database (Denmark)

    Novak, Ivana; Wang, Jing; Henriksen, Katrine L.

    2011-01-01

    Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium expres...

  6. Reward acts as a signal to control delay-period activity in delayed-response tasks.

    Science.gov (United States)

    Ichihara-Takeda, Satoe; Takeda, Kazuyoshi; Funahashi, Shintaro

    2010-03-31

    Prefrontal delay-period activity represents a neural mechanism for the active maintenance of information and needs to be controlled by some signal to appropriately operate working memory. To examine whether reward-delivery acts as this signal, the effects of delay-period activity in response to unexpected reward-delivery were examined by analyzing single-neuron activity recorded in the primate dorsolateral prefrontal cortex. Among neurons that showed delay-period activity, 34% showed inhibition of this activity in response to unexpected reward-delivery. The delay-period activity of these neurons was affected by the expectation of reward-delivery. The strength of the reward signal in controlling the delay-period activity is related to the strength of the effect of reward information on the delay-period activity. These results indicate that reward-delivery acts as a signal to control delay-period activity.

  7. Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

    Science.gov (United States)

    Miller, L A; Cochrane, D E; Feldberg, R S; Carraway, R E

    1998-06-01

    Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

  8. Study on acoustic emission signals of active defect in pressure piping under hydraulic pressure

    International Nuclear Information System (INIS)

    Ai Qiong; Liu Caixue; Wang Yao; He Pan; Song Jian

    2009-01-01

    Experimental investigations of acoustic emission (AE) of active defect in pressure piping with a prefabricated crack under hydraulic pressure tester were conducted. AE signals of fatigue-crack-growth in pressure piping were monitored incessantly in all processes, and all signals recorded were analyzed and processed. The result of signal processing show that the amplitude and energy of acoustic emission signals from defect in pressure pipeline increase gradually with the load time, and thus the active defects in pipeline can be identified; the amplitude, energy and count of acoustic emission signals increase sharply before the defect runs through, and we can forecast the penetrated leakage of pipeline. (authors)

  9. LcrG secretion is not required for blocking of Yops secretion in Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Matson Jyl S

    2008-02-01

    Full Text Available Abstract Background LcrG, a negative regulator of the Yersinia type III secretion apparatus has been shown to be primarily a cytoplasmic protein, but is secreted at least in Y. pestis. LcrG secretion has not been functionally analyzed and the relevance of LcrG secretion on LcrG function is unknown. Results An LcrG-GAL4AD chimera, originally constructed for two-hybrid analyses to analyze LcrG protein interactions, appeared to be not secreted but the LcrG-GAL4AD chimera retained the ability to regulate Yops secretion. This result led to further investigation to determine the significance of LcrG secretion on LcrG function. Additional analyses including deletion and substitution mutations of amino acids 2–6 in the N-terminus of LcrG were constructed to analyze LcrG secretion and LcrG's ability to control secretion. Some changes to the N-terminus of LcrG were found to not affect LcrG's secretion or LcrG's secretion-controlling activity. However, substitution of poly-isoleucine in the N-terminus of LcrG did eliminate LcrG secretion but did not affect LcrG's secretion controlling activity. Conclusion These results indicate that secretion of LcrG, while observable and T3SS mediated, is not relevant for LcrG's ability to control secretion.

  10. Hedgehog signaling activation induces stem cell proliferation and hormone release in the adult pituitary gland

    OpenAIRE

    Joanna Pyczek; Rolf Buslei; David Schult; Annett Hölsken; Michael Buchfelder; Ina Heß; Heidi Hahn; Anja Uhmann

    2016-01-01

    Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2(+) and S...

  11. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  12. High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

    Science.gov (United States)

    Koizume, Shiro; Ito, Shin; Yoshioka, Yusuke; Kanayama, Tomohiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Yamada, Roppei; Ochiya, Takahiro; Ruf, Wolfram; Miyagi, Etsuko; Hirahara, Fumiki; Miyagi, Yohei

    2016-01-01

    Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

  13. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

    Science.gov (United States)

    Maza, Paloma K; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

  14. Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

    Science.gov (United States)

    Siese, A; Jaros, P P; Willig, A

    1999-02-01

    In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

  15. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    DEFF Research Database (Denmark)

    Owens, T

    1988-01-01

    on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

  16. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta; Mariotto, Sofia; Dal Bosco, Martina; Darra, Elena; Vaiana, Nadia; Shoji, Kazuo; Safwat, Abdel Azeim; Marechal, Jean Didier; Perahia, David; Suzuki, Hisanori; Romeo, Sergio

    2013-01-01

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins

  17. Bile Formation and Secretion

    Science.gov (United States)

    Boyer, James L.

    2014-01-01

    Bile is a unique and vital aqueous secretion of the liver that is formed by the hepatocyte and modified down stream by absorptive and secretory properties of the bile duct epithelium. Approximately 5% of bile consists of organic and inorganic solutes of considerable complexity. The bile-secretory unit consists of a canalicular network which is formed by the apical membrane of adjacent hepatocytes and sealed by tight junctions. The bile canaliculi (~1 μm in diameter) conduct the flow of bile countercurrent to the direction of portal blood flow and connect with the canal of Hering and bile ducts which progressively increase in diameter and complexity prior to the entry of bile into the gallbladder, common bile duct, and intestine. Canalicular bile secretion is determined by both bile salt-dependent and independent transport systems which are localized at the apical membrane of the hepatocyte and largely consist of a series of adenosine triphosphate-binding cassette transport proteins that function as export pumps for bile salts and other organic solutes. These transporters create osmotic gradients within the bile canalicular lumen that provide the driving force for movement of fluid into the lumen via aquaporins. Species vary with respect to the relative amounts of bile salt-dependent and independent canalicular flow and cholangiocyte secretion which is highly regulated by hormones, second messengers, and signal transduction pathways. Most determinants of bile secretion are now characterized at the molecular level in animal models and in man. Genetic mutations serve to illuminate many of their functions. PMID:23897680

  18. Effecf of pH and some cations on activity of acid phosphatase secreted from Ustilago sp. isolated from acid sulphate soil

    Directory of Open Access Journals (Sweden)

    Chairatana Nilnond

    2007-03-01

    Full Text Available Acid phosphatase secreted from Ustilago sp. is able to hydrolyze organic phosphorus. These soil yeast microorganisms were isolated from rice roots grown in acid sulphate soil that generally contains highamount of aluminum (Al, iron (Fe and manganese (Mn ions. Therefore, the objectives of this study were to examine the effect of pH and some cations on acid phosphatase activity. Two isolates of Ustilago sp., AR101and AR102, were cultured in 100 mL of modified Pikovskaya's broth containing Na-phytate, pH 4, and acid phosphatase activity was determined at pH 2.0-7.0. Effect of Al, Fe, and Mn, including calcium (Ca ions,on growth of AR101 and AR102, secreted acid phosphatase activity, and the ability of acid phosphatase on the phosphorus release from Na-phytate by Ustilago sp. were investigated. It was found that the optimum pH for acid phosphatase activity was 3.5-4.5. The activity of acid phosphatase secreted from AR101 (3,690nmol min-1 mL-1 was remarkably higher than that from AR102 (956 nmol min-1 mL-1. Aluminum, iron, manganese and calcium ions in the medium did not affect the growth of either isolate. The activity of secretedacid phosphatase of AR101 was inhibited by Al and Ca ion, and synthesis of acid phosphatase of Ustilago sp. AR102 was possibly stimulated by Fe ion. Both AR101 and AR102 solubilized Na-phytate, resulting in therelease of P. However, some amount of released P was then precipitated with Al and Fe ions as the highly insoluble Fe- or Al- phosphate.

  19. Digital signaling decouples activation probability and population heterogeneity

    DEFF Research Database (Denmark)

    Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz

    2015-01-01

    Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamic...

  20. Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Wen-Hsiung Chan

    2008-05-01

    Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

  1. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    International Nuclear Information System (INIS)

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-01-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals

  2. Orphan nuclear receptor TLX activates Wnt/β-catenin signalling to stimulate neural stem cell proliferation and self-renewal

    Science.gov (United States)

    Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T.; Gage, Fred H.; Evans, Ronald M.; Shi, Yanhong

    2010-01-01

    The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/β-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/β-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active β-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a β-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active β-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active β-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active β-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode. PMID:20010817

  3. Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

    Science.gov (United States)

    Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

    2010-01-01

    The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

  4. The role of glypicans in Wnt inhibitory factor-1 activity and the structural basis of Wif1's effects on Wnt and Hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    Andrei Avanesov

    Full Text Available Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh signals. Members of the Wnt Inhibitory Factor-1 (WIF1 family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding "EGF-like" domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the "WIF" domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.

  5. Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

    Science.gov (United States)

    De Nisco, Nicole J.; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-01-01

    Bacterial effectors are potent manipulators of host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para), delivers effectors into host cells through two type three secretion systems (T3SS). The ubiquitous T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate non-apoptotic cell death. Much is known about how T3SS1 effectors function in isolation, but we wanted to understand how their concerted action globally affects host cell signaling. To assess the host response to T3SS1, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1+) to those in cells infected with V. para lacking T3SS1 (T3SS1−). Overall, the host transcriptional response to both T3SS1+ and T3SS1− V. para was rapid, robust, and temporally dynamic. T3SS1 re-wired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors target host cells at the posttranslational level to cause cytotoxicity, network analysis indicated that V. para T3SS1 also precipitates a host transcriptional response that initially activates cell survival and represses cell death networks. The increased expression of several key pro-survival transcripts mediated by T3SS1 was dependent on a host signaling pathway that is silenced later in infection by the posttranslational action of T3SS1. Taken together, our analysis reveals a complex interplay between roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. PMID:28512145

  6. Maturation processes and structures of small secreted peptides in plants

    Directory of Open Access Journals (Sweden)

    Ryo eTabata

    2014-07-01

    Full Text Available In the past decade, small secreted peptides have proven to be essential for various aspects of plant growth and development, including the maintenance of certain stem cell populations. Most small secreted peptides identified in plants to date are recognised by membrane-localized receptor kinases, the largest family of receptor proteins in the plant genome. This peptide-receptor interaction is essential for initiating intracellular signalling cascades. Small secreted peptides often undergo post-translational modifications and proteolytic processing to generate the mature peptides. Recent studies suggest that, in contrast to the situation in mammals, the proteolytic processing of plant peptides involves a number of complex steps. Furthermore, NMR-based structural analysis demonstrated that post-translational modifications induce the conformational changes needed for full activity. In this mini review, we summarise recent advances in our understanding of how small secreted peptides are modified and processed into biologically active peptides and describe the mature structures of small secreted peptides in plants.

  7. Danger Signals Activating the Immune Response after Trauma

    Directory of Open Access Journals (Sweden)

    Stefanie Hirsiger

    2012-01-01

    Full Text Available Sterile injury can cause a systemic inflammatory response syndrome (SIRS that resembles the host response during sepsis. The inflammatory response following trauma comprises various systems of the human body which are cross-linked with each other within a highly complex network of inflammation. Endogenous danger signals (danger-associated molecular patterns; DAMPs; alarmins as well as exogenous pathogen-associated molecular patterns (PAMPs play a crucial role in the initiation of the immune response. With popularization of the “danger theory,” numerous DAMPs and PAMPs and their corresponding pathogen-recognition receptors have been identified. In this paper, we highlight the role of the DAMPs high-mobility group box protein 1 (HMGB1, interleukin-1α (IL-1α, and interleukin-33 (IL-33 as unique dual-function mediators as well as mitochondrial danger signals released upon cellular trauma and necrosis.

  8. ROS-activated calcium signaling mechanisms regulating endothelial barrier function.

    Science.gov (United States)

    Di, Anke; Mehta, Dolly; Malik, Asrar B

    2016-09-01

    Increased vascular permeability is a common pathogenic feature in many inflammatory diseases. For example in acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS), lung microvessel endothelia lose their junctional integrity resulting in leakiness of the endothelial barrier and accumulation of protein rich edema. Increased reactive oxygen species (ROS) generated by neutrophils (PMNs) and other inflammatory cells play an important role in increasing endothelial permeability. In essence, multiple inflammatory syndromes are caused by dysfunction and compromise of the barrier properties of the endothelium as a consequence of unregulated acute inflammatory response. This review focuses on the role of ROS signaling in controlling endothelial permeability with particular focus on ALI. We summarize below recent progress in defining signaling events leading to increased endothelial permeability and ALI. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

    Science.gov (United States)

    Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

    2012-08-01

    Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

  10. Inflammation Activates the Interferon Signaling Pathways in Taste Bud Cells

    OpenAIRE

    Wang, Hong; Zhou, Minliang; Brand, Joseph; Huang, Liquan

    2007-01-01

    Patients with viral and bacterial infections or other inflammatory illnesses often experience taste dysfunctions. The agents responsible for these taste disorders are thought to be related to infection-induced inflammation, but the mechanisms are not known. As a first step in characterizing the possible role of inflammation in taste disorders, we report here evidence for the presence of interferon (IFN)-mediated signaling pathways in taste bud cells. IFN receptors, particularly the IFN-γ rece...

  11. Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

    Science.gov (United States)

    Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

    2011-06-01

    Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

    Directory of Open Access Journals (Sweden)

    Farhad Dehkhoda

    2018-02-01

    Full Text Available The growth hormone receptor (GHR, although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK–signal transducer and activator of transcription (STAT signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

  13. Citrus tachibana Leaves Ethanol Extract Alleviates Airway Inflammation by the Modulation of Th1/Th2 Imbalance via Inhibiting NF-κB Signaling and Histamine Secretion in a Mouse Model of Allergic Asthma.

    Science.gov (United States)

    Bui, Thi Tho; Piao, Chun Hua; Kim, Soo Mi; Song, Chang Ho; Shin, Hee Soon; Lee, Chang-Hyun; Chai, Ok Hee

    2017-07-01

    Asthma is a chronic inflammatory disease of bronchial airway, which is characterized by chronic airway inflammation, airway edema, goblet cell hyperplasia, the aberrant production of the Th2 cytokines, and eosinophil infiltration in the lungs. In this study, the therapeutic effect and the underlying mechanism of Citrus tachibana leaves ethanol extract (CTLE) in the ovalbumin (OVA)-induced allergic asthma and compound 48/80-induced anaphylaxis were investigated. Oral administration of CTLE inhibited OVA-induced asthmatic response by reducing airway inflammation, OVA-specific IgE and IgG1 levels, and increasing OVA-specific IgG2a levels. CTLE restored Th1/Th2 balance through an increase in Th2 cytokines tumor necrosis factor-α, interleukin (IL)-4, and IL-6 and decreases in Th1 cytokines interferon-γ and IL-12. Furthermore, CTLE inhibited the total level of NF-κB and the phosphorylation of IκB-α and NF-κB by OVA. In addition, CTLE dose-dependently inhibited compound 48/80-induced anaphylaxis via blocking histamine secretion from mast cells. The anti-inflammatory mechanism of CTLE may involve the modulation of Th1/Th2 imbalance via inhibiting the NF-κB signaling and histamine secretion. Taken together, we suggest that CTLE could be used as a therapeutic agent for patients with Th2-mediated or histamine-mediated allergic asthma.

  14. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

    Science.gov (United States)

    Folders, J; Algra, J; Roelofs, M S; van Loon, L C; Tommassen, J; Bitter, W

    2001-12-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

  15. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    Directory of Open Access Journals (Sweden)

    Kumar N Alagramam

    2014-01-01

    Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  16. The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

    Science.gov (United States)

    Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

    2000-04-01

    Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

  17. AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jaguin, Marie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes Cedex (France); Lecureur, Valérie, E-mail: valerie.lecureur@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France)

    2015-06-15

    Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantly increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants. - Highlights: • PDGF-B expression and secretion are increased by DEPe exposure in human M1 and M2 MΦ. • DEPe-induced PDGF-B expression is aryl-hydrocarbon-dependent. • DEPe-exposed M1 MΦ secrete sufficient PDGF-B to increase lung fibroblast proliferation.

  18. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

    2013-12-01

    Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

  19. Intercellular signaling pathways active during and after growth and differentiation of the lumbar vertebral growth plate.

    Science.gov (United States)

    Dahia, Chitra Lekha; Mahoney, Eric J; Durrani, Atiq A; Wylie, Christopher

    2011-06-15

    Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways. To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate. The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis. Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGFβ, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling. We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate. During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

  20. Proinflammatory Cytokine Infusion Attenuates LH's Feedforward on Testosterone Secretion: Modulation by Age

    Science.gov (United States)

    Yang, Rebecca; Roelfsema, Ferdinand; Takahashi, Paul

    2016-01-01

    Context: In the experimental animal, inflammatory signals quench LH's feedforward drive of testosterone (T) secretion and appear to impair GnRH-LH output. The degree to which such suppressive effects operate in the human is not known. Objective: To test the hypothesis that IL-2 impairs LH's feedforward drive on T and T's feedback inhibition of LH secretion in healthy men. Setting: Mayo Center for Translational Science Activities. Patients or Other Participants: A total of 35 healthy men, 17 young and 18 older. Interventions: Randomized prospective double-blind saline-controlled study of IL-2 infusion in 2 doses with concurrent 10-minute blood sampling for 24 hours. Main Outcome Measures: Deconvolution analysis of LH and T secretion. Results: After saline injection, older compared with young men exhibited reduced LH feedforward drive on T secretion (P feedback inhibition of LH secretion (P feedforward onto T secretion declined markedly especially in young subjects (P feedback on LH secretion especially in older volunteers. Conclusion: This investigation confirms combined feedforward and feedback deficits in older relative to young men given saline and demonstrates 1) joint mechanisms by which IL-2 enforces biochemical hypogonadism, viz, combined feedforward block and feedback amplification; and 2) unequal absolute inhibition of T and LH secretion by IL-2 in young and older men. These outcomes establish that the male gonadal axis is susceptible to dual-site suppression by a prototypic inflammatory mediator. Thus, we postulate that selected ILs might also enforce male hypogonadism in chronic systemic inflammation. PMID:26600270

  1. TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns.

    Directory of Open Access Journals (Sweden)

    Elaine Cristina Cardoso

    Full Text Available Mother-to-child transmission (MTCT of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs (TLR2, TLR4 and TLR5 and intracellular TLRs (TLR7, TLR7/8 and TLR9. Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs and plasmacytoid DCs (pDCs to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097 stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7, IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7

  2. TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns.

    Science.gov (United States)

    Cardoso, Elaine Cristina; Pereira, Nátalli Zanete; Mitsunari, Gabrielle Eimi; Oliveira, Luanda Mara da Silva; Ruocco, Rosa Maria S A; Francisco, Rossana Pulcineli Vieira; Zugaib, Marcelo; da Silva Duarte, Alberto José; Sato, Maria Notomi

    2013-01-01

    Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway

  3. Angiogenic activity of sesamin through the activation of multiple signal pathways

    International Nuclear Information System (INIS)

    Chung, Byung-Hee; Lee, Jung Joon; Kim, Jong-Dai; Jeoung, Dooil; Lee, Hansoo; Choe, Jongseon; Ha, Kwon-Soo; Kwon, Young-Geun; Kim, Young-Myeong

    2010-01-01

    The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125 FAK -, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.

  4. Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

    NARCIS (Netherlands)

    Herranz, C; Driessen, AJM

    Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal

  5. Design of excitation signals for active system monitoring in a performance assessment setup

    DEFF Research Database (Denmark)

    Green, Torben; Izadi-Zamanabadi, Roozbeh; Niemann, Hans Henrik

    2011-01-01

    This paper investigates how the excitation signal should be chosen for a active performance setup. The signal is used in a setup where the main purpose is to detect whether a parameter change of the controller has changed the global performance significantly. The signal has to be able to excite...... the dynamics of the subsystem under investigation both before and after the parameter change. The controller is well know, but there exists no detailed knowledge about the dynamics of the subsystem....

  6. Ixeris dentata extract regulates salivary secretion through the activation of aquaporin-5 and prevents diabetes-induced xerostomia.

    Science.gov (United States)

    Bhattarai, Kashi Raj; Lee, Sang-Won; Kim, Seung Hyun; Kim, Hyung-Ryong; Chae, Han-Jung

    2017-01-01

    The aim of this study was to investigate the effects of Ixeris dentata (IXD) extract to improve the salivation rate in dry mouth induced by diabetes. Both control and diabetic rats were treated with a sublingual spray of either water or IXD extract to determine the effects of IXD on salivation. During the study, we observed that IXD extract treatment increased the salivary flow rate in diabetic rats. The expression of α-amylase was increased significantly in both saliva and glandular tissue lysates of IXD-treated diabetic rats. Aquaporin-5 protein expression was abnormally low in the salivary glands of diabetic rats, which increased hyposalivation and led to salivary dysfunction. However, a single oral spray of IXD extract drastically increased the expression of aquaporin-5 in salivary gland acinar and ductal cells in diabetic rats. Moreover, IXD extract induced expression of Na + /H + exchangers in the salivary gland, which suggests that Na + /H + exchangers modulate salivary secretions and aid in the fluid-secretion mechanism. Furthermore, transient treatment with IXD extract increased the intracellular calcium in human salivary gland cells. Taken together, these results suggest the potential value of an IXD extract for the treatment of diabetes-induced hyposalivation and xerostomia.

  7. Catechin secretion and phytotoxicity

    Science.gov (United States)

    Kaushik, Shail

    2010-01-01

    Research indicates that the invasiveness of Centaurea stoebe is attributed to the stronger allelopathic effects on the native North American species than on the related European species, which is one of the unquestionable aspects of the “novel weapons hypothesis (NWH).” Studies originating from controlled to field conditions have shown that C. stoebe utilizes its biochemical potential to exert its invasiveness. The roots of C. stoebe secrete a potent phytotoxin, catechin, which has a detrimental effect on the surrounding plant species. Although, studies on catechin secretion and phytotoxicity represent one of the most well studied systems describing negative plant-plant interactions, it has also sparked controversies lately due to its phytotoxicity dosages and secretion effluxes. Previous reports negate the phytotoxic and pro-oxidant nature of catechin.1–3 In our recent study we have shown that catechin is highly phytotoxic against Arabidopsis thaliana and Festuca idahoensis. We also show that (±) catechin applied to roots of A. thaliana induces reactive oxygen species (ROS) confirming the pro-oxidant nature of catechin. In addition, activation of signature cell death genes such as acd2 and cad1 post catechin treatment in A. thaliana ascertains the phytotoxic nature of catechin. PMID:21057643

  8. Extracellular signal-regulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation.

    Science.gov (United States)

    Pascoli, Vincent; Cahill, Emma; Bellivier, Frank; Caboche, Jocelyne; Vanhoutte, Peter

    2014-12-15

    Addiction is a chronic and relapsing psychiatric disorder that is thought to occur in vulnerable individuals. Synaptic plasticity evoked by drugs of abuse in the so-called neuronal circuits of reward has been proposed to underlie behavioral adaptations that characterize addiction. By increasing dopamine in the striatum, addictive drugs alter the balance of dopamine and glutamate signals converging onto striatal medium-sized spiny neurons (MSNs) and activate intracellular events involved in long-term behavioral alterations. Our laboratory contributed to the identification of salient molecular changes induced by administration of addictive drugs to rodents. We pioneered the observation that a common feature of addictive drugs is to activate, by a double tyrosine/threonine phosphorylation, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the striatum, which control a plethora of substrates, some of them being critically involved in cocaine-mediated molecular and behavioral adaptations. Herein, we review how the interplay between dopamine and glutamate signaling controls cocaine-induced ERK1/2 activation in MSNs. We emphasize the key role of N-methyl-D-aspartate receptor potentiation by D1 receptor to trigger ERK1/2 activation and its subsequent nuclear translocation where it modulates both epigenetic and genetic processes engaged by cocaine. We discuss how cocaine-induced long-term synaptic and structural plasticity of MSNs, as well as behavioral adaptations, are influenced by ERK1/2-controlled targets. We conclude that a better knowledge of molecular mechanisms underlying ERK1/2 activation by drugs of abuse and/or its role in long-term neuronal plasticity in the striatum may provide a new route for therapeutic treatment in addiction. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  9. Regulation of ghrelin secretion and action.

    Science.gov (United States)

    Camiña, Jesus P; Carreira, Marcos C; Micic, Dragan; Pombo, Manuel; Kelestimur, Fahrettin; Dieguez, Carlos; Casanueva, Felipe F

    2003-10-01

    The pulsatile release of growth hormone (GH) from anterior pituitary gland is regulated by the interplay of at least two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. Furthermore, release of GH in vivo may also be controlled by a third type of receptor, the growth hormone secretagogue receptor, a G-protein-coupled receptor, called GHS receptor type 1a (GHSR1a), which was identified in the pituitary and the hypothalamus in humans using a nonpeptidyl growth hormone secretagogue (MK-0677). Ghrelin, the endogenous ligand for the GHS-R1a, is a 28-amino-acid peptide isolated from human stomach that is modified by a straight chain octanoyl group covalently linked to Ser3, which is essential for its endocrine activity. This hormone, predominantly expressed and secreted by the stomach, has a dual action on GH secretion and food intake, showing interdependency between these actions. The finding that fasting and food intake, respectively, increase and decrease the secretion of ghrelin suggests that this hormone may be the bridge connecting somatic growth and body composition with energy metabolism, and appears to play a role in the alteration of energy homeostasis and body weight in pathophysiological states such as hypothyroidism and hyperthyroidism. Despite this, little is known about the intracellular signaling through which ghrelin exerts its regulatory actions. Activation of intracellular calcium mobilization is one of the earliest known cellular signals elicited by ghrelin. In HEK- 293 cells expressing the GHS-R1a, ghrelin induces a biphasic cytosolic calcium elevation characterized by a spike phase of the response, which reflects Ins(1,4,5)P3- dependent calcium mobilization of intracellular stores, and a sustained phase of the response, which is due to calcium influx across the plasma membrane triggered by aperture of capacitative calcium channels

  10. Chemical signal activation of an organocatalyst enables control over soft material formation.

    Science.gov (United States)

    Trausel, Fanny; Maity, Chandan; Poolman, Jos M; Kouwenberg, D S J; Versluis, Frank; van Esch, Jan H; Eelkema, Rienk

    2017-10-12

    Cells can react to their environment by changing the activity of enzymes in response to specific chemical signals. Artificial catalysts capable of being activated by chemical signals are rare, but of interest for creating autonomously responsive materials. We present an organocatalyst that is activated by a chemical signal, enabling temporal control over reaction rates and the formation of materials. Using self-immolative chemistry, we design a deactivated aniline organocatalyst that is activated by the chemical signal hydrogen peroxide and catalyses hydrazone formation. Upon activation of the catalyst, the rate of hydrazone formation increases 10-fold almost instantly. The responsive organocatalyst enables temporal control over the formation of gels featuring hydrazone bonds. The generic design should enable the use of a large range of triggers and organocatalysts, and appears a promising method for the introduction of signal response in materials, constituting a first step towards achieving communication between artificial chemical systems.Enzymes regulated by chemical signals are common in biology, but few such artificial catalysts exist. Here, the authors design an aniline catalyst that, when activated by a chemical trigger, catalyses formation of hydrazone-based gels, demonstrating signal response in a soft material.

  11. Discovery of novel small molecule activators of β-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Folkert Verkaar

    Full Text Available Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β-catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling.

  12. Information Theoretic Secret Key Generation: Structured Codes and Tree Packing

    Science.gov (United States)

    Nitinawarat, Sirin

    2010-01-01

    This dissertation deals with a multiterminal source model for secret key generation by multiple network terminals with prior and privileged access to a set of correlated signals complemented by public discussion among themselves. Emphasis is placed on a characterization of secret key capacity, i.e., the largest rate of an achievable secret key,…

  13. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

    Science.gov (United States)

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  14. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

    Directory of Open Access Journals (Sweden)

    Peng Li

    2017-09-01

    Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

  15. Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity

    Directory of Open Access Journals (Sweden)

    Perdigón Gabriela

    2007-09-01

    Full Text Available Background Fermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by L. helveticus R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6 to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of Salmonella typhimurium infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by L. helveticus R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by L. helveticus R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut. Results We observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance

  16. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function

    DEFF Research Database (Denmark)

    Indo, Hiroko P.; Hawkins, Clare L; Nakanishi, Ikuo

    2017-01-01

    -κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA...

  17. Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

    Directory of Open Access Journals (Sweden)

    Hanselman Keaton B

    2006-10-01

    Full Text Available Abstract Background In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109 animals. Results This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C phenotype of age-1(mg109. Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109;akt-1(mg247 animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109;pdk-1(mg261 animals was dependent on akt-1. However, reproductive development in age-1(mg109; mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109 animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247 and pdk-1(mg261 did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these

  18. Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling.

    Science.gov (United States)

    Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

    2006-10-04

    In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals. This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes. A screen for suppressors of PI3K

  19. Ghrelin: ghrelin as a regulatory Peptide in growth hormone secretion.

    Science.gov (United States)

    Khatib, Nazli; Gaidhane, Shilpa; Gaidhane, Abhay M; Khatib, Mahanaaz; Simkhada, Padam; Gode, Dilip; Zahiruddin, Quazi Syed

    2014-08-01

    Ghrelin is a type of growth hormone (GH) secretagogue that stimulates the release of GH. It is a first hormone linking gastrointestinal-pituitary axis. This review highlights the interaction of ghrelin with GHRH and somatostatin to regulate the secretion of GH and intends to explore the possible physiological role of the ghrelin-pituitary-GH axis linkage system. Ghrelin is highly conserved among species and is classified into octanoylated (C8:0), decanoylated (C10:0), decenoylated (C10:1) and nonacylated,ghrelin. Acylated ghrelin is the major active form of human ghrelin. The primary production site of ghrelin is the stomach, and it interacts with stomach ghrelin as well as hypothalamic GHRH and somatostatin in the regulation of pituitary GH secretion. Ghrelin stimulate GH release through the GHS receptor to increase intracellular Ca2+ ([Ca2+] levels via IP3 signal transduction pathway. Ghrelin is a specific endogenous ligand for the GHS receptor and provides a definitive proof of the occurance of a GHS-GHS receptor signalling system in the regulation of GH secretion. Studies suggests that ghrelin is a powerful pharmacological agent that exerts a potent, time-dependent stimulation of pulsatile secretion of GH.

  20. Information transmission and signal permutation in active flow networks

    Science.gov (United States)

    Woodhouse, Francis G.; Fawcett, Joanna B.; Dunkel, Jörn

    2018-03-01

    Recent experiments show that both natural and artificial microswimmers in narrow channel-like geometries will self-organise to form steady, directed flows. This suggests that networks of flowing active matter could function as novel autonomous microfluidic devices. However, little is known about how information propagates through these far-from-equilibrium systems. Through a mathematical analogy with spin-ice vertex models, we investigate here the input–output characteristics of generic incompressible active flow networks (AFNs). Our analysis shows that information transport through an AFN is inherently different from conventional pressure or voltage driven networks. Active flows on hexagonal arrays preserve input information over longer distances than their passive counterparts and are highly sensitive to bulk topological defects, whose presence can be inferred from marginal input–output distributions alone. This sensitivity further allows controlled permutations on parallel inputs, revealing an unexpected link between active matter and group theory that can guide new microfluidic mixing strategies facilitated by active matter and aid the design of generic autonomous information transport networks.

  1. Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

    Science.gov (United States)

    Wood, Chris M; Kajimura, Makiko; Mommsen, Thomas P; Walsh, Patrick J

    2008-01-01

    Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as

  2. TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    International Nuclear Information System (INIS)

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-01-01

    Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  3. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  4. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    International Nuclear Information System (INIS)

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C.; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A.; Cardozo, Christopher P.

    2011-01-01

    Highlights: → Nerve transection increased Notch signaling in paralyzed muscle. → Nandrolone prevented denervation-induced Notch signaling. → Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. → Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  5. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Yao, Shen; Qiao, Rui-Fang; Levine, Alice C. [Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Kirschenbaum, Alexander [Department of Urology, Mount Sinai School of Medicine, New York, NY 10029 (United States); Pan, Jiangping; Wu, Yong [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Qin, Weiping [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Bauman, William A. [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Rehabilitation Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Cardozo, Christopher P., E-mail: chris.cardozo@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States); Rehabilitation Medicine, Mount Sinai School of Medicine, New York, NY 10029 (United States)

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  6. Towards brain-activity-controlled information retrieval: Decoding image relevance from MEG signals.

    Science.gov (United States)

    Kauppi, Jukka-Pekka; Kandemir, Melih; Saarinen, Veli-Matti; Hirvenkari, Lotta; Parkkonen, Lauri; Klami, Arto; Hari, Riitta; Kaski, Samuel

    2015-05-15

    We hypothesize that brain activity can be used to control future information retrieval systems. To this end, we conducted a feasibility study on predicting the relevance of visual objects from brain activity. We analyze both magnetoencephalographic (MEG) and gaze signals from nine subjects who were viewing image collages, a subset of which was relevant to a predetermined task. We report three findings: i) the relevance of an image a subject looks at can be decoded from MEG signals with performance significantly better than chance, ii) fusion of gaze-based and MEG-based classifiers significantly improves the prediction performance compared to using either signal alone, and iii) non-linear classification of the MEG signals using Gaussian process classifiers outperforms linear classification. These findings break new ground for building brain-activity-based interactive image retrieval systems, as well as for systems utilizing feedback both from brain activity and eye movements. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2006-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways which in turn results in the loss of growth control in prostate cancer cells...

  8. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2004-01-01

    .... The experiments proposed in this application are based upon the hypothesis that stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  9. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2002-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  10. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2005-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  11. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2003-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  12. Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat.

    Science.gov (United States)

    Lepist, Eve-Irene; Zhang, Xuexiang; Hao, Jia; Huang, Jane; Kosaka, Alan; Birkus, Gabriel; Murray, Bernard P; Bannister, Roy; Cihlar, Tomas; Huang, Yong; Ray, Adrian S

    2014-08-01

    Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

  13. Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat

    Science.gov (United States)

    Lepist, Eve-Irene; Zhang, Xuexiang; Hao, Jia; Huang, Jane; Kosaka, Alan; Birkus, Gabriel; Murray, Bernard P; Bannister, Roy; Cihlar, Tomas; Huang, Yong; Ray, Adrian S

    2014-01-01

    Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition. PMID:24646860

  14. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates a biologically active peptide, prepro-TRH-(160-169), which regulates TRH-induced thyrotropin secretion

    International Nuclear Information System (INIS)

    Bulant, M.; Vaudry, H.; Roussel, J.P.; Astier, H.; Nicolas, P.

    1990-01-01

    Rat thyrotropin-releasing hormone (TRH) prohormone contains five copies of the TRH progenitor sequence Gln-His-Pro-Gly linked together by connecting sequences whose biological activity is unknown. Both the predicted connecting peptide prepro-TRH-(160-169) (Ps4) and TRH are predominant storage forms of TRH precursor-related peptides in the hypothalamus. To determine whether Ps4 is co-released with TRH, rat median eminence slices were perfused in vitro. Infusion of depolarizing concentrations of KCl induced stimulation of release of Ps4- and TRH-like immunoreactivity. The possible effect of Ps4 on thyrotropin release was investigated in vitro using quartered anterior pituitaries. Infusion of Ps4 alone had no effect on thyrotropin secretion but potentiated TRH-induced thyrotropin release in a dose-dependent manner. In addition, the occurrence of specific binding sites for 125 I-labeled Tyr-Ps4 in the distal lobe of the pituitary was demonstrated by binding analysis and autoradiographic localization. These findings indicate that these two peptides that arise from a single multifunctional precursor, the TRH prohormone, act in a coordinate manner on the same target cells to promote hormonal secretion. These data suggest that differential processing of the TRH prohormone may have the potential to modulate the biological activity of TRH

  15. Signaling pathways activation profiles make better markers of cancer than expression of individual genes

    OpenAIRE

    Borisov, Nikolay M.; Terekhanova, Nadezhda V.; Aliper, Alexander M.; Venkova, Larisa S.; Smirnov, Philip Yu; Roumiantsev, Sergey; Korzinkin, Mikhail B.; Zhavoronkov, Alex A.; Buzdin, Anton A.

    2014-01-01

    Identification of reliable and accurate molecular markers remains one of the major challenges of contemporary biomedicine. We developed a new bioinformatic technique termed OncoFinder that for the first time enables to quantatively measure activation of intracellular signaling pathways basing on transcriptomic data. Signaling pathways regulate all major cellular events in health and disease. Here, we showed that the Pathway Activation Strength (PAS) value itself may serve as the biomarker for...

  16. EGF stimulates the activation of EGF receptors and the s