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Sample records for active tubular secretion

  1. Renal tubular secretion of pramipexole.

    PubMed

    Knop, Jana; Hoier, Eva; Ebner, Thomas; Fromm, Martin F; Müller, Fabian

    2015-11-15

    The dopamine agonist pramipexole is cleared predominantly by the kidney with a major contribution of active renal secretion. Previously the organic cation transporter 2 (OCT2) was shown to be involved in the uptake of pramipexole by renal tubular cells, while the mechanism underlying efflux into tubular lumen remains unclear. Cimetidine, a potent inhibitor of multidrug and toxin extrusion proteins 1 (MATE1) and 2-K (MATE2-K), decreases renal pramipexole clearance in humans. We hypothesized that, in addition to OCT2, pramipexole may be a substrate of MATE-mediated transport. Pramipexole uptake was investigated using MDCK or HEK cells overexpressing OCT2, MATE1 or MATE2-K and the respective vector controls (Co). Transcellular pramipexole transport was investigated in MDCK cells single- or double-transfected with OCT2 and/or MATE1 and in Co cells, separating a basal from an apical compartment in a model for renal tubular secretion. Pramipexole uptake was 1.6-, 1.1-, or 1.6-folds in cells overexpressing OCT2, MATE1 or MATE2-K, respectively as compared to Co cells (p<0.05). In transcellular transport experiments, intracellular pramipexole accumulation was 1.7-folds in MDCK-OCT2 (p<0.001), and transcellular pramipexole transport was 2.2- and 4.0-folds in MDCK-MATE1 and MDCK-OCT2-MATE1 cells as compared to Co cells (p<0.001). Transcellular pramipexole transport was pH dependent and inhibited by cimetidine with IC50 values of 12μM and 5.5μM in MATE1 and OCT2-MATE1 cells, respectively. Taken together, coordinate activity of OCT2-mediated uptake and MATE-mediated efflux determines pramipexole renal secretion. Reduced OCT2 or MATE transport activity due to genetic variation or drug-drug interactions may affect pramipexole renal secretion.

  2. Renal tubular secretion of glutathione (GSH)

    SciTech Connect

    Scott, R.D.; Curthoys, N.P.

    1986-05-01

    The rapid turnover of renal GSH may require its secretion into the tubular lumen. Renal clearance of plasma GSH was measured in rats anesthetized with Inactin and infused with (/sup 3/H)inulin. Renal ..gamma..-glutamyltranspeptidase (..gamma..GT) was then inactivated (> 97%) by infusion of acivicin and samples were collected for 6-7 h. By 4.5 h arterial and urinary GSH increased from 5..mu..M and 1.3 n mol/h to 23 ..mu..M and 2400-7000 nmol/h, respectively. The ratio of urinary GSH to filtered load increased from < 0.01 to 0.7-2.6. When renal GSH was decreased to 30% of normal by pretreating rats with buthionine sulfoximine (BSO), the subsequent inactivation of ..gamma..GT caused only a slight increase in arterial GSH and urinary GSH increased to only 400-600 nmol/h (60-70% of filtered load). The amount of GSH filtered by the kidney was reduced by initially treating a rat with acivicin and 3 h later infusing purified ..gamma..GT (0.2 mg/h) to degrade plasma GSH. Just before infusion of ..gamma..GT, arterial GSH was 23 ..mu..M and urinary GSH was equal to 90% of the filtered load. At 1 h after infusion of ..gamma..GT, arterial GSH decreased to 0.3 ..mu..M, whereas urinary GSH remained elevated (1200-1800 nmol/h) and now equalled 10-20 times the filtered load. When similar experiments were carried out in BSO treated rats, maximal urinary GSH was reduced to 200 nmol/h, a value that was still 10 times the filtered load. Therefore, secreted GSH constitutes a significant portion of the GSH that is normally catabolized within the tubular lumen.

  3. Loss of tubular creatinine secretion as the only sign of tubular proximal cell dysfunction in light chain proximal tubulopathy

    PubMed Central

    Stehlé, Thomas; Vignon, Marguerite; Flamant, Martin; Figueres, Marie-Lucile; Rabant, Marion; Rodenas, Anita; Noël, Laure-Hélène; Arnulf, Bertrand; Vidal-Petiot, Emmanuelle

    2016-01-01

    Abstract Light chain proximal tubulopathy (LCPT) is a rare disease, characterized by cytoplasmic inclusions of light chain (usually kappa) immunoglobulins. Clinical presentation is usually a Fanconi syndrome. The proximal tubular dysfunction can be incomplete, and exceptional cases of LCPT without any tubular dysfunction have even been described. Here, we report a case of LCPT in which the only sign of proximal tubulopathy is the absence of secretion of creatinine, as assessed by the simultaneous measurement of renal clearance of creatinine and 51CrEDTA. The loss of tubular creatinine secretion as a sign of tubular proximal cell dysfunction ought to be identified in patients with light chain proximal tubulopathy as it leads to a clinically relevant underestimation of GFR by the creatinine-derived equations. The prevalence and prognostic significance of this particular proximal tubular damage in LCPT remain to be determined. PMID:27367983

  4. Glomerular filtration and tubular secretion of MAG-3 in the rat kidney

    SciTech Connect

    Mueller-Suur, R.M.; Mueller-Suur, C. )

    1989-12-01

    Technetium-99m mercaptoacetyltriglycine (MAG-3) has recently been introduced as a new radiopharmaceutical for dynamic renal scintigraphy. To elucidate the mechanism of renal excretion, micropuncture experiments were performed in rat kidneys for direct measurements of glomerular filtration and tubular secretory capacity. Fluid of Bowman space was collected from superficial glomeruli and analyzed for its contents of (99mTc)MAG-3, (125I)hippurate and (3H)inulin during constant infusion of these compounds. The ratio of activity of ultrafiltrate to that of arterial plasma was 0.23 for MAG-3, 0.68 for hippurate and 1.04 for inulin which demonstrates that the filtrated amount of MAG-3 is only 23% of that of inulin, presumably because of higher plasma protein binding which was also measured in vitro and found to be 80 +/- 1.5% for MAG-3 and 32 +/- 2% for (125I)hippurate. Proximal and distal tubules were also micropunctured and their tubular fluid as well as the final urine analyzed for the activity of hippurate and MAG-3. The tubular fluid to plasma ratio values along the nephron and in the final urine were all lower for MAG-3 than for hippurate, indicating a lower secretory capacity. From measurements of whole renal clearance, GFR and plasma protein binding the filtered amount of MAG-3 was 0.26 and of hippurate 0.87 ml/min.g kidney weight (p less than 0.001) and the secreted amount 2.01 and 2.38 ml/min.g kidney weight (p less than 0.05), respectively. We conclude that MAG-3 is predominantly excreted by tubular secretion and that the lower renal clearance of MAG-3 as compared with that of hippurate is a result both of a substantially decreased glomerular filtration and of a lower tubular secretion.

  5. Tubular fluid secretion in the seminiferous epithelium: ion transporters and aquaporins in Sertoli cells.

    PubMed

    Rato, Luís; Socorro, Sílvia; Cavaco, José E B; Oliveira, Pedro F

    2010-07-01

    Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K(+) than the blood and express various membrane and water transporters (Na(+)/K(+)-ATPase; Ca(2+)-ATPase; V-type ATPase; Cl(-) channels; CFTR Cl(-) channels; K(+) channels; L-, T- and N-type Ca(2+) channels; Na(+)/H(+) exchangers; Na(+)-driven HCO(3) (-)/Cl(-) exchangers (NDCBEs); Na(+)/HCO(3) (-) cotransporters (NBCes); Na(+)-K(+)-2Cl(-) cotransporter; Na(+)/Ca(2+) exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.

  6. Competitive inhibition of renal tubular secretion of ciprofloxacin and metabolite by probenecid

    PubMed Central

    Landersdorfer, Cornelia B; Kirkpatrick, Carl M J; Kinzig, Martina; Bulitta, Jürgen B; Holzgrabe, Ulrike; Jaehde, Ulrich; Reiter, Andreas; Naber, Kurt G; Rodamer, Michael; Sörgel, Fritz

    2010-01-01

    AIMS Probenecid influences transport processes of drugs at several sites in the body and decreases elimination of several quinolones. We sought to explore extent, time course, and mechanism of the interaction between ciprofloxacin and probenecid at renal and nonrenal sites. METHODS A randomized, two-way crossover study was conducted in 12 healthy volunteers (in part previously published Clin Pharmacol Ther 1995; 58: 532–41). Subjects received 200 mg ciprofloxacin as 30-min intravenous infusion without and with 3 g probenecid divided into five oral doses. Drug concentrations were analysed by liquid chromatography–tandem mass spectrometry and high-performance liquid chromatography. Ciprofloxacin and its 2-aminoethylamino-metabolite (M1) in plasma and urine with and without probenecid were modelled simultaneously with WinNonlin®. RESULTS Data are ratio of geometric means (90% confidence intervals). Addition of probenecid reduced the median renal clearance from 23.8 to 8.25 l h−1[65% reduction (59, 71), P < 0.01] for ciprofloxacin and from 20.5 to 8.26 l h−1 (66% reduction (57, 73), P < 0.01] for M1 (estimated by modelling). Probenecid reduced ciprofloxacin nonrenal clearance by 8% (1, 14) (P < 0.08). Pharmacokinetic modelling indicated competitive inhibition of the renal tubular secretion of ciprofloxacin and M1 by probenecid. The affinity for the renal transporter was 4.4 times higher for ciprofloxacin and 3.6 times higher for M1 than for probenecid, based on the molar ratio. Probenecid did not affect volume of distribution of ciprofloxacin or M1, nonrenal clearance or intercompartmental clearance of ciprofloxacin. CONCLUSIONS Probenecid inhibited the renal tubular secretion of ciprofloxacin and M1, probably by a competitive mechanism and due to reaching >100-fold higher plasma concentrations. Formation of M1, nonrenal clearance and distribution of ciprofloxacin were not affected. PMID:20233180

  7. Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

    PubMed

    Rodriguez, Stéphane; Rudloff, Stefan; Koenig, Katrin Franziska; Karthik, Swapna; Hoogewijs, David; Huynh-Do, Uyen

    2016-08-01

    Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular

  8. Tubular dielectric elastomer actuator for active fluidic control

    NASA Astrophysics Data System (ADS)

    McCoul, David; Pei, Qibing

    2015-10-01

    We report a novel low-profile, biomimetic dielectric elastomer tubular actuator capable of actively controlling hydraulic flow. The tubular actuator has been established as a reliable tunable valve, pinching a secondary silicone tube completely shut in the absence of a fluidic pressure bias or voltage, offering a high degree of resistance against fluidic flow, and able to open and completely remove this resistance to flow with an applied low power actuation voltage. The system demonstrates a rise in pressure of ∼3.0 kPa when the dielectric elastomer valve is in the passive, unactuated state, and there is a quadratic fall in this pressure with increasing actuation voltage, until ∼0 kPa is reached at 2.4 kV. The device is reliable for at least 2000 actuation cycles for voltages at or below 2.2 kV. Furthermore, modeling of the actuator and fluidic system yields results consistent with the observed experimental dependence of intrasystem pressure on input flow rate, actuator prestretch, and actuation voltage. To our knowledge, this is the first actuator of its type that can control fluid flow by directly actuating the walls of a tube. Potential applications may include an implantable artificial sphincter, part of a peristaltic pump, or a computerized valve for fluidic or pneumatic control.

  9. The role played by endocytosis in albumin-induced secretion of TGF-beta1 by proximal tubular epithelial cells.

    PubMed

    Diwakar, Ramaswamy; Pearson, Alex L; Colville-Nash, Paul; Brunskill, Nigel J; Dockrell, Mark E C

    2007-05-01

    Proteinuria predicts the decline of renal function in chronic kidney disease. Reducing albuminuria has been shown to be associated with a reduction in this rate of decline. Proximal tubular epithelial cells (PTECs), when exposed to albumin produce matrix proteins, proinflammatory and profibrotic cytokines like TGF-beta(1). Some of these effects are dependent on endocytosis of albumin by PTECs. However, conditions like diabetic nephropathy, believed to be associated with reduced albumin endocytosis, are associated with interstitial fibrosis. Moreover, megalin, the putative albumin binding receptor in PTECs, has potential signaling motifs in its cytoplasmic domain, suggesting its ability to signal in response to ligand binding from the apical surface of PTECs. Hence, we looked to see whether albumin-induced secretion of TGF-beta(1) by PTECs is dependent on albumin endocytosis or whether it could occur in the absence of albumin endocytosis. We studied the production of TGF-beta(1) in two accepted models of PTECs, opossum kidney cells and human kidney cell clone-8 cells, with widely varying degrees of endocytosis. We then studied the effect of inhibiting albumin endocytosis with various inhibitors on albumin-induced TGF-beta(1) secretion. Our results indicate that albumin-induced TGF-beta(1) secretion by PTECs does not require albumin endocytosis and therefore the mechanism for the induction of some profibrotic responses by albumin may differ from those required for some of the inflammatory responses. Moreover, we found that albumin-induced TGF-beta(1) secretion by PTECs is not dependent on its interaction with megalin. PMID:17213467

  10. Calcium oxalate crystals increased enolase-1 secretion from renal tubular cells that subsequently enhanced crystal and monocyte invasion through renal interstitium

    PubMed Central

    Chiangjong, Wararat; Thongboonkerd, Visith

    2016-01-01

    Calcium oxalate monohydrate (COM) crystals cause kidney stone disease by still unclear mechanisms. The present study aimed to characterize changes in secretion of proteins from basolateral compartment of renal tubular epithelial cells after exposure to COM crystals and then correlated them with the stone pathogenesis. Polarized MDCK cells were cultivated in serum-free medium with or without 100 μg/ml COM crystals for 20 h. Secreted proteins collected from the lower chamber (basolateral compartment) were then resolved in 2-D gels and visualized by Deep Purple stain (n = 5 gels/group). Spot matching and intensity analysis revealed six protein spots with significantly altered levels in COM-treated samples. These proteins were then identified by tandem mass spectrometry (Q-TOF MS/MS), including enolase-1, phosphoglycerate mutase-1, actinin, 14-3-3 protein epsilon, alpha-tubulin 2, and ubiquitin-activating enzyme E1. The increased enolase-1 level was confirmed by Western blot analysis. Functional analysis revealed that enolase-1 dramatically induced COM crystal invasion through ECM migrating chamber in a dose-dependent manner. Moreover, enolase-1 bound onto U937 monocytic cell surface markedly enhanced cell migration through the ECM migrating chamber. In summary, our data indicated that the increased secretory enolase-1 induced by COM crystals played an important role in crystal invasion and inflammatory process in renal interstitium. PMID:27045290

  11. Activated microglia enhance neurogenesis via trypsinogen secretion.

    PubMed

    Nikolakopoulou, Angeliki M; Dutta, Ranjan; Chen, Zhihong; Miller, Robert H; Trapp, Bruce D

    2013-05-21

    White matter neurons in multiple sclerosis brains are destroyed during demyelination and then replaced in some chronic multiple sclerosis lesions that exhibit a morphologically distinct population of activated microglia [Chang A, et al. (2008) Brain 131(Pt 9):2366-2375]. Here we investigated whether activated microglia secrete factors that promote the generation of neurons from white matter cells. Adult rat brain microglia (resting or activated with lipopolysaccharide) were isolated by flow cytometry and cocultured with neonatal rat optic nerve cells in separate but media-connected chambers. Optic nerve cells cocultured with activated microglia showed a significant increase in the number of cells of neuronal phenotype, identified by neuron-specific class III beta-tubulin (TUJ-1) labeling, compared with cultures with resting microglia. To investigate the possible source of the TUJ-1-positive cells, A2B5-positive oligodendrocyte progenitor cells and A2B5-negative cells were isolated and cocultured with resting and activated microglia. Significantly more TUJ-1-positive cells were generated from A2B5-negative cells (∼70%) than from A2B5-positive cells (~30%). Mass spectrometry analysis of microglia culture media identified protease serine 2 (PRSS2) as a factor secreted by activated, but not resting, microglia. When added to optic nerve cultures, PRSS2 significantly increased neurogenesis, whereas the serine protease inhibitor, secretory leukocyte protease inhibitor, decreased activated microglia-induced neurogenesis. Collectively our data provide evidence that activated microglia increase neurogenesis through secretion of PRSS2.

  12. Chronicity following ischaemia-reperfusion injury depends on tubular-macrophage crosstalk involving two tubular cell-derived CSF-1R activators: CSF-1 and IL-34.

    PubMed

    Sanchez-Niño, Maria Dolores; Sanz, Ana Belen; Ortiz, Alberto

    2016-09-01

    Two structurally unrelated ligands activate the macrophage colony stimulating factor receptor (CSF-1R, c-fms, CD115): M-CSF/CSF-1 and interleukin-34 (IL-34). Both ligands promote macrophage proliferation, survival and differentiation. IL-34 also activates the protein-tyrosine phosphatase ζ receptor (PTP-ζ, PTPRZ1). Both receptors and cytokines are increased during acute kidney injury. While tubular cell-derived CSF-1 is required for kidney repair, Baek et al (J Clin Invest 2015; 125: 3198-3214) have now identified tubular epithelial cell-derived IL-34 as a promoter of kidney neutrophil and macrophage infiltration and tubular cell destruction during experimental kidney ischaemia-reperfusion, leading to chronic injury. IL-34 promoted proliferation of both intrarenal macrophages and bone marrow cells, increasing circulating neutrophils and monocytes and their kidney recruitment. Thus, injured tubular cells release two CSF-1R activators, one (CSF-1) that promotes tubular cell survival and kidney repair and another (IL-34) that promotes chronic kidney damage. These results hold promise for the development of IL-34-targeting strategies to prevent ischaemia-reperfusion kidney injury in contexts such as kidney transplantation. However, careful consideration should be given to the recent characterization by Bezie et al. (J Clin Invest 2015; 125: 3952-3964) of IL-34 as a T regulatory cell (Treg) cytokine that modulates macrophage responses so that IL-34-primed macrophages potentiate the immune suppressive capacity of Tregs and promote graft tolerance. PMID:27190368

  13. A tubular dielectric elastomer actuator: Fabrication, characterization and active vibration isolation

    NASA Astrophysics Data System (ADS)

    Sarban, R.; Jones, R. W.; Mace, B. R.; Rustighi, E.

    2011-11-01

    This contribution reviews the fabrication, characterization and active vibration isolation performance of a core-free rolled tubular dielectric elastomer (DE) actuator, which has been designed and developed by Danfoss PolyPower A/S. PolyPower DE material, PolyPower TM, is produced in thin sheets of 80 μm thickness with corrugated metallic electrodes on both sides. Tubular actuators are manufactured by rolling the DE sheets in a cylindrical shape. The electromechanical characteristics of such actuators are modeled based on equilibrium pressure equation. The model is validated with experimental measurements from 3 actuators. The dynamic characteristics of three tubular actuators fabricated from the same batch of manufactured DE material are presented and compared to: (a) provide insight into the ability of the fabrication process to produce actuators with similar characteristics and (b) highlight the dominant dynamic characteristics of the core-free tubular actuator. It has been observed that all actuators have similar dynamic characteristics in a frequency range up to 1 kHz. A tubular actuator is then used to provide active vibration isolation (AVI) of a 250 g mass subject to shaker generated 'ground vibration'. An adaptive feedforward control approach is used to achieve this. The tubular actuator is shown to provide excellent isolation against harmonic vibratory disturbances with attenuation of the resulting 5 and 10 Hz harmonics being 66 and 23 dB, respectively. AVI against a narrow band vibratory disturbance with frequency content 2-8 Hz, produced an attenuation of 20 dB across the frequency band.

  14. Active Wnt proteins are secreted on exosomes.

    PubMed

    Gross, Julia Christina; Chaudhary, Varun; Bartscherer, Kerstin; Boutros, Michael

    2012-10-01

    Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

  15. Cisplatin Nephrotoxicity Involves Mitochondrial Injury with Impaired Tubular Mitochondrial Enzyme Activity

    PubMed Central

    Ellezian, Lena; Brown, Dan; Horváth, Béla; Mukhopadhyay, Partha; Kalyanaraman, Balaraman; Parikh, Samir M.; Karumanchi, S. Ananth; Stillman, Isaac E.; Pacher, Pál

    2012-01-01

    Cisplatin is a widely used antineoplastic agent. However, its major limitation is dose-dependent nephrotoxicity whose precise mechanism is poorly understood. Recent studies have suggested that mitochondrial dysfunction in tubular epithelium contributes to cisplatin-induced nephrotoxicity. Here the authors extend those findings by describing the role of an important electron transport chain enzyme, cytochrome c oxidase (COX). Immunohistochemistry for COX 1 protein demonstrated that, in response to cisplatin, expression was mostly maintained in focally damaged tubular epithelium. In contrast, COX enzyme activity in proximal tubules (by light microscopy) was decreased. Ultrastructural analysis of the cortex and outer stripe of the outer medulla showed decreased mitochondrial mass, disruption of cristae, and extensive mitochondrial swelling in proximal tubular epithelium. Functional electron microscopy showed that COX enzyme activity was decreased in the remaining mitochondria in the proximal tubules but maintained in distal tubules. In summary, cisplatin-induced nephrotoxicity is associated with structural and functional damage to the mitochondria. More broadly, using functional electron microscopy to measure mitochondrial enzyme activity may generate mechanistic insights across a spectrum of renal disorders. PMID:22511597

  16. Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity.

    PubMed

    Zsengellér, Zsuzsanna K; Ellezian, Lena; Brown, Dan; Horváth, Béla; Mukhopadhyay, Partha; Kalyanaraman, Balaraman; Parikh, Samir M; Karumanchi, S Ananth; Stillman, Isaac E; Pacher, Pál

    2012-07-01

    Cisplatin is a widely used antineoplastic agent. However, its major limitation is dose-dependent nephrotoxicity whose precise mechanism is poorly understood. Recent studies have suggested that mitochondrial dysfunction in tubular epithelium contributes to cisplatin-induced nephrotoxicity. Here the authors extend those findings by describing the role of an important electron transport chain enzyme, cytochrome c oxidase (COX). Immunohistochemistry for COX 1 protein demonstrated that, in response to cisplatin, expression was mostly maintained in focally damaged tubular epithelium. In contrast, COX enzyme activity in proximal tubules (by light microscopy) was decreased. Ultrastructural analysis of the cortex and outer stripe of the outer medulla showed decreased mitochondrial mass, disruption of cristae, and extensive mitochondrial swelling in proximal tubular epithelium. Functional electron microscopy showed that COX enzyme activity was decreased in the remaining mitochondria in the proximal tubules but maintained in distal tubules. In summary, cisplatin-induced nephrotoxicity is associated with structural and functional damage to the mitochondria. More broadly, using functional electron microscopy to measure mitochondrial enzyme activity may generate mechanistic insights across a spectrum of renal disorders. PMID:22511597

  17. Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells

    PubMed Central

    Gui, Ting; Gai, Zhibo

    2015-01-01

    To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly induced. Here we describe in details the contents and quality controls for the gene expression and related results associated with the data uploaded to Gene Expression Omnibus (accession number GSE70296). PMID:26697325

  18. Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy

    PubMed Central

    Liu, Hong; Gu, Liu-bao; Tu, Yue; Hu, Hao; Huang, Yan-ru; Sun, Wei

    2016-01-01

    Aim: A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro. Methods: The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. Results: Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects. Conclusion: Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity. PMID:26775661

  19. Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

    PubMed

    Yiu, Wai Han; Wong, Dickson W L; Chan, Loretta Y Y; Leung, Joseph C K; Chan, Kwok Wah; Lan, Hui Yao; Lai, Kar Neng; Tang, Sydney C W

    2014-01-01

    Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation. PMID:24586431

  20. Tissue Kallikrein Mediates Pro-Inflammatory Pathways and Activation of Protease-Activated Receptor-4 in Proximal Tubular Epithelial Cells

    PubMed Central

    Yiu, Wai Han; Wong, Dickson W. L.; Chan, Loretta Y. Y.; Leung, Joseph C. K.; Chan, Kwok Wah; Lan, Hui Yao; Lai, Kar Neng; Tang, Sydney C. W.

    2014-01-01

    Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation. PMID:24586431

  1. IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway

    PubMed Central

    Wu, Zengbin; Yu, Yang; Niu, Lei; Fei, Aihua; Pan, Shuming

    2016-01-01

    Injury of renal tubular epithelial cells can induce acute renal failure and obstructive nephropathy. Previous studies have shown that administration of insulin-like growth factor-1 (IGF-1) ameliorates the renal injury in a mouse unilateral ureteral obstruction (UUO) model, whereas the underlying mechanisms are not completely understood. Here, we addressed this question. We found that the administration of IGF-1 significantly reduced the severity of the renal fibrosis in UUO. By analyzing purified renal epithelial cells, we found that IGF-1 significantly reduced the apoptotic cell death of renal epithelial cells, seemingly through upregulation of anti-apoptotic protein Bcl-2, at protein but not mRNA level. Bioinformatics analyses and luciferase-reporter assay showed that miR-429 targeted the 3′-UTR of Bcl-2 mRNA to inhibit its protein translation in renal epithelial cells. Moreover, IGF-1 suppressed miR-429 to increase Bcl-2 in renal epithelial cells to improve survival after UUO. Furthermore, inhibition of ERK/MAPK signaling pathway in renal epithelial cells abolished the suppressive effects of IGF-1 on miR-429 activation, and then the enhanced effects on Bcl-2 in UUO. Thus, our data suggest that IGF-1 may protect renal tubular epithelial cells via activation of ERK/MAPK signaling pathway during renal injury. PMID:27301852

  2. Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

    PubMed Central

    Böhm, Johann; Chevessier, Frédéric; De Paula, André Maues; Koch, Catherine; Attarian, Shahram; Feger, Claire; Hantaï, Daniel; Laforêt, Pascal; Ghorab, Karima; Vallat, Jean-Michel; Fardeau, Michel; Figarella-Branger, Dominique; Pouget, Jean; Romero, Norma B.; Koch, Marc; Ebel, Claudine; Levy, Nicolas; Krahn, Martin; Eymard, Bruno; Bartoli, Marc; Laporte, Jocelyn

    2013-01-01

    Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca2+ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca2+-binding EF hands. Ca2+ stores are refilled through a process called store-operated Ca2+ entry (SOCE). Upon Ca2+-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca2+ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca2+ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca2+ level in TAM cells and a dysregulation of intracellular Ca2+ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function. PMID:23332920

  3. Effects of cytokines on potassium channels in renal tubular epithelia.

    PubMed

    Nakamura, Kazuyoshi; Komagiri, You; Kubokawa, Manabu

    2012-02-01

    Renal tubular potassium (K(+)) channels play important roles in the formation of cell-negative potential, K(+) recycling, K(+) secretion, and cell volume regulation. In addition to these physiological roles, it was reported that changes in the activity of renal tubular K(+) channels were involved in exacerbation of renal cell injury during ischemia and endotoxemia. Because ischemia and endotoxemia stimulate production of cytokines in immune cells and renal tubular cells, it is possible that cytokines would affect K(+) channel activity. Although the regulatory mechanisms of renal tubular K(+) channels have extensively been studied, little information is available about the effects of cytokines on these K(+) channels. The first report was that tumor necrosis factor acutely stimulated the single channel activity of the 70 pS K(+) channel in the rat thick ascending limb through activation of tyrosine phosphatase. Recently, it was also reported that interferon-γ (IFN-γ) and interleukin-1β (IL-1β) modulated the activity of the 40 pS K(+) channel in cultured human proximal tubule cells. IFN-γ exhibited a delayed suppression and an acute stimulation of K(+) channel activity, whereas IL-1β acutely suppressed the channel activity. Furthermore, these cytokines suppressed gene expression of the renal outer medullary potassium channel. The renal tubular K(+) channels are functionally coupled to the coexisting transporters. Therefore, the effects of cytokines on renal tubular transporter activity should also be taken into account, when interpreting their effects on K(+) channel activity. PMID:22042037

  4. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  5. The molecular interactions between filtered proteins and proximal tubular cells in proteinuria.

    PubMed

    Baines, Richard J; Brunskill, Nigel J

    2008-01-01

    Proteinuria is associated with progressive chronic kidney disease and poor cardiovascular outcomes. Exposure of proximal tubular epithelial cells to excess proteins leads to the development of proteinuric nephropathy with tubular atrophy, interstitial inflammation and scarring. Numerous signalling pathways are activated in proximal tubular epithelial cells under proteinuric conditions resulting in gene transcription, altered growth and the secretion of inflammatory and profibrotic mediators. Megalin, the proximal tubular scavenger receptor for filtered macromolecules, has intrinsic signalling functions and may also link albumin to growth factor receptor signalling via regulated intramembrane proteolysis. It now seems that endocytosis is not always a prerequisite for albumin-evoked alterations in proximal tubular cell phenotype. Recent evidence shows the presence of other potential receptors for proteins, such as the neonatal Fc receptor and CD36, in the proximal tubular epithelium. PMID:18849618

  6. Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney

    SciTech Connect

    Rogers, S.A.; Hammerman, M.R. )

    1989-08-01

    To delineate pathways for signal transduction by growth hormone (GH) in proximal tubule, the authors incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP{sub 3}) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP{sub 3} and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP{sub 3} were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP{sub 3} in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP{sub 3} were detectable in membranes suspended in 0, 0.1, and 0.2 {mu}M calcium but not in 0.3 or 1.0 {mu}M calcium. {sup 125}I-labeled hGH-receptor complexes with M{sub r} values of 66,000 and 140,000 were identified in isolated basolateral membranes. The findings establish that GH activates phospholipase C in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate signal transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.

  7. Mitochondrial reactive oxygen species-mediated NLRP3 inflammasome activation contributes to aldosterone-induced renal tubular cells injury

    PubMed Central

    Ding, Wei; Guo, Honglei; Xu, Chengyan; Wang, Bin; Zhang, Minmin; Ding, Feng

    2016-01-01

    Aldosterone (Aldo) is an independent risk factor for chronic kidney disease (CKD), and although Aldo directly induces renal tubular cell injury, the underlying mechanisms remain unclear. NLRP3 inflammasome and mitochondrial reactive oxygen species (ROS) have recently been implicated in various kinds of CKD. The present study hypothesized that mitochondrial ROS and NLRP3 inflammasome mediated Aldo–induced tubular cell injury. The NLRP3 inflammasome is induced by Aldo in a dose- and time-dependent manner, as evidenced by increased NLRP3, ASC, caspase-1, and downstream cytokines, such as interleukin (IL)-1β and IL-18. The activation of the NLRP3 inflammasome was significantly prevented by the selective mineralocorticoid receptor (MR) antagonist eplerenone (EPL) (P < 0.01). Mice harboring genetic knock-out of NLRP3 (NLRP3−/−) showed decreased maturation of renal IL-1β and IL-18, reduced renal tubular apoptosis, and improved renal epithelial cell phenotypic alternation, and attenuated renal function in response to Aldo-infusion. In addition, mitochondrial ROS was also increased in Aldo-stimulated HK-2 cells, as assessed by MitoSOXTM red reagent. Mito-Tempo, the mitochondria-targeted antioxidant, significantly decreased HK-2 cell apoptosis, oxidative stress, and the activation of NLRP3 inflammasome. We conclude that Aldo induces renal tubular cell injury via MR dependent, mitochondrial ROS-mediated NLRP3 inflammasome activation. PMID:27014913

  8. The secrets of highly active older adults.

    PubMed

    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). PMID:24300060

  9. A supramolecular tubular nanoreactor.

    PubMed

    Li, Zhi-Qiang; Zhang, Ying-Ming; Chen, Yong; Liu, Yu

    2014-07-01

    The extremely strong noncovalent complexation between the rigid host of phthalocyanine-bridged β-cyclodextrins and the amphiphilic guest carboxylated porphyrin is employed to construct a hollow tubular structure as a supramolecular nanoreactor. A representative coupling reaction occurs in the hydrophobic interlayers of the tubular walls in pure water at room temperature, leading to an enhancement of ten times higher reaction rate without any adverse effect on catalytic activity and conversion. PMID:24890802

  10. Induction of interleukin 1 secretion by adjuvant-active peptidoglycans.

    PubMed Central

    Vacheron, F; Guenounou, M; Nauciel, C

    1983-01-01

    The ability of differently structured, purified peptidoglycans (PG) to induce interleukin 1 (IL1) secretion was compared. PG from Bacillus megaterium and Staphylococcus aureus stimulated the production of IL1 by mouse peritoneal macrophages and human adherent mononuclear cells, whereas PG from Micrococcus lysodeikticus and Corynebacterium poinsettiae were inactive. There was a correlation between the ability of PG to induce IL1 secretion and previously demonstrated immunoenhancing activities (adjuvant effect, increase of resistance to tumor growth) of PG. PG solubilization by lysozyme decreased but did not abolish the PG effect on IL1 secretion. Active PG induced IL1 production in nude mice and in the C3H/HeJ strain (which is unresponsive to lipopolysaccharides). PMID:6605929

  11. Secrets of Lost Empires: Family Activity Book.

    ERIC Educational Resources Information Center

    Whitman, John D.; Gaffney, Dennis

    This family activity book features information on the background and filming of five stories. "Pharaoh's Obelisk" questions how ancient Egyptians transported and raised stone obelisks. "Roman Bath" studies how Romans built bathhouses and attempts to build a working Roman bath. "China Bridge" investigates the structure of the China Bridge and…

  12. Secretion of platelet-activating factor by periovulatory ovine follicles

    SciTech Connect

    Alexander, B.M.; Van Kirk, E.A.; Murdoch, W.J. )

    1990-01-01

    Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.

  13. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2014-05-27

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  14. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2013-10-01

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  15. Methods of increasing secretion of polypeptides having biological activity

    SciTech Connect

    Merino, Sandra

    2014-10-28

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  16. Methods of increasing secretion of polypeptides having biological activity

    SciTech Connect

    Merino, Sandra

    2015-04-14

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  17. Biodegradation of wastewater pollutants by activated sludge encapsulated inside calcium-alginate beads in a tubular packed bed reactor.

    PubMed

    Sergio, A Martinez D; Bustos, T Yaneth

    2009-09-01

    The wastewater treatment plants produce large quantities of biomass (sludge) that require about one-third of the total inversion and operation plant costs for their treatment. By the microorganisms immobilization it is possible to handle high cell concentration in the reactor, increasing its efficiency, reducing the loss of biomass and the wash out is avoided. Moreover, there is no cell growth then the sludge production is reduced. In this study, the COD removal and VSS variation were modeled in a tubular reactor with activated sludge immobilized in Ca-alginate. Moreover, two aspects that are commonly not considered in the performance of the actual reactors of this kind were introduced; the performance in non-steady state and the dispersion effect. The model was calibrated with an actual wastewater taken out from a Mexican wastewater treatment plant. The results of the performance of the tubular bioreactor at different scenarios (i.e., different residence time and VSS in the reactor) are presented. With longer residence times and higher VSS concentration in the Ca-alginate beads in the tubular bioreactor it is possible to increase the time operation of the bioreactor and to treat higher volumes of wastewater. During the process, the sludge generation was drastically reduced and it is possible to remove nitrogen form the wastewater making this process more attractive.

  18. Neuronal Activity Promotes Glioma Growth through Neuroligin-3 Secretion.

    PubMed

    Venkatesh, Humsa S; Johung, Tessa B; Caretti, Viola; Noll, Alyssa; Tang, Yujie; Nagaraja, Surya; Gibson, Erin M; Mount, Christopher W; Polepalli, Jai; Mitra, Siddhartha S; Woo, Pamelyn J; Malenka, Robert C; Vogel, Hannes; Bredel, Markus; Mallick, Parag; Monje, Michelle

    2015-05-01

    Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). By using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma xenograft model, we demonstrate that active neurons similarly promote HGG proliferation and growth in vivo. Conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures, indicating secretion of activity-regulated mitogen(s). The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen, and soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feedforward expression of NLGN3 in glioma cells. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

  19. Antifungal activity of lectins against yeast of vaginal secretion

    PubMed Central

    Gomes, Bruno Severo; Siqueira, Ana Beatriz Sotero; de Cássia Carvalho Maia, Rita; Giampaoli, Viviana; Teixeira, Edson Holanda; Arruda, Francisco Vassiliepe Sousa; do Nascimento, Kyria Santiago; de Lima, Adriana Nunes; Souza-Motta, Cristina Maria; Cavada, Benildo Sousa; Porto, Ana Lúcia Figueiredo

    2012-01-01

    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. PMID:24031889

  20. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  1. Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells

    PubMed Central

    Wu, Yiqing; Zhang, Min; Liu, Rui

    2016-01-01

    Purpose Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. Materials and Methods Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. Results HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. Conclusion Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells. PMID:27401659

  2. Simultaneous recording of t-tubular electrical activity and Ca2+-release in heart failure

    NASA Astrophysics Data System (ADS)

    Crocini, C.; Coppini, R.; Ferrantini, C.; Yan, P.; Loew, L.; Tesi, C.; Poggesi, C.; Cerbai, E.; Pavone, F. S.; Sacconi, L.

    2014-05-01

    T-tubules (TT) are invaginations of the surface sarcolemma (SS) that mediate the rapid propagation of the action potential (AP) to the cardiomyocyte core. We employed the advantages of an ultrafast random access multi-photon (RAMP) microscope (Sacconi et al., PNAS 2012) with a double staining approach to optically record t-tubular AP and, simultaneously, the corresponding local Ca2+-release in different positions across the cardiomyocytes. Despite a uniform AP between SS and TT at steady-state stimulation, in control cardiomyocytes we observed a non-negligible be variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced by applying 0.1μM Isoproterenol, which increases the opening probability of Ca2+-release units. In the rat heart failure model (HF), we previously demonstrated that some tubular elements fail to propagate AP. We found that the tubules unable to propagate AP, displayed a reduced correspondent Ca2+-transient amplitude as well as a slower Ca2+ rise compared to electrically coupled tubules. Moreover variability of Ca2+-transient kinetics were increased in HF. Finally, TT that did not show AP, occasionally exhibited spontaneous depolarizations that were never accompanied by local Ca2+-release in the absence of any pro-arrhythmogenic stimulation. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodeled tubules. This work was funded by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162).

  3. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

    SciTech Connect

    González-Guerrero, Cristian; Ocaña-Salceda, Carlos; Berzal, Sergio; Carrasco, Susana; Fernández-Fernández, Beatriz; and others

    2013-11-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory

  4. Secretion of active bovine somatotropin in Escherichia coli.

    PubMed

    Klein, B K; Hill, S R; Devine, C S; Rowold, E; Smith, C E; Galosy, S; Olins, P O

    1991-09-01

    We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.

  5. Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

    PubMed Central

    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.

    2015-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a 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 signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling 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 types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems. PMID:25731164

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

    PubMed

    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.

  7. The Mitogen-Activated Protein Kinase p38α Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis

    PubMed Central

    Müller, Ralf; Daniel, Christoph; Hugo, Christian; Amann, Kerstin; Mielenz, Dirk; Endlich, Karlhans; Braun, Tobias; van der Veen, Betty; Heeringa, Peter; Schett, Georg; Zwerina, Jochen

    2013-01-01

    p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38α conditional knockout mice (MxCre-p38αΔ/Δ) were used to examine the role of p38α in anti-GBM induced nephritis. Both wild type and MxCre-p38αΔ/Δ mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38αΔ/Δ mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38αΔ/Δ mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38αΔ/Δ mice. In conclusion, p38α is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs. PMID:23441175

  8. [The role of disorders in the tubular transport of water and osmotically active substances in the progression of chronic kidney failure].

    PubMed

    Esaian, A M; Kaiukov, I G

    1993-01-01

    Lithium clearance served the criterion for measuring proximal and distal tubular transport of water, sodium and osmotically active substances (OAS) in 41 patients with chronic renal failure (CRF) of various degree. Distal reabsorption of water and sodium occurs early in CRF. In moderate disease the authors observed a drastic rise in distal excreted fraction of OAS which surpassed that of sodium threefold. Proximal fractional excretion of water acquired defects in advanced CRF only. In the range of creatinine clearance 30-65 ml/min an inverse relationship appeared between the clearance and the ratio of urea of OAS concentrations to chlorine concentrations. Distal fractional OAS excretion correlated with relevant parameters directly. It is suggested that a decline in relative chlorine ions content in the tubular fluid at the site of the thick spot entails hypoactivity of the tubular-glomerular feedback mechanisms resultant in hypertension and hyperfiltration in the intact nephrons which start to perish because of overloading.

  9. Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

    PubMed

    Higazi, Tarig B; Pearlman, Eric; Whikehart, David R; Unnasch, Thomas R

    2003-06-01

    Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

  10. Wnt-dependent beta-catenin signaling is activated after unilateral ureteral obstruction, and recombinant secreted frizzled-related protein 4 alters the progression of renal fibrosis.

    PubMed

    Surendran, Kameswaran; Schiavi, Susan; Hruska, Keith A

    2005-08-01

    beta-Catenin functions as a transducer of Wnt signals to the nucleus, where it interacts with the T cell factor (TCF) family of DNA binding proteins to regulate gene expression. On the basis of the genes regulated by beta-catenin and TCF in various biologic settings, two predicted functions of beta-catenin/TCF-dependent transcription are to mediate the loss of epithelial polarity and to promote fibroblast activities, such as the increased synthesis of fibronectin during chronic renal disease. These predictions were tested by determination of the expression and function of an inhibitor of Wnt signaling, secreted frizzled-related protein 4 (sFRP4), during renal tubular epithelial injury initiated by unilateral ureteral obstruction (UUO). Despite increased sFRP4 gene expression in perivascular regions of injured kidneys, total sFRP4 protein levels decreased after injury. The decreased sFRP4 protein levels after UUO accompanied increased Wnt-dependent beta-catenin signaling in tubular epithelial and interstitial cells, along with increased expression of markers of fibrosis. Administration of recombinant sFRP4 protein caused a reduction in tubular epithelial beta-catenin signaling and suppressed the progression of renal fibrosis, as evidenced by a partial maintenance of E-cadherin mRNA expression and a reduction in the amount of fibronectin and alpha-smooth muscle actin proteins. Furthermore, recombinant sFRP4 reduced the number of myofibroblasts, a central mediator of fibrosis. It is concluded that beta-catenin signaling is activated in tubular epithelial and interstitial cells after renal injury, and recombinant sFRP4 can interfere with epithelial de-differentiation and with fibroblast differentiation and function during progression of renal fibrosis.

  11. Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma.

    PubMed

    Arici, Mustafa; Chana, Ravinder; Lewington, Andrew; Brown, Jez; Brunskill, Nigel John

    2003-01-01

    In nephrotic syndrome, large quantities of albumin enter the kidney tubule. This albumin carries with it a heavy load of fatty acids to which the proximal tubule cells are exposed at high concentration. It is postulated that exposure to fatty acids in this way is injurious to proximal tubule cells. This study has examined the ability of fatty acids to interact with peroxisome proliferator-activated receptors (PPAR) in primary cultures of human proximal tubule cells. Luciferase reporter assays in transiently transfected human proximal tubule cells were used to show that albumin bound fatty acids and other agonists activate PPARgamma in a dose-dependent manner. One of the consequences of this activation is apoptosis of the cells as determined by changes in cell morphology, evidence of PARP cleavage, and appearance of DNA laddering. Overexpression of PPARgamma in these cells also results in enhanced apoptosis. Both fatty acid-induced PPAR activation and apoptosis in these cells can be blocked by PPAR response element decoy oligonucleotides. Activation of PPARgamma by the specific agonist PGJ(2) is associated with inhibition of cell proliferation, whereas activation by albumin bound fatty acids is accompanied by increased proliferation. However, the net balance of apoptosis/proliferation favors deletion of cells. These results implicate albumin-bound fatty acids as important mediators of tubular injury in nephrosis and provide fresh impetus for pursuit of lipid-lowering strategies in proteinuric renal disease. PMID:12506134

  12. Calpain secreted by activated human lymphoid cells degrades myelin.

    PubMed

    Deshpande, R V; Goust, J M; Hogan, E L; Banik, N L

    1995-10-01

    Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination. PMID:8568927

  13. Tubular toxicity of proteinuria.

    PubMed

    Baines, Richard J; Brunskill, Nigel J

    2011-03-01

    Proteinuria is a prognostic indicator of progressive kidney disease and poor cardiovascular outcomes. Abnormally filtered bioactive macromolecules interact with proximal tubular epithelial cells (PTECs), which results in the development of proteinuric nephropathy. This condition is characterized by alterations in PTEC growth, apoptosis, gene transcription and inflammatory cytokine production as a consequence of dysregulated signaling pathways that are stimulated by proteinuric tubular fluid. The megalin-cubilin complex mediates the uptake of several proteins, including albumin, into PTECs. Megalin might also possess intrinsic signaling properties and the ability to regulate cell signaling pathways and gene transcription after processing regulated intramembrane proteolysis. Megalin could, therefore, link abnormal PTEC albumin exposure with altered growth factor receptor activation, proinflammatory and profibrotic signaling, and gene transcription. Evidence now suggests that other PTEC pathways for protein reabsorption of (patho)physiological importance might be mediated by the neonatal Fc receptor and CD36. PMID:21151210

  14. Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity.

    PubMed

    Smith, Philip; Fallon, Rosie E; Mangan, Niamh E; Walsh, Caitriona M; Saraiva, Margarida; Sayers, Jon R; McKenzie, Andrew N J; Alcami, Antonio; Fallon, Padraic G

    2005-11-21

    The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been selected to modulate the host to tolerate chronic worm infections, often for decades, without overt morbidity. The modulation of immunity by schistosomes has been shown to prevent a range of immune-mediated diseases, including allergies and autoimmunity. Individual immune-modulating schistosome molecules have, therefore, therapeutic potential as selective manipulators of the immune system to prevent unrelated diseases. Here we show that S. mansoni eggs secrete a protein into host tissues that binds certain chemokines and inhibits their interaction with host chemokine receptors and their biological activity. The purified recombinant S. mansoni chemokine binding protein (smCKBP) suppressed inflammation in several disease models. smCKBP is unrelated to host proteins and is the first described chemokine binding protein encoded by a pathogenic human parasite and may have potential as an antiinflammatory agent.

  15. Fluid Flow Stimulates Tissue Plasminogen Activator Secretion by Cultured Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Diamond, S. L.; Eskin, S. G.; McIntire, L. V.

    1989-03-01

    Wall shear stress generated by blood flow may regulate the expression of fibrinolytic proteins by endothelial cells. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) secretion by cultured human endothelial cells were not affected by exposure to venous shear stress (4 dynes/cm2). However, at arterial shear stresses of 15 and 25 dynes/cm2, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unaffected by shear stress over the entire physiological range.

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

    PubMed

    van der Plas, Mariena J A; Andersen, Anders S; Nazir, Sheresma; van Tilburg, Nico H; Oestergaard, Peter R; Krogfelt, Karen A; van Dissel, Jaap T; Hensbergen, Paul J; Bertina, Rogier M; Nibbering, Peter H

    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 plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel serine protease within secretions, designated Sericase, cleaved plasminogen to several fragments. Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis by Sericase. We conclude from our in vitro studies that the novel serine protease Sericase, with the aid of a non-proteolytic cofactor, enhances plasminogen activator-induced fibrinolysis.

  17. Secreted dipeptidyl peptidase IV activity in the dimorphic fungal pathogen Histoplasma capsulatum.

    PubMed

    Cooper, Kendal G; Woods, Jon P

    2009-06-01

    Dipeptidyl peptidase type IV (DppIV) enzymes are broadly distributed phylogenetically and display diverse functions, including intercellular signaling, immunomodulation, protein maturation and processing, metabolism, and nutrient acquisition. We identified a secreted proteolytic activity in Histoplasma capsulatum effective toward DppIV-specific substrates. In order to determine the gene(s) that encodes this activity, we identified two putative DPPIV homologs (HcDPPIVA and HcDPPIVB) in H. capsulatum based on a homology search with Aspergillus fumigatus DppIV. Comparative sequence analysis revealed that HcDppIVA is similar to secreted DppIV enzymes, while HcDppIVB clusters with intracellular DapB-like enzymes. Unexpectedly, silencing of HcDPPIVA by RNA interference (RNAi) had no effect on secreted DppIV activity and an HcDPPIVA-null deletion mutant also showed no abrogation of secreted DppIV activity. In contrast, RNAi silencing of HcDPPIVB significantly reduced the level of secreted DppIV activity. RNAi silencing of HcDPPIVB in the HcDPPIVA-null mutant had no additional effect on secreted DppIV activity, indicating that HcDPPIVA does not contribute to secreted activity. RNAi silencing of HcDPPIVB did not affect the ability to kill a murine macrophage-like cell line, RAW 264.7, indicating that this gene is not required for infection of macrophages. PMID:19349421

  18. Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition

    PubMed Central

    Wang, Jehng-Kang; Lee, Ming-Shyue; Tseng, I-Chu; Chou, Feng-Pai; Chen, Ya-Wen; Fulton, Amy; Lee, Herng-Sheng; Chen, Cheng-Jueng; Johnson, Michael D.; Lin, Chen-Yong

    2009-01-01

    Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in the in vivo secretion were also observed in polarized Caco-2 intestinal epithelial cells. The cells target latent matriptase to the basolateral plasma membrane where activation, inhibition, and secretion of matriptase appear to take place. However, a proportion of matriptase-HAI-1 complexes, but not the latent matriptase, appears to undergo transcytosis to the apical plasma membrane for secretion. When epithelial cells lose their polarity, they secrete both latent and activated matriptase. Although most epithelial cells retain very low levels of matriptase-HAI-1 complex by rapidly secreting the complex, gastric chief cells may activate matriptase and store matriptase-HAI-1 complexes in the pepsinogen-secretory granules, suggesting an intracellular activation and regulated secretion in these cells. Taken together, while zymogen activation and closely coupled HAI-1-mediated inhibition are common features for matriptase regulation, the cellular location of matriptase activation and

  19. Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury.

    PubMed

    Jang, Hee-Seong; Han, Sang Jun; Kim, Jee In; Lee, Sanggyu; Lipschutz, Joshua H; Park, Kwon Moo

    2013-12-01

    Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.

  20. Active endocannabinoids are secreted on extracellular membrane vesicles.

    PubMed

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

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

    SciTech Connect

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

    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. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  3. Peroxisome Proliferator–Activated Receptor α Protects Renal Tubular Cells from Gentamicin-Induced Apoptosis via Upregulating Na+/H+ Exchanger NHE1

    PubMed Central

    Chen, Cheng-Hsien; Chen, Tso-Hsiao; Wu, Mei-Yi; Chen, Jia-Rung; Hong, Li-Yu; Zheng, Cai-Mei; Chiu, I-Jen; Lin, Yuh-Feng; Hsu, Yung-Ho

    2015-01-01

    Peroxisome proliferator–activated receptor (PPAR)-α is a transcription factor that has been reported to inhibit gentamicin-induced apoptosis in renal tubular cells. However, the antiapoptotic mechanism of PPARα is still unknown. In this study, we found that PPARα overexpression induced Na+/H+ exchanger-1 (NHE1) expression in the rat renal tubular cells NRK-52E. Beraprost, a PPARα ligand, also increased NHE1 expression in the renal tubules in normal mice, but not in PPARα knockout mice. Chromatin immunoprecipitation assays revealed that two PPARα binding elements were located in the rat NHE1 promoter region. Na+/H+ exchanger activity also increased in the PPARα-overexpressed cells. Flow cytometry showed that the PPARα-overexpressed cells were resistant to apoptosis-induced shrinkage. Cariporide, a selective NHE1 inhibitor, inhibited the antiapoptotic effect of PPARα in the gentamicin-treated cells. The interaction between NHE1 and ezrin/radixin/moesin (ERM) and between ERM and phosphatidylinositol 4,5-bisphosphate in the PPARα-overexpressed cells was more than in the control cells. ERM short interfering RNA (siRNA) transfection inhibited the PPARα-induced antiapoptotic effect. PPARα overexpression also increased the phosphoinositide 3-kinase (PI3K) expression, which is dependent on NHE1 activity. Increased PI3K further increased the phosphorylation of the prosurvival kinase Akt in the PPARα-overexpressed cells. Wortmannin, a PI3K inhibitor, inhibited PPARα-induced Akt activity and the antiapoptotic effect. We conclude that PPARα induces NHE1 expression and then recruits ERM to promote PI3K/Akt-mediated cell survival in renal tubular cells. The application of PPARα activation reduces the nephrotoxicity of gentamicin and may expand the clinical use of gentamicin. PMID:26623927

  4. Unchaining the beast; insights from structural and evolutionary studies on TGFβ secretion, sequestration, and activation

    PubMed Central

    Robertson, Ian B; Rifkin, Daniel B

    2013-01-01

    TGFβ is secreted in a latent state and must be “activated” by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFβ mobilization in vivo. Here we review the process of TGFβ secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFβ is secreted, sequestered and released. This allows the separation of more ancient TGFβ activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms. PMID:23849989

  5. In vivo stimulation of AMP-activated protein kinase enhanced tubuloglomerular feedback but reduced tubular sodium transport during high dietary NaCl intake.

    PubMed

    Huang, Dan Yang; Gao, Huanhuan; Boini, Krishna M; Osswald, Hartmut; Nürnberg, Bernd; Lang, Florian

    2010-06-01

    AMP-activated protein kinase (AMPK) is expressed in the apical membrane of cortical thick ascending limb, distal, and collecting tubules as well as macula densa cells of the kidneys. AMPK is an active modulator of epithelial Na(+) channels, Na(+)-2Cl(-)-K(+) cotransporter, and the ATP-dependent potassium channel. The present experiments explored whether AMPK participates in the regulation of tubuloglomerular feedback (TGF) and renal tubular sodium handling. To this end, renal clearance and micropuncture experiments were performed in anesthetized rats. Under normal NaCl diet, neither TGF response nor renal fluid and sodium excretion were altered by pharmacological activation of AMPK in vivo. However, under high NaCl diet, the TGF response was significantly enhanced after intravenous or intratubular application of the AMPK activator AICAR. Moreover, AICAR application significantly increased fractional delivery of fluid and sodium to the end of the proximal tubule. High dietary NaCl intake increased the renal transcript levels encoding the AMPK-alpha1 subunit, while it decreased the expression of AMPK-beta1 and AMPK-gamma2 subunits. Immunoblots revealed that high dietary NaCl intake reduced renal expression of activated AMPK by about three times compared to normal NaCl diet whereas additional AICAR application increased AMPK activity. Our results suggest that AMPK regulates tubuloglomerular balance as well as tubular transport upon change of renal work load.

  6. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

    PubMed Central

    Verhoeven, A J; Neve, B P; Jansen, H

    1999-01-01

    Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked. PMID:9854035

  7. Glucose stimulates calcium-activated chloride secretion in small intestinal cells.

    PubMed

    Yin, Liangjie; Vijaygopal, Pooja; MacGregor, Gordon G; Menon, Rejeesh; Ranganathan, Perungavur; Prabhakaran, Sreekala; Zhang, Lurong; Zhang, Mei; Binder, Henry J; Okunieff, Paul; Vidyasagar, Sadasivan

    2014-04-01

    The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

  8. Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes.

    PubMed

    Nagasawa, Takahiro; Somamoto, Tomonori; Nakao, Miki

    2015-10-01

    Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

  9. Aldosterone secretion, measurements of membrane potential and intracellular potassium activity in the isolated adrenal zone glomerulosa.

    PubMed

    Wiederholt, M; Hampel, J; Belkien, L; Oelkers, W

    1984-09-01

    Cell membrane potential and intracellular potassium activity (microelectrodes filled with ion-sensitive liquid ion exchanger) were measured in the zona glomerulosa of superfused hemi-adrenals of rats kept on different diets. Simultaneously, samples of the superfusate were collected and analyzed by radioimmunoassay for aldosterone content. Cell membrane potential and intracellular potassium activity were not influenced by high sodium, low sodium or high potassium diet. However, aldosterone secretion significantly changed. These results suggest that membrane potential and intracellular potassium activity per se may not be linked to changes in aldosterone secretion.

  10. Correlation between secretion and phospholipase D activation in differentiated HL60 cells.

    PubMed Central

    Stutchfield, J; Cockcroft, S

    1993-01-01

    Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself. PMID:8352731

  11. A Novel Serine Protease Secreted by Medicinal Maggots Enhances Plasminogen Activator-Induced Fibrinolysis

    PubMed Central

    van der Plas, Mariena J. A.; Andersen, Anders S.; Nazir, Sheresma; van Tilburg, Nico H.; Oestergaard, Peter R.; Krogfelt, Karen A.; van Dissel, Jaap T.; Hensbergen, Paul J.

    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 plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel serine protease within secretions, designated Sericase, cleaved plasminogen to several fragments. Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis by Sericase. We conclude from our in vitro studies that the novel serine protease Sericase, with the aid of a non-proteolytic cofactor, enhances plasminogen activator-induced fibrinolysis. PMID:24647546

  12. Berberine induces GLP-1 secretion through activation of bitter taste receptor pathways.

    PubMed

    Yu, Yunli; Hao, Gang; Zhang, Quanying; Hua, Wenyan; Wang, Meng; Zhou, Wenjia; Zong, Shunlin; Huang, Ming; Wen, Xiaozhou

    2015-09-15

    Our previous studies revealed that berberine-mediated GLP-1 secretion was a possible mechanism for berberine exerting good effects on hyperglycemia. This study was designed to ascertain whether berberine-induced secretion of GLP-1 was related with activation of bitter taste receptors expressed in gastrointestinal tract. Western blotting results showed that TAS2R38, a subtype of bitter taste receptor, was expressed on human enteroendocrine NCI-H716 cells. GLP-1 secretion induced by berberine from NCI-H716 cells was inhibited by incubation with anti-TAS2R38 antibody. We further performed gene silencing using siRNA to knockdown TAS2R38 from NCI-H716 cells, which showed that siRNA knockdown of the TAS2R38 reduced berberine-mediated GLP-1 secretion. We adopted inhibitors of PLC and TRPM5 known to be involved in bitter taste transduction to investigate the underlying pathways mediated in berberine-induced GLP-1 secretion. It was found that PLC inhibitor U73122 inhibited berberine-induced GLP-1 release in NCI-H716 cells, while TRPM5 blocker quinine failed to attenuate berberine-induced secretion of GLP-1. The present results demonstrated that berberine stimulated GLP-1 secretion via activation of gut-expressed bitter taste receptors in a PLC-dependent manner. Because berberine was found to be a ligand of bitter taste receptor, the results of present study may provide an explanation for some bitter taste substance obtain hypoglycemic effect.

  13. Pulsatile secretion of LH in relation to the resumption of ovarian activity post partum.

    PubMed

    Glasier, A; McNeilly, A S; Howie, P W

    1984-04-01

    Changes in the pulsatile pattern of LH secretion in relation to the resumption of ovarian activity post partum have been studied in breast feeding mothers. Basal concentrations of LH were significantly lower than normal both when ovarian activity was completely suppressed and when there was evidence of some follicular development prior to the resumption of menstruation. Once menstruation resumed basal concentrations of LH were unchanged whether ovulation occurred or not. No difference in the frequency or amplitude of pulses in LH secretion could be found at any stage post partum in either breast or bottle feeding women. FSH levels remained constant throughout the post partum period while mean concentrations of prolactin fell as ovarian activity returned to normal. However, during the period of complete suppression of ovarian activity in breast feeding women, two patterns of pulsatile secretion of LH were observed. In 76% of observation periods, basal levels of LH were lower than normal and were characterized by low frequency and low amplitude pulses of LH. In contrast, in the remaining 24% of observation periods, basal levels and pulse amplitude and frequency of LH were similar to those in the follicular phase of normal menstrual cycles in these women. Individual women showed either or both of these patterns of secretion during the period of ovarian suppression post partum. The data suggests that the failure to maintain ovarian follicular development post partum in breast feeding women may be due to: (1) a direct block of LH action at ovarian level, perhaps by the high levels of prolactin associated with lactation and/or (2) an inability of the hypothalamic-pituitary axis-as a result of the suckling stimulus and/or prolactin-to maintain pulsatile secretion of LH in the face of the negative feedback effects of the increased oestrogen secretion resulting from the initiation of follicular development.

  14. The Secrets of the Iceman. Technology Learning Activity.

    ERIC Educational Resources Information Center

    Deal, Walter F., III

    1993-01-01

    This learning activity asks students to use critical thinking skills to imagine life in the late stone age, including the tools and technology that would have existed. Presents the context, the challenge, objectives, resources, material and equipment needs, and evaluation methods. (SK)

  15. Chlamydial infection of monocytes stimulates IL-1beta secretion through activation of the NLRP3 inflammasome.

    PubMed

    Abdul-Sater, Ali A; Saïd-Sadier, Najwane; Padilla, Eduardo V; Ojcius, David M

    2010-08-01

    Chlamydia trachomatis infections represent the leading cause of bacterial sexually-transmitted disease in the United States and can cause serious tissue damage leading to infertility and ectopic pregnancies in women. Inflammation and hence the innate immune response to chlamydial infection contributes significantly to tissue damage, particularly by secreting proinflammatory cytokines such as interleukin (IL)-1beta from monocytes, macrophages and dendritic cells. Here we demonstrate that C. trachomatis or Chlamydia muridarum infection of a monocytic cell line leads to caspase-1 activation and IL-1beta secretion through a process requiring the NLRP3 inflammasome. Thus, secretion of IL-1beta decreased significantly when cells were depleted of NLRP3 or treated with the anti-inflammatory inhibitors parthenolide or Bay 11-7082, which inhibit inflammasomes and the transcription factor NF-kappaB. As for other infections causing NRLP3 inflammasome assembly, caspase-1 activation in monocytes is triggered by potassium efflux and reactive oxygen species production. However, anti-oxidants inhibited IL-1beta secretion only partially. Atypically for a bacterial infection, caspase-1 activation during chlamydial infection also involves partially the spleen tyrosine kinase (Syk), which is usually associated with a pathogen recognition receptor for fungal pathogens. Secretion of IL-1beta during infection by many bacteria requires both microbial products from the pathogen and an exogenous danger signal, but chlamydial infection provides both the pathogen-associated molecular patterns and danger signals necessary for IL-1beta synthesis and its secretion from human monocytes. Use of inhibitors that target the inflammasome in animals should therefore dampen inflammation during chlamydial infection.

  16. Detection of antidiabetic activity by crude paratoid gland secretions from common Indian toad (bufomelano stictus)

    PubMed Central

    Neerati, Prasad

    2015-01-01

    Background: Amphibians have provided a remarkable array of biological active compounds, which are secreted from socalled granular skin glands which serve to protect the amphibians from predators due to its noxious effects on buccal tissue and at least in the case of some peptides, to protect from bacterial (or) protozoan infections. Given the respiratory and antimicrobial functions of amphibian skin, it is likely that some of the novel molecules found in amphibian granular gland secretions might be of use in the treatment of skin and respiratory infections. Secretions from common Indian toad (Bufo melanostictus) a member of Bufonidae family has the history of medicinal use however the anti-diabetic activity is not reported. The present study is aimed to determine whether paratoid gland extract have any influence on the diabetes and the pharmacokinetics and pharmacodynamics of glimepiride (GLM) in normal and diabetic rats. Materials and Methods: An aqueous and methanolic extracts of paratoid glandular secretions were prepared, air dried and used to determine the antidiabetic activity in rats. The blood sampling was done at preset time intervals between 0, 0.5, 1, 2, 4, 6, 8 and 12 h, using heparinized capillaries. The blood glucose levels are estimated by glucose oxidase-peroxidase method, and reversed-phase high-performance liquid chromatography is used to determine the pharmacokinetic parameters of GLM using glibenclamide as an internal standard. Results: Both the aqueous and methanolic extracts produced better glycemic control in diabetic rats, and methanolic extract is better than the aqueous extract. Serum concentrations of GLM increased at 2nd h, and the percentage glucose reduction is maximal at the 4th h with both aqueous and methanolic extracts of paratoid secretions of common Indian toad. Conclusions: Paratoid gland secretions of the common Indian toad is antidiabetic, in addition it has beneficial effects in combination with GLM. Further, it requires the

  17. Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

    DOE PAGES

    Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; Connolly, Lynn; Bendebury, Anastasia; Finer-Moore, Janet; Holton, James; Cheng, Yifan; Stroud, Robert M.; Cox, Jeffery S.

    2015-04-09

    We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less

  18. Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

    SciTech Connect

    Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; Connolly, Lynn; Bendebury, Anastasia; Finer-Moore, Janet; Holton, James; Cheng, Yifan; Stroud, Robert M.; Cox, Jeffery S.

    2015-04-09

    We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

  19. Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

    PubMed Central

    2013-01-01

    Background The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine). Results Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity. Conclusions The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane. PMID:24499077

  20. Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway

    PubMed Central

    Liang, Lihua; MacDonald, Kelvin; Schwiebert, Erik M.; Zeitlin, Pamela L.; Guggino, William B.

    2009-01-01

    Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl− channel. Its dysfunction limits Cl− secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl− channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca2+ activate the epithelial CaCCs, which provides an alternative Cl− secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl− secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl− secretion in CF via a pathway independent of CFTR. PMID:18987251

  1. Inhibition of Colony-spreading Activity of Staphylococcus aureus by Secretion of δ-Hemolysin*

    PubMed Central

    Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2012-01-01

    Staphylococcus aureus spreads on the surface of soft agar, a phenomenon we termed “colony spreading.” Here, we found that S. aureus culture supernatant inhibited colony spreading. We purified δ-hemolysin (Hld, δ-toxin), a major protein secreted from S. aureus, as a compound that inhibits colony spreading. The culture supernatants of hld-disrupted mutants had 30-fold lower colony-spreading inhibitory activity than those of the parent strain. Furthermore, hld-disrupted mutants had higher colony-spreading ability than the parent strain. These results suggest that S. aureus negatively regulates colony spreading by secreting δ-hemolysin. PMID:22411996

  2. Broadening the Spectrum of Actin-Based Protrusive Activity Mediated by Arp2/3 Complex-Facilitated Polymerization: Motility of Cytoplasmic Ridges and Tubular Projections

    PubMed Central

    Henson, John H.; Gianakas, Anastasia D.; Henson, Lauren H.; Lakin, Christina L.; Voss, Meagen K.; Bewersdorf, Joerg; Oldenbourg, Rudolf; Morris, Robert L.

    2014-01-01

    Arp2/3 complex-facilitated actin polymerization plays an essential role in a variety of cellular functions including motility, adherence, endocytosis and trafficking. In the present study we employ the sea urchin coelomocyte experimental model system to test the hypotheses that Arp2/3 complex-nucleated actin assembly mediates the motility of two unusual cellular protrusions; the cytoplasmic ridges present during coelomocyte spreading, and inducible, tubular-shaped, and neurite-like projections. Our investigations couple pharmacological manipulation employing inhibitors of actin polymerization and the Arp2/3 complex with a wide array of imaging methods including digitally enhanced phase contrast, DIC and polarization light microscopy of live cells; conventional, confocal and super-resolution light microscopy of fluorescently labeled cells; and scanning and transmission electron microscopy. Taken together, the results of this study indicate that Arp2/3 complex-facilitated actin polymerization underlies the motility of coelomocyte cytoplasmic ridges and tubular projections, that these processes are related to each other, and that they have been preliminarily identified in other cell types. The results also highlight the broad spectrum of actin-based protrusive activities dependent on the Arp2/3 complex and provide additional insights into the pervasive nature of this ubiquitous actin nucleator. Furthermore we provide the first evidence of a possible mechanistic difference between the impacts of the small molecule drugs BDM and CK666 on the Arp2/3 complex. PMID:25111797

  3. Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

    PubMed

    Loera-Arias, María J; Villatoro-Hernández, Julio; Parga-Castillo, Miguel A; Salcido-Montenegro, Alejandro; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Montes-de-Oca-Luna, Roberto; Saucedo-Cárdenas, Odila

    2014-12-01

    Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications.

  4. Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

    PubMed

    Loera-Arias, María J; Villatoro-Hernández, Julio; Parga-Castillo, Miguel A; Salcido-Montenegro, Alejandro; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Montes-de-Oca-Luna, Roberto; Saucedo-Cárdenas, Odila

    2014-12-01

    Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications. PMID:25214209

  5. Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation

    PubMed Central

    Silverman, Julie M.; Austin, Laura S.; Hsu, FoSheng; Hicks, Kevin G.; Hood, Rachel D.; Mougous, Joseph D.

    2013-01-01

    Summary Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the hemolysin co-regulated secretion island I (HSI-I)-encoded type VI secretion system (H1-T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact-dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S-associated protein, TagF, as a posttranslational repressor of the H1-T6SS. Strains activated by TagF derepression or stimulation of a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that the pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1-T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact-promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors. PMID:22017253

  6. Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats.

    PubMed

    Veloso, Roberto V; Latorraca, Márcia Q; Arantes, Vanessa C; Reis, Marise A B; Ferreira, Fabiano; Boschero, Antonio C; Carneiro, Everardo M

    2008-11-01

    Maternal malnutrition leads to permanent alterations in insulin secretion of offspring and the soybean diet contributes to improve insulin release. At least a soy component, genistein, seems to increase the insulin secretion by activating the cAMP/PKA and PLC/PKC pathways. Here, we investigated the effect of the soybean diet on the expression of PKAalpha and PKCalpha, and insulin secretion in response to glucose and activators of adenylate cyclase and PKC in adult pancreatic rat islets. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet until 90 days of life. The soybean diet improved the insulin response to a physiological concentration of glucose in control islets, but only in the presence of supra-physiological concentrations of glucose in islets from CR and SR groups. PMA also improved the insulin response in islets of SC and SR groups. The expression of PKCalpha was similar in all groups. Forskolin increased the insulin secretion; however, the magnitude of the increment was lower in islets from CR and SR groups than in control animals and in those from rats maintained with soybean diet than in rats fed with casein diet. The PKAalpha expression was similar between SR and CR groups and lower in SC than in CC islets. Thus, soybean diet improved the secretory pattern of beta cells, at least in part, by activating the cAMP/PKA-signaling cascade. PMID:18430554

  7. Micro-Tubular Fuel Cells

    NASA Technical Reports Server (NTRS)

    Kimble, Michael C.; Anderson, Everett B.; Jayne, Karen D.; Woodman, Alan S.

    2004-01-01

    Micro-tubular fuel cells that would operate at power levels on the order of hundreds of watts or less are under development as alternatives to batteries in numerous products - portable power tools, cellular telephones, laptop computers, portable television receivers, and small robotic vehicles, to name a few examples. Micro-tubular fuel cells exploit advances in the art of proton-exchange-membrane fuel cells. The main advantage of the micro-tubular fuel cells over the plate-and-frame fuel cells would be higher power densities: Whereas the mass and volume power densities of low-pressure hydrogen-and-oxygen-fuel plate-and-frame fuel cells designed to operate in the targeted power range are typically less than 0.1 W/g and 0.1 kW/L, micro-tubular fuel cells are expected to reach power densities much greater than 1 W/g and 1 kW/L. Because of their higher power densities, micro-tubular fuel cells would be better for powering portable equipment, and would be better suited to applications in which there are requirements for modularity to simplify maintenance or to facilitate scaling to higher power levels. The development of PEMFCs has conventionally focused on producing large stacks of cells that operate at typical power levels >5 kW. The usual approach taken to developing lower-power PEMFCs for applications like those listed above has been to simply shrink the basic plate-and-frame configuration to smaller dimensions. A conventional plate-and-frame fuel cell contains a membrane/electrode assembly in the form of a flat membrane with electrodes of the same active area bonded to both faces. In order to provide reactants to both electrodes, bipolar plates that contain flow passages are placed on both electrodes. The mass and volume overhead of the bipolar plates amounts to about 75 percent of the total mass and volume of a fuel-cell stack. Removing these bipolar plates in the micro-tubular fuel cell significantly increases the power density.

  8. Association of bactericidal activity of genital tract secretions with Escherichia coli colonization in pregnancy

    PubMed Central

    GHARTEY, Jeny P.; CARPENTER, Colleen; GIALANELLA, Philip; RISING, Charlotte; McANDREW, Thomas C.; MHATRE, Mohak; TUGETMAN, Jessica; EINSTEIN, Mark H.; CHAZOTTE, Cynthia; HEROLD, Betsy C.

    2012-01-01

    Objective Genital tract secretions exhibit bactericidal activity against Escherichia coli. We hypothesized that this defense may be modulated during pregnancy. Study Design Secretions were collected by vaginal swab from 70 pregnant women (35–37 weeks gestation) and 35 non-pregnant controls. Escherichia coli were mixed with swab eluants or control buffer and colonies enumerated to measure bactericidal activity. Cytokines, chemokines and antimicrobial peptides were quantified by Luminex or ELISA. Results Pregnant women had significantly greater bactericidal activity, higher concentrations of pro-inflammatory cytokines and lower levels of beta defensins compared to controls. Seven (10%) pregnant and 8 (23%) non-pregnant women were vaginally colonized with Escherichia coli; colonization was inversely associat ed with bactericidal activity. Conclusion The soluble mucosal immune environment is altered in pregnancy. We speculate that the observed changes may protect against colonization and ascending infection and could provide a biomarker to identify pregnant women at risk for infectious complications including preterm birth. PMID:22867687

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  11. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells.

  12. Constitutively active heat shock factor 1 enhances glucose-driven insulin secretion.

    PubMed

    Uchiyama, Tsuyoshi; Tomono, Shoichi; Utsugi, Toshihiro; Ohyama, Yoshio; Nakamura, Tetsuya; Tomura, Hideaki; Kawazu, Shoji; Okajima, Fumikazu; Kurabayashi, Masahiko

    2011-06-01

    Weak pancreatic β-cell function is a cause of type 2 diabetes mellitus. Glucokinase regulates insulin secretion via phosphorylation of glucose. The present study focused on a system for the self-protection of pancreatic cell by expressing heat shock factor (HSF) and heat shock protein (HSP) to improve insulin secretion without inducing hypoglycemia. We previously generated a constitutively active form of human HSF1 (CA-hHSF1). An adenovirus expressing CA-hHSF1 using the cytomegalovirus promoter was generated to infect mouse insulinoma cells (MIN6 cells). An adenovirus expressing CA-hHSF1 using a human insulin promoter (Ins-CA-hHSF1) was also generated to infect rats. We investigated whether CA-hHSF1 induces insulin secretion in MIN6 cells and whether Ins-CA-hHSF1 can improve blood glucose and serum insulin levels in healthy Wister rats and type 2 diabetes mellitus model rats. CA-hHSF1 expression increased insulin secretion 1.27-fold compared with the overexpression of wild-type hHSF1 in MIN6 cells via induction of HSP90 expression and subsequent activation of glucokinase. This mechanism is associated with activation of both glucokinase and neuronal nitric oxide synthase. Ins-CA-hHSF1 improved blood glucose levels in neonatal streptozotocin-induced diabetic rats. Furthermore, Ins-CA-hHSF1 reduced oral glucose tolerance testing results in healthy Wister rats because of an insulin spike at 15 minutes; however, it did not induce hypoglycemia. CA-hHSF1 induced insulin secretion both in vitro and in vivo. These findings suggest that gene therapy with Ins-CA-hHSF1 will be able to be used to treat patients with type 2 diabetes mellitus and impaired glucose tolerance without causing hypoglycemia at fasting. PMID:20817212

  13. The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function.

    PubMed

    Faria, Diana; Rock, Jason R; Romao, Ana M; Schweda, Frank; Bandulik, Sascha; Witzgall, Ralph; Schlatter, Eberhard; Heitzmann, Dirk; Pavenstädt, Hermann; Herrmann, Edwin; Kunzelmann, Karl; Schreiber, Rainer

    2014-06-01

    The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

  14. BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP‐12

    PubMed Central

    Yakhontova, K.; Lu, M.; Manzetti, J.

    2015-01-01

    BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV‐specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)‐ and calcineurin‐inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR–SP6‐kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC‐1 kinase inhibitor torin‐1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP‐12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP‐12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation. PMID:26639422

  15. Selective optogenetic activation of arcuate kisspeptin neurons generates pulsatile luteinizing hormone secretion.

    PubMed

    Han, Su Young; McLennan, Timothy; Czieselsky, Katja; Herbison, Allan E

    2015-10-20

    Normal reproductive functioning in mammals depends upon gonadotropin-releasing hormone (GnRH) neurons generating a pulsatile pattern of gonadotropin secretion. The neural mechanism underlying the episodic release of GnRH is not known, although recent studies have suggested that the kisspeptin neurons located in the arcuate nucleus (ARN) may be involved. In the present experiments we expressed channelrhodopsin (ChR2) in the ARN kisspeptin population to test directly whether synchronous activation of these neurons would generate pulsatile luteinizing hormone (LH) secretion in vivo. Characterization studies showed that this strategy targeted ChR2 to 70% of all ARN kisspeptin neurons and that, in vitro, these neurons were activated by 473-nm blue light with high fidelity up to 30 Hz. In vivo, the optogenetic activation of ARN kisspeptin neurons at 10 and 20 Hz evoked high amplitude, pulse-like increments in LH secretion in anesthetized male mice. Stimulation at 10 Hz for 2 min was sufficient to generate repetitive LH pulses. In diestrous female mice, only 20-Hz activation generated significant increments in LH secretion. In ovariectomized mice, 5-, 10-, and 20-Hz activation of ARN kisspeptin neurons were all found to evoke LH pulses. Part of the sex difference, but not the gonadal steroid dependence, resulted from differential pituitary sensitivity to GnRH. Experiments in kisspeptin receptor-null mice, showed that kisspeptin was the critical neuropeptide underlying the ability of ARN kisspeptin neurons to generate LH pulses. Together these data demonstrate that synchronized activation of the ARN kisspeptin neuronal population generates pulses of LH. PMID:26443858

  16. Selective optogenetic activation of arcuate kisspeptin neurons generates pulsatile luteinizing hormone secretion.

    PubMed

    Han, Su Young; McLennan, Timothy; Czieselsky, Katja; Herbison, Allan E

    2015-10-20

    Normal reproductive functioning in mammals depends upon gonadotropin-releasing hormone (GnRH) neurons generating a pulsatile pattern of gonadotropin secretion. The neural mechanism underlying the episodic release of GnRH is not known, although recent studies have suggested that the kisspeptin neurons located in the arcuate nucleus (ARN) may be involved. In the present experiments we expressed channelrhodopsin (ChR2) in the ARN kisspeptin population to test directly whether synchronous activation of these neurons would generate pulsatile luteinizing hormone (LH) secretion in vivo. Characterization studies showed that this strategy targeted ChR2 to 70% of all ARN kisspeptin neurons and that, in vitro, these neurons were activated by 473-nm blue light with high fidelity up to 30 Hz. In vivo, the optogenetic activation of ARN kisspeptin neurons at 10 and 20 Hz evoked high amplitude, pulse-like increments in LH secretion in anesthetized male mice. Stimulation at 10 Hz for 2 min was sufficient to generate repetitive LH pulses. In diestrous female mice, only 20-Hz activation generated significant increments in LH secretion. In ovariectomized mice, 5-, 10-, and 20-Hz activation of ARN kisspeptin neurons were all found to evoke LH pulses. Part of the sex difference, but not the gonadal steroid dependence, resulted from differential pituitary sensitivity to GnRH. Experiments in kisspeptin receptor-null mice, showed that kisspeptin was the critical neuropeptide underlying the ability of ARN kisspeptin neurons to generate LH pulses. Together these data demonstrate that synchronized activation of the ARN kisspeptin neuronal population generates pulses of LH.

  17. Biological activities of skin and parotoid gland secretions of bufonid toads (Bufo bufo, Bufo verrucosissimus and Bufotes variabilis) from Turkey.

    PubMed

    Nalbantsoy, Ayse; Karış, Mert; Yalcin, Husniye Tansel; Göçmen, Bayram

    2016-05-01

    Toad glandular secretions and skin extractions contain numerous natural agents which may provide unique resources for novel drug development. Especially the skin-parotoid gland secretions of toads from genus Bufo contain as many as 86 different types of active compounds, each with the potential of becoming a potent drug. In the present study, crude skin-parotoid gland secretions from Bufo bufo, Bufo verrucosissimus and Bufotes variabilis from Turkey were screened against various cancer cells together with normal cells using MTT assay. Furthermore, the antimicrobial properties of skin secretions were tested on selected bacterial and fungal species for assessing the possible medical applications. Antimicrobial activity of skin secretions was studied by determining minimal inhibitory concentration (MIC) in broth dilution method. Hemolytic activity of each skin-secretion was also estimated for evaluating pharmaceutical potential. Both skin-parotoid gland secretions showed high cytotoxic effect on all cancerous and non-cancerous cell lines with IC50 values varying between <0.1μg/ml and 6.02μg/ml. MIC results of antimicrobial activity tests were found to be between 3.9μg/ml and 250μg/ml. No hemolytic activities on rabbit red blood cells at concentrations between 0.5μg/ml and 50μg/ml were observed. In conclusion, skin-parotoid secretions of bufonid toads might be remarkable candidates for anti-cancer and antimicrobial agents without hemolytic activities. PMID:27133069

  18. Review of the micro-tubular solid oxide fuel cell. Part I. Stack design issues and research activities

    NASA Astrophysics Data System (ADS)

    Lawlor, V.; Griesser, S.; Buchinger, G.; Olabi, A. G.; Cordiner, S.; Meissner, D.

    Fuel cells are devices that convert chemical energy in hydrogen enriched fuels into electricity electrochemically. Micro-tubular solid oxide fuel cells (MT-SOFCs), the type pioneered by K. Kendall in the early 1990s, are a variety of SOFCs that are on the scale of millimetres compared to their much larger SOFC relatives that are typically on the scale of tens of centimetres. The main advantage of the MT-SOFC, over its larger predecessor, is that it is smaller in size and is more suitable for rapid start up. This may allow the SOFC to be used in devices such as auxiliary power units, automotive power supplies, mobile electricity generators and battery re-chargers. The following paper is Part I of a two part series. Part I will introduce the reader to the MT-SOFC stack and its applications, indicating who is researching what in this field and also specifically investigate the design issues related to multi-cell reactor systems called stacks. Part II will review in detail the combinations of materials and methods used to produce the electrodes and electrolytes of MT-SOFC's. Also the role of modelling and validation techniques used in the design and improvement of the electrodes and electrolytes will be investigated. A broad range of scientific and engineering disciplines are involved in a stack design. Scientific and engineering content has been discussed in the areas of thermal-self-sustainability and efficiency, sealing technologies, manifold design, electrical connections and cell performance optimisation.

  19. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.

    PubMed

    Rigg, Rachel A; Healy, Laura D; Nowak, Marie S; Mallet, Jérémy; Thierheimer, Marisa L D; Pang, Jiaqing; McCarty, Owen J T; Aslan, Joseph E

    2016-04-01

    Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.

  20. Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions.

    PubMed

    Raqib, Rubhana; Rahman, Jubayer; Kamaluddin, A K M; Kamal, S M Mostafa; Banu, Fauzia A; Ahmed, Shakeel; Rahim, Zeaur; Bardhan, Pradip K; Andersson, Jan; Sack, David A

    2003-08-01

    In the present study, we investigated the tuberculosis (TB) diagnostic performance of an assay on the basis of detection of TB-specific antibodies from peripheral blood mononuclear cells (PBMCs), to determine whether antibodies in lymphocyte secretions obtained from PBMCs would better reflect active disease than antibodies in serum. PBMCs from patients with and without TB cultured in various concentrations for different times were assessed. Immunoglobulin G (IgG) specific for antigen (bacille Calmette-Guérin [BCG] vaccine and purified protein derivative [PPD]) was measured in lymphocyte secretions. Patients with active TB had higher BCG- or PPD-specific IgG antibody responses than patients without TB or healthy subjects (P=.001). This method can be used as a quick diagnostic aid to facilitate rapid detection of TB cases.

  1. Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency.

    PubMed

    Higa, Ataru; Miyamoto, Erika; ur Rahman, Laiq; Kitamura, Yoshie

    2008-04-01

    Hyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron-deficient condition secreted riboflavin from the root tips into the culture medium and the productivity depended on the number and size of root tips among the clones. A decline of pH was observed before riboflavin production and root development. By studying effects of proton-pump inhibitors, medium acidification with external organic acid, and riboflavin addition upon pH change and riboflavin productivity, we indicate that riboflavin efflux is not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs as a response to an internal requirement in H. albus hairy roots under iron deficiency. PMID:18367404

  2. ANTIMICROBIAL ACTIVITY FOR EXCRETION AND SECRETION OF THE GREENBOTTLE FLY LARVAE LUCILIA SERICATA (MEIGEN) (DIPTERA: CALLIPHORIDAE).

    PubMed

    Hassan, Mostafa I; Amer, Mounir S; Hammad, Kotb M; Zidan, Mahmoud M

    2016-04-01

    Sterile larval excretion/secretion (ES) exhibited antibacterial activity against some species of bacteria. They were shown to inhibit the growth of Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae and Fungi Geotricum candidum and Aspergillus fumigatus thus exhibited limited inhibitory effect towards Gram-positive bacteria Streptococcus pyogenes and Staphylococcus epidermidis and Gram-negative Proteous vulgaris and Fungi Syncephalastrum racemosum, Candida albicans, that effect was slowed down when challenged with secretion on a solid media but no zone of complete inhibition was detectqd. Growth inhibiting activity was determined in liquid growth media using the Gram-positive, Gram-negative bacterial and fungal strains as indicator organisms. PMID:27363054

  3. [Lysozyme activity of the salivary gland secretion of the medicinal leech H. verbana, H. medicinalis and H. orientalis].

    PubMed

    Baskova, I P; Kharitonova, O V; Zavalova, L L

    2011-01-01

    Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-beta-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).

  4. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

    PubMed

    Välimäki, Elina; Miettinen, Juho J; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A

    2013-03-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  5. Lipoxin A4 activates ALX/FPR2 Receptor to Regulate Conjunctival Goblet Cell Secretion

    PubMed Central

    Hodges, Robin R.; Li, Dayu; Shatos, Marie A.; Bair, Jeffrey A.; Lippestad, Marit; Serhan, Charles N.; Dartt, Darlene A.

    2016-01-01

    Conjunctival goblet cells play a major role in maintaining the mucous layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis.. Resolution of inflammation is mediated by pro-resolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i) and identify signaling pathways activated by LXA4. ALX/FPR was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and ERK 1/2 activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared to its initial response. LXA4 activated phospholipase C, -D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases. PMID:27072607

  6. Secreted proteases. Regulation of their activity and their possible role in metastasis.

    PubMed

    Goldberg, G I; Frisch, S M; He, C; Wilhelm, S M; Reich, R; Collier, I E

    1990-01-01

    Extracellular matrix metalloproteases are secreted by the resident cells of the tissue in a proenzyme form, and their extracellular activity is regulated at the level of gene expression, proenzyme activation, and interaction with inhibitors. To understand the molecular mechanisms that control the activity of ECM metalloproteases and their effect on the cellular phenotype, we have established cell lines in which the transcription of the protease genes is repressed. We also have undertaken a detailed study of the pathway of extracellular activation of interstitial procollagenase. Stable transfection of three human tumor cell lines--H-ras-transformed bronchial epithelial cells TBE-1, fibrosarcoma cells HT1080, and melanoma cells A2058--with the adenovirus E1A gene dramatically repressed the expression of the secreted proteases, type IV and interstitial collagenases, and urokinase-type plasminogen activator. Concomitantly, E1A-expressing cells showed reduced metastatic activity in vivo and reduced ability to traverse a reconstituted basement membrane in vitro. Monospecific anti-type IV collagenase antibody inhibited the invasive activity of parental tumor cell lines in the in vitro system, suggesting a possible causal relationship between the effect of E1A on the expression of secreted proteases and the reduced metastatic potential of the E1A-expressing transformants. We have also studied the mechanism of regulation of metalloprotease activity at the level of extracellular activation by investigating the cascade of proteolytic events that results in the activation of interstitial procollagenase. Cocultivation of the major cellular components of skin, dermal fibroblasts, and epidermal keratinocytes induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a uPA-plasmin-dependent pathway in which plasmin catalyzes the first step in activation of both collagenase and stromelysin by amino

  7. Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator.

    PubMed

    Gualandris, A; Jones, T E; Strickland, S; Tsirka, S E

    1996-04-01

    Tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to active plasmin, is produced in the rat and mouse hippocampus and participates in neuronal plasticity. To help define the role of tPA in the nervous system, we have analyzed the regulation of its expression in the neuronal cell line PC12. In control cultures, tPA activity is exclusively cell-associated, and no activity is measurable in the culture medium. When the cells are treated with depolarizing agents, such as KCI, tPA activity becomes detectable in the medium. The increased secreted tPA activity is not accompanied by an increase in tPA mRNA levels, and it is not blocked by protein synthesis inhibitors. In contrast, tPA release is abolished by Ca2+ channel blockers, suggesting that chemically induced membrane depolarization stimulates the secretion of preformed enzyme. Moreover, KCI has a similar effect in vivo when administered to the murine brain via an osmotic pump: tPA activity increases along the CA2-CA3 regions and dentate gyrus of the hippocampal formation. These results demonstrate a neuronal activity-dependent secretory mechanism that can rapidly increase the amount of tPA in neuronal tissue.

  8. Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies.

    PubMed

    Savkovic, S D; Koutsouris, A; Wu, G; Hecht, G

    2000-09-01

    Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.

  9. Hormonal control in a state of decreased activation: potentiation of arginine vasopressin secretion.

    PubMed

    O'Halloran, J P; Jevning, R; Wilson, A F; Skowsky, R; Walsh, R N; Alexander, C

    1985-10-01

    Behaviorally induced stress is associated with increased arginine vasopressin (AVP) secretion. In this report we describe a phasic conditioned response of AVP secretion yielding 2.6-7.1 times normal plasma concentration of this hormone in association with a physiological state of decreased activation, that associated with the mental technique of "transcendental meditation" (TM) in long-term practitioners (6-8 years of regular elicitation). Such a very large phasic response of AVP was previously unknown in the normal physiology of AVP. This elevation was not accompanied by elevation of plasma osmolality. Unstylized ordinary eyes closed rest in a separate group of subjects studied in the same manner was associated with normal plasma AVP concentration. Galvanic skin resistance (GSR) increased during both TM and rest with significantly larger increase associated with TM. Other measures of activation, including muscle metabolism, and the Spielberger Anxiety Inventory indicated marked relaxation in association with TM. In previous research it has been shown that blood pressure does not change acutely during this behavior. These observations indicate that neither stress nor operation of other usual homeostatic control mechanisms are responsible for elevated for AVP in the meditators. It is speculated that the apparently unique mechanism of TM-induced AVP secretion may be more specifically related to the behavioral effects of meditation.

  10. A mathematical method for extracting cell secretion rate from affinity biosensors continuously monitoring cell activity.

    PubMed

    Gao, Yandong; Zhou, Qing; Matharu, Zimple; Liu, Ying; Kwa, Timothy; Revzin, Alexander

    2014-03-01

    Our laboratory has previously developed miniature aptasensors that may be integrated at the site of a small group of cells for continuous detection of cell secreted molecules such as inflammatory cytokine interferon gamma (IFN-γ). In a system such as this, the signal measured at the sensor surfaces is a complex function of transport, reaction, as well as of cellular activity. Herein, we report on the development of a mathematical framework for extracting cell production rates from binding curves generated with affinity biosensors. This framework consisted of a diffusion-reaction model coupled to a root finding algorithm for determining cell production rates values causing convergence of a predetermined criterion. To experimentally validate model predictions, we deployed a microfluidic device with an integrated biosensor for measuring the IFN-γ release from CD4 T cells. We found close agreement between secretion rate observed theoretically and those observed experimentally. After taking into account the differences in sensor geometry and reaction kinetics, the method for cell secretion rate determination described in this paper may be broadly applied to any biosensor continuously measuring cellular activity.

  11. Tubular shear stress and phenotype of renal proximal tubular cells.

    PubMed

    Essig, Marie; Friedlander, Gérard

    2003-06-01

    Phenotypic alterations resulting from flow-induced mechanical strains is a growing field of research in many cell types such as vascular endothelial and smooth muscle cells, chondrocytes, and osteocytes. Because renal mass reduction is followed by a dramatic increase in GFR in the remaining nephron, modulation of tubular cell phenotype by flow-induced mechanical strains could be one of the events initiating the deleterious pathways that lead to the destruction of renal parenchyma after renal mass reduction. This study demonstrates that increased flow induced, in vitro and in vivo, a reinforcement of the apical domain of actin cytoskeleton and an inhibition of plasminogen activator expression. These effects of flow on plasminogen activator expression were prevented by blocking the reorganization of actin cytoskeleton and were associated with an increase in a shear-stress responsive element binding activity. These results confirm that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases. PMID:12761236

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

    PubMed

    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. PMID:27000261

  13. Activation of human pro-urokinase by unrelated proteases secreted by Pseudomonas aeruginosa.

    PubMed

    Beaufort, Nathalie; Seweryn, Paulina; de Bentzmann, Sophie; Tang, Aihua; Kellermann, Josef; Grebenchtchikov, Nicolai; Schmitt, Manfred; Sommerhoff, Christian P; Pidard, Dominique; Magdolen, Viktor

    2010-06-15

    Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted bacterial proteases might contribute to the activation of this major extracellular proteolytic system, thereby participating in bacterial dissemination. We report that LasB, a thermolysin-like metalloprotease secreted by Ps. aeruginosa, converts the human uPA zymogen into its active form (kcat=4.9 s-1, Km=8.9 microM). Accordingly, whereas the extracellular secretome from the LasB-expressing pseudomonal strain PAO1 efficiently activates pro-uPA, the secretome from the isogenic LasB-deficient strain PDO240 is markedly less potent in pro-uPA activation. Still, both secretomes induce some metalloprotease-independent activation of the human zymogen. The latter involves a serine protease, which we identified via both recombinant protein expression in Escherichia coli and purification from pseudomonal cultures as protease IV (PIV; kcat=0.73 s-1, Km=6.2 microM). In contrast, neither secretomes nor the pure proteases activate Plg. Along with this, LasB converts Plg into mini-Plg and angiostatin, whereas, as reported previously, it processes the uPA receptor, inactivates the plasminogen activator inhibitor 1, and activates pro-matrix metalloproteinase 2. PIV does not target these factors at all. To conclude, LasB and PIV, although belonging to different protease families and displaying quite different substrate specificities, both activate the urokinase-type precursor of the Plg activation cascade. Direct pro-uPA activation, as also reported for other bacterial proteases, might be a frequent phenomenon that contributes to bacterial virulence.

  14. Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma.

    PubMed Central

    Brieva, J A; Villar, L M; Leoro, G; Alvarez-Cermeño, J C; Roldán, E; Gonzalez-Porqué, P

    1990-01-01

    HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins. PMID:2122936

  15. Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA

    PubMed Central

    Cunha, Larissa D.; Ribeiro, Juliana M.; Fernandes, Talita D.; Massis, Liliana M.; Khoo, Chen Ai; Moffatt, Jennifer H.; Newton, Hayley J.; Roy, Craig R.; Zamboni, Dario S.

    2015-01-01

    Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA- mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome. PMID:26687278

  16. Engineering of pseudoislets: effect on insulin secretion activity by cell number, cell population, and microchannel networks.

    PubMed

    Kojima, N; Takeuchi, S; Sakai, Y

    2014-05-01

    Engineered pseudoislets reconstituted from a suspension of pancreatic α and β cells have the potential to relieve the shortage of donor islets for transplantation in the treatment of type 1 diabetes. However, the methods to fabricate pseudoislets are not well developed. In this study, we attempted to generate pseudoislets, which show a higher potential for glucose-induced insulin secretion, by altering total cell number, adjusting the cell ratio of pancreatic α and β cells, and fabricating microchannel networks with the use of alginate hydrogel beads. To effectively aggregate α and β cells and hydrogel beads, we used a previously established rapid aggregation method. When pseudoislets were reconstituted with 8,000 cells in a 1:8 α/β-cell ratio, we observed that the glucose-induced insulin secretion was enhanced by 3.1 times compared with the pseudoislets formed with β cells only. In addition, embedding of microchannel networks increased the insulin secretion rate by 4.4 times compared with the pseudoislets without the microstructures. These findings demonstrated that active modification was effective in reconstituting higher functional pseudoislets, which may be useful for islet transplantation.

  17. Engineering of pseudoislets: effect on insulin secretion activity by cell number, cell population, and microchannel networks.

    PubMed

    Kojima, N; Takeuchi, S; Sakai, Y

    2014-05-01

    Engineered pseudoislets reconstituted from a suspension of pancreatic α and β cells have the potential to relieve the shortage of donor islets for transplantation in the treatment of type 1 diabetes. However, the methods to fabricate pseudoislets are not well developed. In this study, we attempted to generate pseudoislets, which show a higher potential for glucose-induced insulin secretion, by altering total cell number, adjusting the cell ratio of pancreatic α and β cells, and fabricating microchannel networks with the use of alginate hydrogel beads. To effectively aggregate α and β cells and hydrogel beads, we used a previously established rapid aggregation method. When pseudoislets were reconstituted with 8,000 cells in a 1:8 α/β-cell ratio, we observed that the glucose-induced insulin secretion was enhanced by 3.1 times compared with the pseudoislets formed with β cells only. In addition, embedding of microchannel networks increased the insulin secretion rate by 4.4 times compared with the pseudoislets without the microstructures. These findings demonstrated that active modification was effective in reconstituting higher functional pseudoislets, which may be useful for islet transplantation. PMID:24815151

  18. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    PubMed

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  19. IL13 activates autophagy to regulate secretion in airway epithelial cells

    PubMed Central

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    ABSTRACT Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent. PMID:26062017

  20. Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

    PubMed

    Wang, Han Chin; Lin, Chun-Chieh; Cheung, Rocky; Zhang-Hooks, YingXin; Agarwal, Amit; Ellis-Davies, Graham; Rock, Jason; Bergles, Dwight E

    2015-12-01

    Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells. PMID:26627734

  1. Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

    PubMed Central

    Kammer, G M; Sapolsky, A I; Malemud, C J

    1985-01-01

    Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus. PMID:3897284

  2. Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.

    PubMed

    Porubsky, Stefan; Federico, Giuseppina; Müthing, Johannes; Jennemann, Richard; Gretz, Norbert; Büttner, Stefan; Obermüller, Nicholas; Jung, Oliver; Hauser, Ingeborg A; Gröne, Elisabeth; Geiger, Helmut; Gröne, Hermann-Josef; Betz, Christoph

    2014-09-01

    The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate

  3. Consequences of individual N-glycan deletions and of proteasomal inhibition on secretion of active BACE.

    PubMed

    Vanoni, Omar; Paganetti, Paolo; Molinari, Maurizio

    2008-10-01

    BACE is an aspartic protease involved in the production of a toxic peptide accumulating in the brain of Alzheimer's disease patients. After attainment of the native structure in the endoplasmic reticulum (ER), BACE is released into the secretory pathway. To better understand the mechanisms regulating protein biogenesis in the mammalian ER, we determined the fate of five variants of soluble BACE with 4, 3, 2, 1, or 0 N-linked glycans. The number of N-glycans displayed on BACE correlated directly with folding and secretion rates and with the yield of active BACE harvested from the cell culture media. Addition of a single N-glycan was sufficient to recruit the calnexin chaperone system and/or for oligosaccharide de-glucosylation by the ER-resident alpha-glucosidase II. Addition of 1-4 N-glycans progressively enhanced the dissociation rate from BiP and reduced the propensity of newly synthesized BACE to enter aberrant soluble and insoluble aggregates. Finally, inhibition of the proteasome increased the yield of active BACE. This shows that active protein normally targeted for destruction can be diverted for secretion, as if for BACE the quality control system would be acting too stringently in the ER lumen, thus causing loss of functional polypeptides. PMID:18632981

  4. 7-hydroxycalamenene Effects on Secreted Aspartic Proteases Activity and Biofilm Formation of Candida spp.

    PubMed Central

    Azevedo, Mariana M. B.; Almeida, Catia A.; Chaves, Francisco C. M.; Rodrigues, Igor A.; Bizzo, Humberto R.; Alviano, Celuta S.; Alviano, Daniela S.

    2016-01-01

    Background: The 7-hydroxycalamenenene-rich essential oil (EO) obtained from the leaves of Croton cajucara (red morphotype) have been described as active against bacteria, protozoa, and fungi species. In this work, we aimed to evaluate the effectiveness of 7-hydroxycalamenenene against Candida albicans and nonalbicans species. Materials and Methods: C. cajucara EO was obtained by hydrodistillation and its major compound, 7-hydroxycalamenene, was purified using preparative column chromatography. The anti-candidal activity was investigated by minimum inhibitory concentration (MIC) and secreted aspartic proteases (SAP) and biofilm inhibition assays. Results: 7-hydroxycalamenene (98% purity) displayed anti-candidal activity against all Candida species tested. Higher activity was observed against Candida dubliniensis, Candida parapsilosis and Candida albicans, showing MIC values ranging from 39.06 μg/ml to 78.12 μg/ml. The purified 7-hydroxycalamenene was able to inhibit 58% of C. albicans ATCC 36801 SAP activity at MIC concentration (pH 7.0). However, 7-hydroxycalamenene demonstrated poor inhibitory activity on C. albicans ATCC 10231 biofilm formation even at the highest concentration tested (2500 μg/ml). Conclusion: The bioactive potential of 7-hydroxycalamenene against planktonic Candida spp. further supports its use for the development of antimicrobials with anti-candidal activity. SUMMARY Croton cajucara Benth. essential oil provides high amounts of 7-hydroxycalamenene7-Hydroxycalameneneisolated from C. cajucarais active against Candida spp7-Hydroxycalameneneinhibits C. albicans aspartic protease activity7-Hydroxycalamenene was not active against C. albicans biofilm formation. Figure PMID:27019560

  5. Cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells.

    PubMed

    Pang, Yue; Wang, Shiyue; Ba, Wei; Li, Qingwei

    2015-01-01

    The supraneural body was identified in the adult lamprey, and its secretions induced the death of a variety of tumor cells but had no effect on normal cells. The cell secretions from different lamprey tissues were separated, and these secretions killed human tumor cells to varying degrees. The cell secretions induced remarkable cell morphological alterations such as cell blebbing, and the plasma membrane was destroyed by the secretions. In addition, the secretions induced morphological alterations of the mitochondria, cytoskeletal structure, and endoplasmic reticulum, eventually leading to cell death. These observations suggest the presence of a novel protein in the lamprey and the possibility of new applications for the protein in the medical field.

  6. Inflammasome activation and IL-1β target IL-1α for secretion as opposed to surface expression

    PubMed Central

    Fettelschoss, Antonia; Kistowska, Magdalena; LeibundGut-Landmann, Salomé; Beer, Hans-Dietmar; Johansen, Pål; Senti, Gabriela; Contassot, Emmanuel; Bachmann, Martin F.; French, Lars E.; Oxenius, Annette; Kündig, Thomas M.

    2011-01-01

    Interleukin-1α (IL-1α) and -β both bind to the same IL-1 receptor (IL-1R) and are potent proinflammatory cytokines. Production of proinflammatory (pro)–IL-1α and pro–IL-1β is induced by Toll-like receptor (TLR)-mediated NF-κB activation. Additional stimulus involving activation of the inflammasome and caspase-1 is required for proteolytic cleavage and secretion of mature IL-1β. The regulation of IL-1α maturation and secretion, however, remains elusive. IL-1α exists as a cell surface-associated form and as a mature secreted form. Here we show that both forms of IL-1α, the surface and secreted form, are differentially regulated. Surface IL-1α requires NF-κB activation only, whereas secretion of mature IL-1α requires additional activation of the inflammasome and caspase-1. Surprisingly, secretion of IL-1α also required the presence of IL-1β, as demonstrated in IL-1β–deficient mice. We further demonstrate that IL-1β directly binds IL-1α, thus identifying IL-1β as a shuttle for another proinflammatory cytokine. These results have direct impact on selective treatment modalities of inflammatory diseases. PMID:22006336

  7. Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst-forming epithelial cells.

    PubMed

    Buchholz, Bjoern; Faria, Diana; Schley, Gunnar; Schreiber, Rainer; Eckardt, Kai-Uwe; Kunzelmann, Karl

    2014-05-01

    Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

  8. Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots

    PubMed Central

    Courty, P. E.; Labbé, J.; Kohler, A.; Marçais, B.; Bastien, C.; Churin, J. L.; Garbaye, J.; Le Tacon, F.

    2011-01-01

    The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F1 individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, β-glucuronidase, cellobiohydrolase, β-glucosidase, β-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner. PMID:20881013

  9. Lipase Activity in Insect Oral Secretions Mediates Defense Responses in Arabidopsis1[C][W][OA

    PubMed Central

    Schäfer, Martin; Fischer, Christine; Meldau, Stefan; Seebald, Eileen; Oelmüller, Ralf; Baldwin, Ian T.

    2011-01-01

    How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses. PMID:21546453

  10. Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

    PubMed Central

    Al-Jallad, Hadil F.; Myneni, Vamsee D.; Piercy-Kotb, Sarah A.; Chabot, Nicolas; Mulani, Amina; Keillor, Jeffrey W.; Kaartinen, Mari T.

    2011-01-01

    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. PMID:21283799

  11. Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets.

    PubMed

    Alenkvist, Ida; Dyachok, Oleg; Tian, Geng; Li, Jia; Mehrabanfar, Saba; Jin, Yang; Birnir, Bryndis; Tengholm, Anders; Welsh, Michael

    2014-12-01

    The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression of Shb in β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereas Shb deficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics in Shb-knockout mice. Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor was Shb deficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca(2+) handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in the Shb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion, Shb deficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

  12. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

    PubMed

    Flores, Alyssa M; Casey, Scott D; Felix, Christian M; Phuan, Puay W; Verkman, A S; Levin, Marc H

    2016-05-01

    Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

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

    PubMed

    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

  14. Ligand-independent activation of peroxisome proliferator-activated receptor-gamma by insulin and C-peptide in kidney proximal tubular cells: dependent on phosphatidylinositol 3-kinase activity.

    PubMed

    Al-Rasheed, Nawal M; Chana, Ravinder S; Baines, Richard J; Willars, Gary B; Brunskill, Nigel J

    2004-11-26

    Peroxisome proliferator-activated receptor gamma (PPARgamma) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARgamma transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARgamma transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARgamma transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARgamma. GW9662, a PPARgamma antagonist, blocked PPARgamma activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARgamma transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARgamma transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARgamma transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARgamma was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARgamma transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARgamma-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARgamma activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in

  15. Does adrenergic activity suppress insulin secretion during surgery? A clinical experiment with halothane anesthesia.

    PubMed Central

    Aärimaa, M; Syvälahti, E; Ovaska, J

    1978-01-01

    Peroperative inhibition of insulin release is widely attributed to increased alpha-adrenergic activity. To test this hypothesis serum insulin and glucose concentrations were measured at short intervals in 11 patients who underwent major surgery. Five patients were anesthetized with halothane and six with general anesthesia without halothane. The results were similar in both patient groups; halothane had no effect on insulin. This suggests that suppression of insulin under operations is probably not due to activation of the alpha-adrenergic receptors of the pancreatic beta-cells. The authors propose that suppression of insulin secretion during surgery may be caused by adrenaline, which, in competing for the glucose receptors, insensitizes the pancreatic beta-cells. PMID:202205

  16. Endogenous opiates modulate the pulsatile secretion of biologically active luteinizing hormone in man.

    PubMed

    Veldhuis, J D; Rogol, A D; Johnson, M L

    1983-12-01

    We studied the secretion of physiological pools of immunoreactive and biologically active luteinizing hormone in response to endogenous pulses of gonadotropin-releasing hormone (GNRH) in eugonadal men. Concentrations of immunoactive and bioactive luteinizing hormone (LH) were determined in blood drawn at 20-min intervals for 8 h in eight normal men under two conditions: (a) after placebo, in order to evaluate spontaneous LH pulsations in the basal state, and (b) after administration of the opiate-receptor antagonist, naltrexone, which is believed to amplify the pulsatile release of endogenous GNRH. Spontaneous and naltrexone-stimulated secretion of LH occurred in pulses of high biological activity, as measured in the RICT (rat interstitial cell testosterone bioassay), i.e., bioactive:immunoactive LH ratios within both spontaneous and naltrexone-stimulated LH pulses were higher than corresponding interpulse ratios (P less than 0.001). Quantitative characterization of the pulsatile release of bioactive LH revealed the following specific effects of opiate-receptor blockade: increased 8-h mean and integrated serum concentrations of bioactive LH (P less than 0.002), enhanced pulse frequency of bioactive LH release (P less than 0.001), and augmented peak amplitude of bio-LH pulses (P less than 0.01). Moreover, this increase in episodic secretion of bioactive LH was associated with increased 8-h mean and integrated serum testosterone concentrations in these men (P less than 0.05). We conclude the following: (a) LH is normally released in spontaneous pulses of high biological activity in men; (b) when the endogenous GNRH signal is amplified by opiate-receptor blockade, the pituitary gland releases more frequent bioactive LH pulses, which are of high amplitude and contain a high bioactive:immunoactive LH ratio. This increase in pulsatile release of bioactive LH quantitated in the RICT assay in vitro is reflected by acutely increased serum testosterone concentrations in vivo

  17. Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

    PubMed Central

    Zakharova, Natalia V.; Artemenko, Elena O.; Podoplelova, Nadezhda A.; Sveshnikova, Anastasia N.; Demina, Irina A.; Ataullakhanov, Fazly I.; Panteleev, Mikhail A.

    2015-01-01

    Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis. PMID:25688860

  18. Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

    PubMed

    LaFramboise, W A; Petrosko, P; Krill-Burger, J M; Morris, D R; McCoy, A R; Scalise, D; Malehorn, D E; Guthrie, R D; Becich, M J; Dhir, R

    2010-03-10

    Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

  19. Novel effects of sphingosylphosphorylcholine on invasion of breast cancer: Involvement of matrix metalloproteinase-3 secretion leading to WNT activation.

    PubMed

    Kim, Hyun Ji; Kang, Gyeoung Jin; Kim, Eun Ji; Park, Mi Kyung; Byun, Hyun Jung; Nam, Seungyoon; Lee, Ho; Lee, Chang Hoon

    2016-09-01

    Sphingosylphosphorylcholine (SPC) participates in several cellular processes including metastasis. SPC induces keratin reorganization and regulates the viscoelasticity of metastatic cancer cells including PANC-1 cancer cells leading to enhanced migration and invasion. The role of SPC and the relevant mechanism in invasion of breast cell are as yet unknown. SPC dose-dependently induces invasion of breast cancer cells or breast immortalized cells. Reverse transcription polymerase chain reaction and Western blot analyses of MCF10A and ZR-75-1 cells indicated that SPC induces expression and secretion of matrix metalloproteinase-3 (MMP3). From online KMPLOT, relapse free survival is high in patients having low MMP3 expressed basal breast cancer (n=581, p=0.032). UK370106 (MMP3 inhibitor) or gene silencing of MMP3 markedly inhibited the SPC-induced invasion of MCF10A cells. An extracellular signal-regulated kinase (ERK) inhibitor, PD98059, significantly suppressed the secretion and the gelatinolytic activity of MMP3, and invasion in MCF10A cells. Over-expression of ERK1 and ERK2 promoted both the expression and secretion of MMP3. In contrast, gene silencing of ERK1 and ERK2 attenuated the secretion of MMP3 in MCF10A cells. The effects of SPC-induced MMP3 secretion on β-catenin and TCF/lymphoid enhancer factor (LEF) promoter activity were examined since MMP3 indirectly activates canonical Wnt signaling. SPC induced translocation of β-catenin to nucleus and increased TCF/LEF promoter activity. These events were suppressed by UK370106 or PD98059. Wnt inhibitor, FH535 inhibited SPC-induced MMP3 secretion and invasion. Taken together, these results suggest that SPC induces MMP3 expression and secretion via ERK leading to Wnt activation. PMID:27216977

  20. Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3

    PubMed Central

    Kim, Jung; Neufeld, Thomas P.

    2015-01-01

    Secreted ligands of the insulin family promote cell growth and maintain sugar homeostasis. Insulin release is tightly regulated in response to dietary conditions, but how insulin producing cells (IPCs) coordinate their responses to distinct nutrient signals is unclear. Here, we show that regulation of insulin secretion in Drosophila larvae has been segregated into distinct branches: whereas amino acids promote secretion of Drosophila insulin-like peptide 2 (Dilp2), circulating sugars promote selective release of Dilp3. Dilp3 is uniquely required for sugar-mediated activation of TOR signaling and suppression of autophagy in the larval fat body. Sugar levels are not sensed directly by the IPCs, but rather by the adipokinetic hormone (AKH)-producing cells of the corpora cardiaca, and we demonstrate that AKH signaling is required in the IPCs for sugar-dependent Dilp3 release. Thus, IPCs integrate multiple cues to regulate secretion of distinct insulin subtypes under varying nutrient conditions. PMID:25882208

  1. Simulation of Cl− Secretion in Epithelial Tissues: New Methodology Estimating Activity of Electro-Neutral Cl− Transporter

    PubMed Central

    Sasamoto, Kouhei; Niisato, Naomi; Taruno, Akiyuki; Marunaka, Yoshinori

    2015-01-01

    Transcellular Cl− secretion is, in general, mediated by two steps; (1) the entry step of Cl− into the cytosolic space from the basolateral space across the basolateral membrane by Cl− transporters, such as Na+-K+-2Cl− cotransporter (NKCC1, an isoform of NKCC), and (2) the releasing step of Cl− from the cytosolic space into the luminal (air) space across the apical membrane via Cl− channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Transcellular Cl− secretion has been characterized by using various experimental techniques. For example, measurements of short-circuit currents in the Ussing chamber and patch clamp techniques provide us information on transepithelial ion movements via transcellular pathway, transepithelial conductance, activity (open probability) of single channel, and whole cell currents. Although many investigators have tried to clarify roles of Cl− channels and transporters located at the apical and basolateral membranes in transcellular Cl− secretion, it is still unclear how Cl− channels/transporters contribute to transcellular Cl− secretion and are regulated by various stimuli such as Ca2+ and cAMP. In the present study, we simulate transcellular Cl− secretion using mathematical models combined with electrophysiological measurements, providing information on contribution of Cl− channels/transporters to transcellular Cl− secretion, activity of electro-neutral ion transporters and how Cl− channels/transporters are regulated. PMID:26779025

  2. Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice

    PubMed Central

    Johansson, Elinn; Rönö, Birgitte; Johansson, Martin; Lindgren, David; Möller, Christina; Axelson, Håkan; Smith, Emma M. K.

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, representing approximately 75% of all renal neoplasms. ccRCC is known to be strongly associated with silencing of the von Hippel Lindau (VHL) tumor suppressor gene, yet VHL deficiency alone does not seem to be sufficient to drive the oncogenic transformation of normal renal epithelium and induce renal tumorigenesis. We, and others, have previously suggested that constitutive activation of the Notch signaling pathway, alongside with VHL loss, contribute to the oncogenic features of ccRCC. Here we report a prevailing hyperactivation of the Notch1 receptor in human ccRCC relative to the healthy counterpart. To explore the consequences of the elevated Notch1 signaling observed in ccRCC patient material, we made use of a conditional mouse model based on concurrent ectopic expression of constitutively active Notch1 (NICD1) and deletion of the Vhl gene. Histological examination of the kidneys of the conditional mice demonstrate the existence of nests of dysplastic cells with a clear cytoplasm as a consequence of lipid accumulation, thus displaying a one important hallmark of human ccRCC. PMID:27491826

  3. Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice.

    PubMed

    Johansson, Elinn; Rönö, Birgitte; Johansson, Martin; Lindgren, David; Möller, Christina; Axelson, Håkan; Smith, Emma M K

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, representing approximately 75% of all renal neoplasms. ccRCC is known to be strongly associated with silencing of the von Hippel Lindau (VHL) tumor suppressor gene, yet VHL deficiency alone does not seem to be sufficient to drive the oncogenic transformation of normal renal epithelium and induce renal tumorigenesis. We, and others, have previously suggested that constitutive activation of the Notch signaling pathway, alongside with VHL loss, contribute to the oncogenic features of ccRCC. Here we report a prevailing hyperactivation of the Notch1 receptor in human ccRCC relative to the healthy counterpart. To explore the consequences of the elevated Notch1 signaling observed in ccRCC patient material, we made use of a conditional mouse model based on concurrent ectopic expression of constitutively active Notch1 (NICD1) and deletion of the Vhl gene. Histological examination of the kidneys of the conditional mice demonstrate the existence of nests of dysplastic cells with a clear cytoplasm as a consequence of lipid accumulation, thus displaying a one important hallmark of human ccRCC. PMID:27491826

  4. Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae

    PubMed Central

    Unterweger, Daniel; Diaz-Satizabal, Laura; Ogg, Stephen; Pukatzki, Stefan

    2015-01-01

    The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen’s arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection. PMID:26317760

  5. Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae.

    PubMed

    Bachmann, Verena; Kostiuk, Benjamin; Unterweger, Daniel; Diaz-Satizabal, Laura; Ogg, Stephen; Pukatzki, Stefan

    2015-01-01

    The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen's arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.

  6. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    PubMed

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

  7. Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

    PubMed

    Blanca, Antonio J; Ruiz-Armenta, María V; Zambrano, Sonia; Salsoso, Rocío; Miguel-Carrasco, José L; Fortuño, Ana; Revilla, Elisa; Mate, Alfonso; Vázquez, Carmen M

    2016-10-01

    Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc.

  8. Protein Secretion in Human Mammary Epithelial Cells following HER1 Receptor Activation: Influence of HER2 and HER3 Expression

    SciTech Connect

    Zhang, Yi; Gonzalez-Hernandez, Rachel M.; Zangar, Richard C.

    2011-02-14

    Background: Secretion of proteins by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a very limited understanding of the cellular regulatory processes that regulate protein secretion. Method: In this study, we utilize an ELISA microarray platform to evaluate the effects of epidermal growth factor receptor (HER) expression on protein secretion in human epithelial mammary cells (HMEC). These secreted proteins included several 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. Result: We utilized HMEC lines that were engineered to express different levels of HER1, HER2 and HER3. We determined the effects of these receptors on the secretion of a variety of growth factors, cytokines, and proteases. Conclusion: Overall, this study suggests that HER overexpression orchestrate broad affects on the tumor microenvironment by altering the secretion of a diverse group of biologically active proteins.

  9. Gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats and cultured rat proximal tubular epithelial cells.

    PubMed

    Ahad, Amjid; Ahsan, Haseeb; Mujeeb, Mohd; Siddiqui, Waseem Ahmad

    2015-10-01

    Diabetic nephropathy (DN) is one of the leading causes of morbidity and mortality in diabetic patients that accounts for about 40% of deaths in type 2 diabetes. p38 mitogen activated protein kinase (p38 MAPK), a serine-threonine kinase, plays an important role in tissue inflammation and is known to be activated under conditions of oxidative stress and hyperglycemia. The role of p38 MAPK has been demonstrated in DN, and its inhibition has been suggested as an alternative approach in the treatment of DN. In the present study, we investigated the nephroprotective effects of an anti-inflammatory phenolic compound, gallic acid (GA, 3,4,5-trihydroxybenzoic acid), in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic wistar albino rats. GA (25 mg/kgbw and 50 mg/kgbw, p.o.) treatment for 16 weeks post induction of diabetes led to a significant reduction in the levels of blood glucose, HbA1c, serum creatinine, blood urea nitrogen and proteinuria as well as a significant reduction in the levels of creatinine clearance. GA significantly inhibited the renal p38 MAPK and nuclear factor kappa B (N-κB) activation as well as significantly reduced the levels of renal transforming growth factor beta (TGF-β) and fibronectin. Treatment with GA resulted in a significant reduction in the serum levels of proinflammatory cytokines viz. interleukin 1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α). Moreover, GA significantly lowered renal pathology and attenuated renal oxidative stress. In cultured rat NRK 52E proximal tubular epithelial cells, GA treatment inhibited high glucose induced activation of p38 MAPK and NF-κB as well as suppressed proinflammatory cytokine synthesis. The results of the present study provide in vivo and in vitro evidences that the p38 MAPK pathway plays an important role in the pathogenesis of DN, and GA attenuates the p38 MAPK-mediated renal dysfunction in HFD/STZ induced type 2 diabetic rats.

  10. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast.

    PubMed

    Liu, Bo; Shi, DanYang; Chang, ShaoHong; Gong, Xin; Yu, YunZhou; Sun, ZhiWei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens. PMID:25567004

  11. Probabilistic secretion of quanta: spontaneous release at active zones of varicosities, boutons, and endplates.

    PubMed Central

    Bennett, M R; Gibson, W G; Robinson, J

    1995-01-01

    The amplitude-frequency histogram of spontaneous miniature endplate potentials follows a Gaussian distribution at mature endplates. This distribution gives the mean and variance of the quantum of transmitter. According to the vesicle hypothesis, this quantum is due to exocytosis of the contents of a single synaptic vesicle. Multimodal amplitude-frequency histograms are observed in varying degrees at developing endplates and at peripheral and central synapses, each of which has a specific active zone structure. These multimodal histograms may be due to the near synchronous exocytosis of more than one vesicle. In the present work, a theoretical treatment is given of the rise of intraterminal calcium after the stochastic opening of a calcium channel within a particular active zone geometry. The stochastic interaction of this calcium with the vesicle-associated proteins involved in exocytosis is then used to calculate the probability of quantal secretions from one or several vesicles at each active zone type. It is shown that this procedure can account for multiquantal spontaneous release that may occur at varicosities and boutons, compared with that at the active zones of motor nerve terminals. PMID:7669909

  12. Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila.

    PubMed

    Fernandez-Funez, Pedro; Sanchez-Garcia, Jonatan; de Mena, Lorena; Zhang, Yan; Levites, Yona; Khare, Swati; Golde, Todd E; Rincon-Limas, Diego E

    2016-08-30

    Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. Here, we used Drosophila to explore a strategy to block Aβ42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aβ42, we added a signal peptide to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aβ42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aβ42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies. PMID:27531960

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

    PubMed

    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. PMID:27377244

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

    PubMed Central

    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

  15. Secreted Thrombospondin-1 Regulates Macrophage Interleukin-1β Production and Activation through CD47

    PubMed Central

    Stein, Erica V.; Miller, Thomas W.; Ivins-O’Keefe, Kelly; Kaur, Sukhbir; Roberts, David D.

    2016-01-01

    Thrombospondin-1 regulates inflammation by engaging several cell surface receptors and by modulating activities of other secreted factors. We have uncovered a novel role of thrombospondin-1 in modulating production and activation of the proinflammatory cytokine IL-1β by human and murine macrophages. Physiological concentrations of thrombospondin-1 limit the induction by lipopolysaccharide of IL-1β mRNA and total protein production by human macrophages. This inhibition can be explained by the ability of thrombospondin-1 to disrupt the interaction between CD47 and CD14, thereby limiting activation of NFκB/AP-1 by lipopolysaccharide. Only the CD47-binding domain of thrombospondin-1 exhibits this activity. In contrast, CD47, CD36, and integrin-binding domains of thrombospondin-1 independently enhance the inflammasome-dependent maturation of IL-1β in human THP-1 monocyte-derived macrophages. Correspondingly, mouse bone marrow-derived macrophages that lack either thrombospondin-1 or CD47 exhibit diminished induction of mature IL-1β in response to lipopolysaccharide. Lack of CD47 also limits lipopolysaccharide induction of IL-1β, NLRP3, and caspase-1 mRNAs. These data demonstrate that thrombospondin-1 exerts CD47-dependent and -independent pro-and anti-inflammatory effects on the IL-1β pathway. Therefore, thrombospondin-1 and its receptor CD47 may be useful targets for limiting the pro-inflammatory effects of lipopolysaccharide and for treating endotoxemia. PMID:26813769

  16. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  17. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

    PubMed Central

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-01-01

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  18. Geomagnetic activity influences the melatonin secretion at latitude 70 degrees N.

    PubMed

    Weydahl, A; Sothern, R B; Cornélissen, G; Wetterberg, L

    2001-01-01

    Factors other than light may affect variations in melatonin, including disturbances in the geomagnetic field. Such a possibility was tested in Alta, Norway, located at latitude 70 degrees N, where the aurora borealis is a result of large changes in the horizontal component (H) of the geomagnetic field. Geomagnetic disturbances are felt more strongly closer to the pole than at lower latitudes. Also noteworthy in Alta is the fact that the sun does not rise above the horizon for several weeks during the winter. To examine whether changes in geomagnetic activity influence the secretion of melatonin, saliva was collected from 25 healthy subjects in Alta several times during the day-night and at different times of the year. Single cosinor analyses yielded individual estimates of.the circadian amplitude and MESOR of melatonin. A 3-hour mean value for the local geomagnetic activity index, K, was used for approximately the same 24-hour span. A circadian rhythm was found to characterize both melatonin and K, the peak in K (23:24) preceding that of melatonin (06:08). During the span of investigation, a circannual variation also characterized both variables. Correlation analyses suggest that changes in geomagnetic activity had to be of a certain magnitude to affect the circadian amplitude of melatonin. If large enough (> 80 nT/3 h), changes in geomagnetic activity also significantly decreased salivary melatonin concentration. PMID:11774869

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

    SciTech Connect

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

    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.

  20. The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

    PubMed Central

    Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In

    2015-01-01

    ABSTRACT The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for

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

    PubMed Central

    Christensen, Louise W; Kuhre, Rune E; Janus, Charlotte; Svendsen, Berit; Holst, Jens J

    2015-01-01

    Glucagon-like peptide 1 (GLP-1) plays a central role in modern treatment of type 2 diabetes (T2DM) in the form of GLP-1 enhancers and GLP-1 mimetics. An alternative treatment strategy is to stimulate endogenous GLP-1 secretion from enteroendocrine L cells using a targeted approach. The G-protein-coupled 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 examined the effect of FFAR1 activation on GLP-1 secretion using isolated, perfused 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 agonists (TAK-875, AMG 837, AM-1638, and AM-5262) were administered through intraluminal and intra-arterial routes, respectively, and dynamic changes in GLP-1 secretion were evaluated. Vascular administration of 10 μmol/L TAK-875, 10 μmol/L AMG 837, 1 μmol/L and 0.1 μmol/L AM-1638, 1 μmol/L AM-6252, and 1 mmol/L LA, all significantly increased GLP-1 secretion compared to basal levels (P < 0.05), whereas luminal administration of LA and FFAR1 agonists was ineffective. Thus, both natural and small-molecule agonists of the FFAR1 receptor appear to require absorption prior to stimulating GLP-1 secretion, indicating that therapies based on activation of nutrient sensing may be more complex than hitherto expected. PMID:26381015

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

    PubMed

    Christensen, Louise W; Kuhre, Rune E; Janus, Charlotte; Svendsen, Berit; Holst, Jens J

    2015-09-01

    Glucagon-like peptide 1 (GLP-1) plays a central role in modern treatment of type 2 diabetes (T2DM) in the form of GLP-1 enhancers and GLP-1 mimetics. An alternative treatment strategy is to stimulate endogenous GLP-1 secretion from enteroendocrine L cells using a targeted approach. The G-protein-coupled 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 examined the effect of FFAR1 activation on GLP-1 secretion using isolated, perfused 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 agonists (TAK-875, AMG 837, AM-1638, and AM-5262) were administered through intraluminal and intra-arterial routes, respectively, and dynamic changes in GLP-1 secretion were evaluated. Vascular administration of 10 μmol/L TAK-875, 10 μmol/L AMG 837, 1 μmol/L and 0.1 μmol/L AM-1638, 1 μmol/L AM-6252, and 1 mmol/L LA, all significantly increased GLP-1 secretion compared to basal levels (P < 0.05), whereas luminal administration of LA and FFAR1 agonists was ineffective. Thus, both natural and small-molecule agonists of the FFAR1 receptor appear to require absorption prior to stimulating GLP-1 secretion, indicating that therapies based on activation of nutrient sensing may be more complex than hitherto expected.

  3. Expandable tubulars for use in geologic structures

    DOEpatents

    Spray, Jeffery A.; Svedeman, Steven; Walter, David; Mckeighan, Peter; Siebanaler, Shane; Dewhurst, Peter; Hobson, Steven; Foss, Doug; Wirz, Holger; Sharpe, Aaron; Apostal, Michael

    2014-08-12

    An expandable tubular includes a plurality of leaves formed from sheet material that have curved surfaces. The leaves extend around a portion or fully around the diameter of the tubular structure. Some of the adjacent leaves of the tubular are coupled together. The tubular is compressed to a smaller diameter so that it can be inserted through previously deployed tubular assemblies. Once the tubular is properly positioned, it is deployed and coupled or not coupled to a previously deployed tubular assembly. The tubular is useful for all types of wells and boreholes.

  4. Tapered, tubular polyester fabric

    NASA Technical Reports Server (NTRS)

    Lapointe, Donat J. E. (Inventor); Wright, Lawrence T. (Inventor); Vincent, Laurence J. (Inventor)

    1987-01-01

    A tapered tubular polyester sleeve is described to serve as the flexible foundation for a spacesuit limb covering. The tube has a large end and a small end with a length to be determined. The ratio of taper is also determined by scale factors. All the warp yarns extend to the large end. A requisite number of warp yarns extend the full length of the sleeve. Other warp yarns extend from the large end but are terminated along the length of the sleeve. It is then woven with a filling yarn which extends in a full circle along the full length of the sleeve to thereby define the tapered sleeve. The sleeve after fabrication is then placed on a mandrel, heated in an oven, and then attached to the arm or other limb of the spacesuit.

  5. Tapered, tubular polyester fabric

    NASA Technical Reports Server (NTRS)

    LaPointe, Donat J. E. (Inventor); Vincent, Laurence J. (Inventor); Wright, Lawrence T. (Inventor)

    1988-01-01

    A tapered tubular polyester sleeve as set forth. It has a large end 12 and a small end 14 with a length to be determined. The ratio of taper is also determined by scale factors. All the warp yarns extend to the large end 12. A requisite number of warp yarns 16 extend the full length of the sleeve. Other warp yarns exemplified at 18, 22, 26, 28, 30 and 32 extend from the large end but are terminated along the length of the sleeve. It is then woven with a filling yarn 40 which extends in a full circle along the full length of the sleeve to thereby define the tapered sleeve. The sleeve after fabrication is then placed on a mandrel 42, heated in an oven 44 and is thereafter placed on the arm or other limb of a space suit exemplified at 50.

  6. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  7. Gallic Acid, the Active Ingredient of Terminalia bellirica, Enhances Adipocyte Differentiation and Adiponectin Secretion.

    PubMed

    Makihara, Hiroko; Koike, Yuka; Ohta, Masatomi; Horiguchi-Babamoto, Emi; Tsubata, Masahito; Kinoshita, Kaoru; Akase, Tomoko; Goshima, Yoshio; Aburada, Masaki; Shimada, Tsutomu

    2016-01-01

    Visceral obesity induces the onset of metabolic disorders such as insulin resistance and diabetes mellitus. Adipose tissue is considered as a potential pharmacological target for treating metabolic disorders. The fruit of Terminalia bellirica is extensively used in Ayurvedic medicine to treat patients with diseases such as diabetes mellitus. We previously investigated the effects of a hot water extract of T. bellirica fruit (TB) on obesity and insulin resistance in spontaneously obese type 2 diabetic mice. To determine the active ingredients of TB and their molecular mechanisms, we focused on adipocyte differentiation using mouse 3T3-L1 cells, which are widely used to study adipocyte physiology. We show here that TB enhanced the differentiation of 3T3-L1 cells to mature adipocytes and that one of the active main components was identified as gallic acid. Gallic acid (10-30 µM) enhanced the expression and secretion of adiponectin via adipocyte differentiation and also that of fatty acid binding protein-4, which is the target of peroxisome proliferator-activated receptor gamma (PPARγ), although it does not alter the expression of the upstream genes PPARγ and CCAAT enhancer binding protein alpha. In the PPARγ ligand assay, the binding of gallic acid to PPARγ was undetectable. These findings indicate that gallic acid mediates the therapeutic effects of TB on metabolic disorders by regulating adipocyte differentiation. Therefore, TB shows promise as a candidate for preventing and treating patients with metabolic syndrome. PMID:27374289

  8. Gallic Acid, the Active Ingredient of Terminalia bellirica, Enhances Adipocyte Differentiation and Adiponectin Secretion.

    PubMed

    Makihara, Hiroko; Koike, Yuka; Ohta, Masatomi; Horiguchi-Babamoto, Emi; Tsubata, Masahito; Kinoshita, Kaoru; Akase, Tomoko; Goshima, Yoshio; Aburada, Masaki; Shimada, Tsutomu

    2016-01-01

    Visceral obesity induces the onset of metabolic disorders such as insulin resistance and diabetes mellitus. Adipose tissue is considered as a potential pharmacological target for treating metabolic disorders. The fruit of Terminalia bellirica is extensively used in Ayurvedic medicine to treat patients with diseases such as diabetes mellitus. We previously investigated the effects of a hot water extract of T. bellirica fruit (TB) on obesity and insulin resistance in spontaneously obese type 2 diabetic mice. To determine the active ingredients of TB and their molecular mechanisms, we focused on adipocyte differentiation using mouse 3T3-L1 cells, which are widely used to study adipocyte physiology. We show here that TB enhanced the differentiation of 3T3-L1 cells to mature adipocytes and that one of the active main components was identified as gallic acid. Gallic acid (10-30 µM) enhanced the expression and secretion of adiponectin via adipocyte differentiation and also that of fatty acid binding protein-4, which is the target of peroxisome proliferator-activated receptor gamma (PPARγ), although it does not alter the expression of the upstream genes PPARγ and CCAAT enhancer binding protein alpha. In the PPARγ ligand assay, the binding of gallic acid to PPARγ was undetectable. These findings indicate that gallic acid mediates the therapeutic effects of TB on metabolic disorders by regulating adipocyte differentiation. Therefore, TB shows promise as a candidate for preventing and treating patients with metabolic syndrome.

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

    PubMed

    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.

  10. The anti-biofilm activity secreted by a marine Pseudoalteromonas strain.

    PubMed

    Klein, Géraldine L; Soum-Soutéra, Emmanuelle; Guede, Zakoua; Bazire, Alexis; Compère, Chantal; Dufour, Alain

    2011-09-01

    Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria. PMID:21895460

  11. Secretion of a lysophospholipase D activity by adipocytes: involvement in lysophosphatidic acid synthesis

    PubMed Central

    Gesta, Stéphane; Simon, Marie-Françoise; Rey, Astrid; Sibrac, David; Girard, Alexia; Lafontan, Max; Valet, Philippe; Saulnier-Blache, Jean Sébastien

    2002-01-01

    The aim of the present work was to depict the metabolic pathways involved in extra-cellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium: CM) of 3T3F442A adipocytes, or human adipose tissue explants, using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could significantly be increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators: EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D- (lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLD-sensitive LPA-precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) as compared to CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra-cellular production of synthesis of LPA by adipocytes. PMID:12032165

  12. Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ.

    PubMed

    Lilo, Sarit; Zheng, Ying; Bliska, James B

    2008-09-01

    Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.

  13. Caspase-1 Activation in Macrophages Infected with Yersinia pestis KIM Requires the Type III Secretion System Effector YopJ▿

    PubMed Central

    Lilo, Sarit; Zheng, Ying; Bliska, James B.

    2008-01-01

    Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1β (IL-1β) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1β was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1β following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1β were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages. PMID:18559430

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

    PubMed Central

    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

  15. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    SciTech Connect

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-06-24

    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} 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 (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  16. Ricinoleic acid stimulation of active anion secretion in colonic mucosa of the rat.

    PubMed Central

    Racusen, L C; Binder, H J

    1979-01-01

    Perfusion of the colon with ricinoleic acid produces fluid and electrolyte accumulation. The mechanism of these changes in water and electrolyte movement is uknown. These studies were designed to determine whether ricinoleic acid effects active ion transport across isolated rat colonic mucosa. 0.5 mM Na ricinoleate produced significant increases in potential difference (3.8 +/- 0.5 mV) and short-circuit current (Isc) (99.2 +/- 10.1 muA/cm2). The increases in Isc produced by Na ricinoleate were inhibited by both removal of bicarbonate and chloride and by the presence of theophylline. The hydroxy fatty acid also resulted in a significant decrease in net Na absorption from 4.7 +/- 0.8 to 0.1 +/- 0.7 mueq/h cm2 and reversed net Cl transport from absorption (+ 4.5 +/- 0.9) to secretion (-2.2 +/- mueq/h cm2). In parallel studies 0.5 mM Na ricinoleate increased mucosal cyclic AMP content by 58%. The concentrations of Na ricinoleate required to produce detectable and maximal increases in both Isc and cyclic AMP were the same. These results provide evidence in support of the concept that hydroxy fatty acid-induced fluid and electrolyte accumulation is driven by an active ion secretory process. PMID:220281

  17. [Intestinal dysbacteriosis promotes intestinal intraepithelial T lymphocyte activation and proinflammatory cytokine secretion in mice].

    PubMed

    Luo, Xia; Luo, Shuang; Zheng, Yanyi; Wen, Ruyan; Deng, Xiangliang; Zhou, Lian

    2016-08-01

    Objective To study the effect of intestinal dysbacteriosis on mouse intestinal intraepithelial T lymphocytes (iIELs). Methods The intestinal dysbacteriosis was induced in mice by oral administration of ceftriaxone sodium. The iIELs were digested with ethylene diaminetetraacetic acid (EDTA) and DL-dithiothreitol (DTT). The phenotype of iIELs and the proportions of subsets of T cells were detected by flow cytometry; the concentrations of cytokines (IL-2, IL-6, IFN-γ) in the intestine were examined by ELISA; the intestinal bacteria were analyzed with selective medium and PCR. Results Compared with the control group, intestinal commensal bacteria in mice were significantly reduced after the administration of ceftriaxone sodium, but fungi and yeasts increased. The proportions of T cell subgroups in ilELs changed, in which the proportion of TCR γδ(+)T cells significantly increased, and the activated CD3(+)T, CD8(+)T and TCR γδ(+)T cells increased. The concentrations of IL-2, IL-6 and IFN-γ were significantly raised in the intestine. Conclusion The dysbacteriosis results in the decrease of commensal bacteria, the increase of the fungus, the damage of microbial barrier, the more activated T cells in ilELs and the promotion of proinflammatory cytokine secretion in the gut. This is probably one of the reasons for inflammatory bowel disease caused by dysbacteriosis. PMID:27412931

  18. Nisin-Triggered Activity of Lys44, the Secreted Endolysin from Oenococcus oeni Phage fOg44▿

    PubMed Central

    Nascimento, João Gil; Guerreiro-Pereira, Maria Carolina; Costa, Sérgio Fernandes; São-José, Carlos; Santos, Mário Almeida

    2008-01-01

    The intrinsic resistance of Oenococcus oeni cells to the secreted endolysin from oenophage fOg44 (Lys44) was investigated. Experiments with several antimicrobials support the hypothesis that the full activity of Lys44 requires sudden ion-nonspecific dissipation of the proton motive force, an event undertaken by the fOg44 holin in the phage infection context. PMID:17981964

  19. Tubular g-C3 N4 Isotype Heterojunction: Enhanced Visible-Light Photocatalytic Activity through Cooperative Manipulation of Oriented Electron and Hole Transfer.

    PubMed

    Tong, Zhenwei; Yang, Dong; Sun, Yuanyuan; Nan, Yanhu; Jiang, Zhongyi

    2016-08-01

    A tubular g-C3 N4 isotype heterojunction (TCNH) photocatalyst was designed for cooperative manipulation of the oriented transfer of photogenerated electrons and holes to pursue high catalytic performance. The adduct of cyanuric acid and melamine (CA·M) is first hydrothermally treated to assemble into hexagonal prism crystals; then the hybrid precursors of urea and CA·M crystals are calcined to form tubular g-C3 N4 isotype heterojunctions. Upon visible-light irradiation, the photogenerated electrons transfer from g-C3 N4 (CA·M) to g-C3 N4 (urea) driven by the conduction band offset of 0.05 eV, while the photogenerated holes transfer from g-C3 N4 (urea) to g-C3 N4 (CA·M) driven by the valence band offset of 0.18 eV, which renders oriented transfer of the charge carriers across the heterojunction interface. Meanwhile, the tubular structure of TCNH is favorable for oriented electron transfer along the longitudinal dimension, which greatly decreases the chance of charge carrier recombination. Consequently, TCNH exhibits a high hydrogen evolution rate of 63 μmol h(-1) (0.04 g, λ > 420 nm), which is nearly five times of the pristine g-C3 N4 and higher than most of the existing g-C3 N4 photocatalysts. This study demonstrates that isotype heterojunction structure and tubular structure can jointly manipulate the oriented transfer of electrons and holes, thus facilitating the visible-light photocatalysis.

  20. Parachute Jumping Induces More Sympathetic Activation Than Cortisol Secretion in First-Time Parachutists

    PubMed Central

    Messina, Giovanni; Chieffi, Sergio; Viggiano, Andrea; Tafuri, Domenico; Cibelli, Giuseppe; Valenzano, Anna; Triggiani, Antonio Ivano; Messina, Antonietta; De Luca, Vincenzo; Monda, Marcellino

    2016-01-01

    Background: The word “stress” describes the status of the body affected by external or internal forces, or “stressors”, threatening to alter its dynamic balance or homeostasis. The adaptive changes which occur in reply to stressors are either behavioral or physical. Once a given threshold is surpassed, a systemic reaction takes place involving the “stress system” in the brain together with its peripheral components, the hypothalamic-pituitary-adrenal axis and autonomic sympathetic. Objectives: Stress induces an activation of the sympathetic nervous system (SNS) and the hypothalamic-pituitary-adrenal (HPA) axis. The purpose of this study was to investigate whether the SNS and the HPA axis would show parallel or divergent stress response patterns in a session of first parachute jump. Patients and Methods: Activation of the SNS was evaluated by dosage of salivary alpha-amylase, galvanic skin responses, and heart rate in seven male novice parachutists. Activation of HPA axis was tested by dosage of cortisol. These variables were measured before and 1 minute and 90 minute after the jump. Results: All variables reached a peak at 1 minute post-jump. Salivary alpha-amylase, galvanic skin responses and heart rate did not return to basal value at 90 minutes post-jump, while cortisol returned to basal value at 90 minutes post-jump. Conclusions: This evidence indicates that parachute jumping is accompanied by a dissociation of SNS and HPA response patterns in novice parachutists, showing a slower recovery in sympathetic activity than in cortisol secretion. PMID:27217924

  1. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-12-11

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

  2. Smart tissue culture: in situ monitoring of the activity of protease enzymes secreted from live cells using nanostructured photonic crystals.

    PubMed

    Kilian, Kristopher A; Lai, Leo M H; Magenau, Astrid; Cartland, Siân; Böcking, Till; Di Girolamo, Nick; Gal, Michael; Gaus, Katharina; Gooding, J Justin

    2009-05-01

    Monitoring enzyme secretion in tissue culture has proved challenging because to date the activity cannot be continuously measured in situ. In this Letter, we present a solution using biopolymer loaded photonic crystals of anodized silicon. Shifts in the optical response by proteolytic degradation of the biopolymer provide label-free sensing with unprecedented low detection limits (1 pg) and calculation of kinetic parameters. The enhancement in sensitivity relative to previous photonic crystal sensors constitutes a change in the sensing paradigm because here the entire pore space is responsive to the secreted enzyme rather than just the pore walls. In situ monitoring is demonstrated by detecting secretion of matrix metalloprotease 9 from stimulated human macrophages.

  3. Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition.

    PubMed

    Casas-Tintó, Sergio; Lolo, Fidel-Nicolás; Moreno, Eduardo

    2015-01-01

    Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue. PMID:26658841

  4. Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma.

    PubMed

    Chen, Xu-Lin; Xia, Zhao-Fan; Wei, Duo; Han, Sheng; Ben, Dao-Feng; Wang, Guang-Qing

    2003-09-01

    This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.

  5. Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris.

    PubMed

    Sakuma, Y; Kimura, M; Takabatake, T; Takeshima, K; Fujimura, H

    1999-09-01

    We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418: His+ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His+ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 micrograms/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH. PMID:10531654

  6. Transient Receptor Potential Vanilloid 1 Activation Enhances Gut Glucagon-Like Peptide-1 Secretion and Improves Glucose Homeostasis

    PubMed Central

    Wang, Peijian; Yan, Zhencheng; Zhong, Jian; Chen, Jing; Ni, Yinxing; Li, Li; Ma, Liqun; Zhao, Zhigang; Liu, Daoyan; Zhu, Zhiming

    2012-01-01

    Type 2 diabetes mellitus (T2DM) is rapidly prevailing as a serious global health problem. Current treatments for T2DM may cause side effects, thus highlighting the need for newer and safer therapies. We tested the hypothesis that dietary capsaicin regulates glucose homeostasis through the activation of transient receptor potential vanilloid 1 (TRPV1)-mediated glucagon-like peptide-1 (GLP-1) secretion in the intestinal cells and tissues. Wild-type (WT) and TRPV1 knockout (TRPV1−/−) mice were fed dietary capsaicin for 24 weeks. TRPV1 was localized in secretin tumor cell-1 (STC-1) cells and ileum. Capsaicin stimulated GLP-1 secretion from STC-1 cells in a calcium-dependent manner through TRPV1 activation. Acute capsaicin administration by gastric gavage increased GLP-1 and insulin secretion in vivo in WT but not in TRPV1−/− mice. Furthermore, chronic dietary capsaicin not only improved glucose tolerance and increased insulin levels but also lowered daily blood glucose profiles and increased plasma GLP-1 levels in WT mice. However, this effect was absent in TRPV1−/− mice. In db/db mice, TRPV1 activation by dietary capsaicin ameliorated abnormal glucose homeostasis and increased GLP-1 levels in the plasma and ileum. The present findings suggest that TRPV1 activation–stimulated GLP-1 secretion could be a promising approach for the intervention of diabetes. PMID:22664955

  7. GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion.

    PubMed

    Chepurny, Oleg G; Holz, George G; Roe, Michael W; Leech, Colin A

    2016-06-01

    GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research. PMID:27082897

  8. Mechanisms of renal tubular defects in old age.

    PubMed Central

    Dontas, A. S.; Marketos, S. G.; Papanayiotou, P.

    1972-01-01

    The mechanisms of renal tubular dysfunction in old age have been examined in twenty-eight clinically healthy elderly subjects without infection, and in fourteen subjects of similar age with laboratory evidence of intrarenal infection. The data were compared with those from thirteen clinically healthy young subjects. Studied were: proximal tubular (Tm(PAH)) and distal tubular (CH2O) activity, minimal and maximal osmolal U/P ratios, maximal osmolal excretion in hydropenia, and GFR levels under standard hydration and under water-loading. The reduction of GFR in old age is evident particularly in men under conditions of standard hydration: it is accentuated in the presence of renal infection. Proximal tubular activity is also significantly lower in elderly men, especially if they have chronic bacteriuria. The reduction is closely related to GFR levels, with identical Tm(PAH):C(in) ratios in all groups. This supports the intact nephron hypothesis for this part of the nephron. Distal tubular activity is depressed in old age in both sexes proportionately more than proximal tubular activity or the GFR. The lower CH2O: GFR ratios imply a selective distal tubular damage. Maximal osmolal U/P ratios in hydropenia are significantly higher in the young (mean 367) than in either the elderly non-infected (mean 279) or the elderly infected subjects (mean 212). Conversely, minimal U/P ratios in water-loading are lower in the young (mean 0.247) than in either elderly group (means 0.418 and 0.668). Osmolal excretion in hydropenia is not different between the groups, but urine flows in water-loading clearly separate them. The data indicate that simple functions of the distal-collecting tubule (e.g. the CH2O), are less affected in old age than are functions involving several medullary structures (as is the maximal U(osm) or U/P ratio). They suggest that the main impairment of the distal tubular cell involves the failure to achieve a proper osmotic gradient between tubular fluid and

  9. Secretion of active recombinant phytase from soybean cell-suspension cultures.

    PubMed Central

    Li, J; Hegeman, C E; Hanlon, R W; Lacy, G H; Denbow, M D; Grabau, E A

    1997-01-01

    Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing. PMID:9232886

  10. Adrenergic and serotoninergic receptors mediate the immunological activation of corticosterone secretion in male rats.

    PubMed

    Guo, A L; Petraglia, F; Criscuolo, M; Ficarra, G; Salvestroni, C; Nappi, R E; Trentini, G P; Genazzani, A R

    1996-06-01

    In order to elucidate the mechanism of action of immune agents on corticosterone secretion, the present study evaluated the possible involvement of some neuronal pathways (serotoninergic, noradrenergic/adrenergic) in the lipopolysaccharide (LPS)-induced corticosterone release in male rats. Serotoninergic antagonists, mianserin (5-HT2C receptor blocker) or pindolol (5HT1A receptor blocker) or noradrenergic/adrenergic antagonists, prazosin (alpha 1-adrenoceptor blocker) or propranolol (beta-adrenoceptor blocker), were intraperitoneally (i.p.) injected before (5 min) the administration of LPS. In each experiment a group of rats i.p. injected with vehicle served as controls. Animals were sacrificed by decapitation 90 min after administration of LPS and trunk blood was collected for corticosterone radioimmunoassay. Results showed that pretreatment with mianserin, but not with pindolol, significantly reduced plasma corticosterone levels following administration of LPS (p < 0.05); prazosin attenuated the plasma corticosterone response to LPS (p < 0.05), while propranolol did not induce significant change. The present study indicated that serotoninergic and noradrenergic/adrenergic pathways are involved in the immunoneuroendocrine modulation of hypothalamus-pituitary-adrenal function in rats. In particular, it is probably mediated by the activation of 5-HT2C receptors and of alpha 1-adrenoceptors, while type 1A serotonin receptors or beta-adrenoceptors do not seem to be involved in such a phenomenon.

  11. MMP-3 secreted from endothelial cells of blood vessels after spinal cord injury activates microglia, leading to oligodendrocyte cell death.

    PubMed

    Lee, Jee Y; Choi, Hae Y; Yune, Tae Y

    2015-10-01

    The activation of microglia after spinal cord injury (SCI) contributes to secondary damage by producing pro-inflammatory cytokines and mediators, leading to cell death of oligodendrocytes and neurons. Here, we show that matrix metalloprotease-3 (MMP-3) produced and secreted in the endothelial cells of blood vessels after SCI mediates microglial activation. MMP-3 was produced and secreted in bEnd.3 cells, a mouse brain-derived endothelial cell line, by oxygen-glucose deprivation/reoxygenation (OGD/RO). OGD/RO-induced MMP-3 expression and activity was also significantly inhibited by ghrelin, which was dependent on the ghrelin receptor GHS-R1a. Furthermore, the secreted MMP-3 from OGD/RO-induced bEnd.3 cells activated BV-2 cells, a murine microglial cell line. We also found that microglial activation after SCI was attenuated in MMP-3 knockout (KO) mice compared with wild type (WT) mice. Both p38 mitogen-activated protein kinase (MAPK) activation and pro-nerve growth factor (proNGF) production were more inhibited in MMP-3 KO than WT mice at 5d after injury. When WT mice were treated with Mmp-3 siRNA after injury, MMP-3 activity, microglial activation, p38MAPK activation and proNGF expression were significantly inhibited. Ghrelin treatment also significantly inhibited MMP-3 expression and activation after SCI, which was dependent on GHS-R1a. Finally, RhoA activation and oligodendrocyte cell death after injury were attenuated by Mmp-3 siRNA or ghrelin treatment compared with vehicle control. Thus, our study indicates that MMP-3 produced in blood vessel endothelial cells after SCI serves as an endogenous molecule for microglial activation followed by p38MAPK activation and proNGF production, and further indicates that the protective effect of ghrelin on oligodendrocytes cell death may be at least partly mediated by the inhibition of MMP-3-induced microglial activation after SCI.

  12. Autophagy and Tubular Cell Death in the Kidney.

    PubMed

    Havasi, Andrea; Dong, Zheng

    2016-05-01

    Many common renal insults such as ischemia and toxic injury primarily target the tubular epithelial cells, especially the highly metabolically active proximal tubular segment. Tubular epithelial cells are particularly dependent on autophagy to maintain homeostasis and respond to stressors. The pattern of autophagy in the kidney has a unique spatial and chronologic signature. Recent evidence has shown that there is complex cross-talk between autophagy and various cell death pathways. This review specifically discusses the interplay between autophagy and cell death in the renal tubular epithelia. It is imperative to review this topic because recent discoveries have improved our mechanistic understanding of the autophagic process and have highlighted its broad clinical applications, making autophagy a major target for drug development. PMID:27339383

  13. Subthreshold α₂-adrenergic activation counteracts glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion.

    PubMed

    Pan, Minglin; Yang, Guang; Cui, Xiuli; Yang, Shao-Nian

    2011-01-01

    The pancreatic β cell harbors α₂-adrenergic and glucagon-like peptide-1 (GLP-1) receptors on its plasma membrane to sense the corresponding ligands adrenaline/noradrenaline and GLP-1 to govern glucose-stimulated insulin secretion. However, it is not known whether these two signaling systems interact to gain the adequate and timely control of insulin release in response to glucose. The present work shows that the α₂-adrenergic agonist clonidine concentration-dependently depresses glucose-stimulated insulin secretion from INS-1 cells. On the contrary, GLP-1 concentration-dependently potentiates insulin secretory response to glucose. Importantly, the present work reveals that subthreshold α₂-adrenergic activation with clonidine counteracts GLP-1 potentiation of glucose-induced insulin secretion. This counteractory process relies on pertussis toxin- (PTX-) sensitive Gi proteins since it no longer occurs following PTX-mediated inactivation of Gi proteins. The counteraction of GLP-1 potentiation of glucose-stimulated insulin secretion by subthreshold α₂-adrenergic activation is likely to serve as a molecular mechanism for the delicate regulation of insulin release.

  14. Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation

    PubMed Central

    Patton, Michael John; McCorrister, Stuart; Grant, Chris; Westmacott, Garrett; Fariss, Robert; Hu, Pingzhao; Zhao, Kaiqiong; Blake, Mary; Whitmire, Bill; Yang, Chunfu

    2016-01-01

    ABSTRACT The chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; however, challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Here, we use a genetic and proteomic approach to identify authentic CPAF targets. Human epithelial cells infected with CPAF-sufficient and CPAF-deficient chlamydiae were lysed using known CPAF-denaturing conditions. Their protein profiles were analyzed using isobaric mass tags and liquid chromatography-tandem mass spectrometry. Comparative analysis of CPAF-sufficient and CPAF-deficient infections identified a limited number of CPAF host and chlamydial protein targets. Host targets were primarily interferon-stimulated gene products, whereas chlamydial targets were type III secreted proteins. We provide evidence supporting a cooperative role for CPAF and type III secreted effectors in blocking NF-κB p65 nuclear translocation, resulting in decreased beta interferon and proinflammatory cytokine synthesis. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). We propose that CPAF and T3SEs cooperate in the inhibition of host innate immunity. PMID:27677792

  15. GLUT1 activity contributes to the impairment of PEDF secretion by the RPE

    PubMed Central

    Calado, Sofia M.; Alves, Liliana S.; Simão, Sónia

    2016-01-01

    Purpose In this study, we aimed to understand whether glucose transporter 1 (GLUT1) activity affects the secretion capacity of antiangiogenic factor pigment epithelium-derived factor (PEDF) by the RPE cells, thus explaining the reduction in PEDF levels observed in patients with diabetic retinopathy (DR). Methods Analysis of GLUT1 expression, localization, and function was performed in vitro in RPE cells (D407) cultured with different glucose concentrations, corresponding to non-diabetic (5 mM of glucose) and diabetic (25 mM of glucose) conditions, further subjected to normoxia or hypoxia. The expression of PEDF was also evaluated in the secretome of the cells cultured in these conditions. Analysis of GLUT1 and PEDF expression was also performed in vivo in the RPE of Ins2Akita diabetic mice and age-matched wild-type (WT) controls. Results We observed an increase in GLUT1 under hypoxia in a glucose-dependent manner, which we found to be directly associated with the translocation and stabilization of GLUT1 in the cell membrane. This stabilization led to an increase in glucose uptake by RPE cells. This increase was followed by a decrease in PEDF expression in RPE cells cultured in conditions that simulated DR. Compared with non-diabetic WT mice, the RPE of Ins2Akita mice showed increased GLUT1 overexpression with a concomitant decrease in PEDF expression. Conclusions Collectively, our data show that expression of GLUT1 is stimulated by hyperglycemia and low oxygen supply, and this overexpression was associated with increased activity of GLUT1 in the cell membrane that contributes to the impairment of the RPE secretory function of PEDF. PMID:27440994

  16. GM-CSF Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury

    PubMed Central

    Huynh, Larry; Marlier, Arnaud; Lee, Yashang; Moeckel, Gilbert W.; Cantley, Lloyd G.

    2015-01-01

    After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4–stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury. PMID:25388222

  17. Identification of Novel Listeria monocytogenes Secreted Virulence Factors following Mutational Activation of the Central Virulence Regulator, PrfA▿ †

    PubMed Central

    Port, Gary C.; Freitag, Nancy E.

    2007-01-01

    Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis. PMID:17938228

  18. 1,25-dihydroxyvitamin D3 stimulates transforming growth factor-beta1 synthesis by mouse renal proximal tubular cells.

    PubMed

    Weinreich, T; Landolt, M; Booy, C; Wüthrich, R; Binswanger, U

    1999-01-01

    1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] is a secosteroid hormone with effects on cell growth, differentiation and immunoregulatory functions in a number of tissues not primarily involved in mineral metabolism. We recently demonstrated growth-regulating effects of 1, 25-(OH)2 D3 on human mesangial cells and proximal tubular cells. To investigate whether 1,25-(OH)2 D3 might also affect the synthesis of cytokines and growth factors in proximal tubular cells, we assessed in the present study the expression and secretion of transforming growth factor-beta1 (TGF-beta1) in a mouse proximal tubular cell line (MCT) in vitro. TGF-beta1 synthesis was measured by a monospecific ELISA in culture supernatant. The secreted TGF-beta1 was proven to be biologically active by means of a bioassay system (CCL-64 mink lung epithelial cell proliferation assay). TGF-beta1 gene expression was assessed by RT-PCR. To analyze whether TGF-beta1 expression mediates the 1,25-(OH)2 D3-induced antiproliferative actions in MCT, proliferation studies in the absence or presence of a blocking monoclonal anti TGF-beta1-3 antibody were performed. 1, 25-(OH)2 D3 (10(-11) to 10(-7) M) specifically increased the TGF-beta1 protein secretion in MCT with a maximum at 10(-8) M. No detectable effect was found with 25 D3 at 10 times higher concentrations. A synthetic 20-epi analogue, MC 1288, increased TGF-beta1 secretion up to similar amounts at equimolar concentrations as the natural hormone 1,25-(OH)2 D3. Steady-state TGF-beta1 mRNA concentration in MCT was transiently increased by 1, 25-(OH)2 D3 between 12 and 24 h, returning to control values at 48 h. Blocking TGF-beta1 did not reduce or abrogate the antiproliferative effect of 1,25-(OH)2 D3. In conclusion, 1,25-(OH)2 D3 stimulates TGF-beta1 expression in renal proximal tubular cells, a growth factor with anti-inflammatory and profibrotic actions which plays an important role in the development and progression of nephrosclerosis. PMID:10394107

  19. Tubular Overexpression of Angiopoietin-1 Attenuates Renal Fibrosis

    PubMed Central

    Lee, Heedoo; Kim, Yeawon; Liu, Tuoen; Guo, Qiusha; Geminiani, Julio J.; Austin, Paul F.; Chen, Ying Maggie

    2016-01-01

    Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor β1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans. PMID:27454431

  20. The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis

    PubMed Central

    Chen, Hengshi; Zhang, Lingling; Chang, Yan; Yan, Shangxue; Dai, Xing; Ma, Yang; Huang, Qiong; Wei, Wei

    2016-01-01

    Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1β, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4+CD69+, CD4+CD154+) and activated B cell subsets (CD19+CD23+, CD19+CD21+, CD19+IgD+ and CD19-CD138+) were increased by IgD. The percentage of unactivated T cell subset (CD4+CD62L+) and immature B cell subset (CD19+IgM+IgD-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA. PMID:26814717

  1. Device for inserting tubular members together

    SciTech Connect

    Milberger, L.J.

    1992-03-17

    This patent describes a well, a lower tubular member with a sealing surface located in the well, an upper tubular member which inserts into engagement with the lower tubular member during running in, the upper and lower tubular members being exposed to well fluid pressure, an improved means for sliding the upper tubular member into engagement with the lower tubular member. It comprises the upper tubular member having a first side and a second side, the second side having a sealing section which mates with the sealing surface of the lower tubular sidewall; axially spaced apart seal means located on the running tool sidewall for sealingly engaging the first side of the upper tubular member above and below the sealing section during running in, for defining a low pressure area between the running tool and the first side which is isolated from the well fluid pressure; the sealing section of the upper tubular member being exposed to well fluid pressure during running in, resulting in a pressure difference across the upper tubular member between the first side of the tubular member and the sealing section, means for eliminating the pressure difference across the upper tubular member between the first side and the sealing section after the upper tubular member has reached its engaged position with the lower tubular member, allowing the sealing section to move radially into engagement with the sealing surface. This patent also describes a method for sliding an upper tubular member into engagement with a sealing surface of a lower tubular member in a well having well fluid pressure, comprising in combination: providing the upper tubular member with a first side and a second side and providing the second side with a sealing section for mating with the sealing surface of the lower tubular member.

  2. Molecular Pathophysiology of Renal Tubular Acidosis

    PubMed Central

    Pereira, P.C.B; Miranda, D.M; Oliveira, E.A; Silva, A.C. Simões e

    2009-01-01

    Renal tubular acidosis (RTA) is characterized by metabolic acidosis due to renal impaired acid excretion. Hyperchloremic acidosis with normal anion gap and normal or minimally affected glomerular filtration rate defines this disorder. RTA can also present with hypokalemia, medullary nephrocalcinosis and nephrolitiasis, as well as growth retardation and rickets in children, or short stature and osteomalacia in adults. In the past decade, remarkable progress has been made in our understanding of the molecular pathogenesis of RTA and the fundamental molecular physiology of renal tubular transport processes. This review summarizes hereditary diseases caused by mutations in genes encoding transporter or channel proteins operating along the renal tubule. Review of the molecular basis of hereditary tubulopathies reveals various loss-of-function or gain-of-function mutations in genes encoding cotransporter, exchanger, or channel proteins, which are located in the luminal, basolateral, or endosomal membranes of the tubular cell or in paracellular tight junctions. These gene mutations result in a variety of functional defects in transporter/channel proteins, including decreased activity, impaired gating, defective trafficking, impaired endocytosis and degradation, or defective assembly of channel subunits. Further molecular studies of inherited tubular transport disorders may shed more light on the molecular pathophysiology of these diseases and may significantly improve our understanding of the mechanisms underlying renal salt homeostasis, urinary mineral excretion, and blood pressure regulation in health and disease. The identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders. PMID:19721811

  3. Roles of Akt and SGK1 in the Regulation of Renal Tubular Transport.

    PubMed

    Satoh, Nobuhiko; Nakamura, Motonobu; Suzuki, Masashi; Suzuki, Atsushi; Seki, George; Horita, Shoko

    2015-01-01

    A serine/threonine kinase Akt is a key mediator in various signaling pathways including regulation of renal tubular transport. In proximal tubules, Akt mediates insulin signaling via insulin receptor substrate 2 (IRS2) and stimulates sodium-bicarbonate cotransporter (NBCe1), resulting in increased sodium reabsorption. In insulin resistance, the IRS2 in kidney cortex is exceptionally preserved and may mediate the stimulatory effect of insulin on NBCe1 to cause hypertension in diabetes via sodium retention. Likewise, in distal convoluted tubules and cortical collecting ducts, insulin-induced Akt phosphorylation mediates several hormonal signals to enhance sodium-chloride cotransporter (NCC) and epithelial sodium channel (ENaC) activities, resulting in increased sodium reabsorption. Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates aldosterone signaling. Insulin can stimulate SGK1 to exert various effects on renal transporters. In renal cortical collecting ducts, SGK1 regulates the expression level of ENaC through inhibition of its degradation. In addition, SGK1 and Akt cooperatively regulate potassium secretion by renal outer medullary potassium channel (ROMK). Moreover, sodium-proton exchanger 3 (NHE3) in proximal tubules is possibly activated by SGK1. This review focuses on recent advances in understanding of the roles of Akt and SGK1 in the regulation of renal tubular transport. PMID:26491696

  4. Roles of Akt and SGK1 in the Regulation of Renal Tubular Transport

    PubMed Central

    Satoh, Nobuhiko; Nakamura, Motonobu; Suzuki, Masashi; Suzuki, Atsushi; Seki, George; Horita, Shoko

    2015-01-01

    A serine/threonine kinase Akt is a key mediator in various signaling pathways including regulation of renal tubular transport. In proximal tubules, Akt mediates insulin signaling via insulin receptor substrate 2 (IRS2) and stimulates sodium-bicarbonate cotransporter (NBCe1), resulting in increased sodium reabsorption. In insulin resistance, the IRS2 in kidney cortex is exceptionally preserved and may mediate the stimulatory effect of insulin on NBCe1 to cause hypertension in diabetes via sodium retention. Likewise, in distal convoluted tubules and cortical collecting ducts, insulin-induced Akt phosphorylation mediates several hormonal signals to enhance sodium-chloride cotransporter (NCC) and epithelial sodium channel (ENaC) activities, resulting in increased sodium reabsorption. Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates aldosterone signaling. Insulin can stimulate SGK1 to exert various effects on renal transporters. In renal cortical collecting ducts, SGK1 regulates the expression level of ENaC through inhibition of its degradation. In addition, SGK1 and Akt cooperatively regulate potassium secretion by renal outer medullary potassium channel (ROMK). Moreover, sodium-proton exchanger 3 (NHE3) in proximal tubules is possibly activated by SGK1. This review focuses on recent advances in understanding of the roles of Akt and SGK1 in the regulation of renal tubular transport. PMID:26491696

  5. Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

    PubMed Central

    Shaffer, Carrie L.; Good, James A. D.; Kumar, Santosh; Krishnan, K. Syam; Gaddy, Jennifer A.; Loh, John T.; Chappell, Joseph; Almqvist, Fredrik

    2016-01-01

    ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. PMID:27118587

  6. Cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and by stimulating insulin secretion in vitro.

    PubMed

    Hafizur, Rahman M; Hameed, Abdul; Shukrana, Mishkat; Raza, Sayed Ali; Chishti, Sidra; Kabir, Nurul; Siddiqui, Rehan A

    2015-02-15

    Although the anti-diabetic activity of cinnamic acid, a pure compound from cinnamon, has been reported but its mechanism(s) is not yet clear. The present study was designed to explore the possible mechanism(s) of anti-diabetic activity of cinnamic acid in in vitro and in vivo non-obese type 2 diabetic rats. Non-obese type 2 diabetes was developed by injecting 90 mg/kg streptozotocin in 2-day-old Wistar pups. Cinnamic acid and cinnamaldehyde were administered orally to diabetic rats for assessing acute blood glucose lowering effect and improvement of glucose tolerance. Additionally, insulin secretory activity of cinnamic acid and cinnamaldehyde was evaluated in isolated mice islets. Cinnamic acid, but not cinnamaldehyde, decreased blood glucose levels in diabetic rats in a time- and dose-dependent manner. Oral administration of cinnamic acid with 5 and 10 mg/kg doses to diabetic rats improved glucose tolerance in a dose-dependent manner. The improvement by 10 mg/kg cinnamic acid was comparable to that of standard drug glibenclamide (5 mg/kg). Further in vitro studies showed that cinnamaldehyde has little or no effect on glucose-stimulated insulin secretion; however, cinnamic acid significantly enhanced glucose-stimulated insulin secretion in isolated islets. In conclusion, it can be said that cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and stimulating insulin secretion in vitro.

  7. Serotonin has kinin-like activity in stimulating secretion by Malpighian tubules of the house cricket Acheta domesticus.

    PubMed

    Coast, Geoffrey

    2011-03-01

    Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC(50) of 9.4 nmol L(-1), although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca(2+) from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT(2)-like receptor.

  8. Albumin Is Recycled from the Primary Urine by Tubular Transcytosis

    PubMed Central

    Tenten, Verena; Menzel, Sylvia; Kunter, Uta; Sicking, Eva-Maria; van Roeyen, Claudia R. C.; Sanden, Silja K.; Kaldenbach, Michaela; Boor, Peter; Fuss, Astrid; Uhlig, Sandra; Lanzmich, Regina; Willemsen, Brigith; Dijkman, Henry; Grepl, Martin; Wild, Klemens; Kriz, Wilhelm; Smeets, Bart; Floege, Jürgen

    2013-01-01

    Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact. PMID:23970123

  9. Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases

    PubMed Central

    2013-01-01

    Background Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. Methods In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. Results B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing

  10. Cadmium effects on hypothalamic activity and pituitary hormone secretion in the male.

    PubMed

    Lafuente, A; Esquifino, A I

    1999-11-22

    Cadmium specifically modify amine metabolism at the central nervous system and pituitary hormone secretions. Thus, the physiological functions controlled by these hormones can be modulated by cadmium. This xenobiotic is associated with deleterious effects on the gonadal function and with changes in the secretory pattern of other pituitary hormones like prolactin, ACTH, GH or TSH. The observed changes in pituitary hormone secretion do not correlate with the modifications of central nervous system metabolism of the neurotransmitters involved in their regulation. The accumulative data indicates the existence of a disruption in the regulatory mechanisms of the hypothalamic-pituitary axis. The physiological significance of these effects remains to be elucidated.

  11. Transcriptional profiling of Vibrio parahaemolyticus exsA reveals a complex activation network for type III secretion

    PubMed Central

    Liu, Aaron C.; Thomas, Nikhil A.

    2015-01-01

    Vibrio parahaemolyticus (Vp) is a marine halophilic bacterium that is commonly associated with oysters and shrimp. Human consumption of contaminated shellfish can result in Vp mediated gastroenteritis and severe diarrheal disease. Vp encodes two type 3 secretion systems (T3SS-1 and T3SS2) that have been functionally implicated in cytotoxicity and enterotoxicity respectively. In this study, we profiled protein secretion and temporal promoter activities associated with exsA and exsB gene expression. exsA is an AraC-like transcriptional activator that is critical for activating multiple operons that encode T3SS-1 genes, whereas exsB is thought to encode an outer membrane pilotin component for T3SS-1. The exsBA genetic locus has two predicted promoter elements. The predicted exsB and exsA promoters were individually cloned upstream of luxCDABE genes in reporter plasmid constructs allowing for in situ, real-time quantitative light emission measurements under many growth conditions. Low calcium growth conditions supported maximal exsB and exsA promoter activation. exsB promoter activity exhibited high basal activity and resulted in an exsBA co-transcript. Furthermore, a separate proximal exsA promoter showed initial low basal activity yet eventually exceeded that of exsB and reached maximal levels after 2.5 h corresponding to an entry into early log phase. exsA promoter activity was significantly higher at 30°C than 37°C, which also coincided with increased secretion levels of specific T3SS-1 effector proteins. Lastly, bioinformatic analyses identified a putative expanded ExsA binding motif for multiple transcriptional operons. These findings suggest a two wave model of Vp T3SS-I induction that integrates two distinct promoter elements and environmental signals into a complex ExsA activation framework. PMID:26539165

  12. The rebirth of interest in renal tubular function.

    PubMed

    Lowenstein, Jerome; Grantham, Jared J

    2016-06-01

    The measurement of glomerular filtration rate by the clearance of inulin or creatinine has evolved over the past 50 years into an estimated value based solely on plasma creatinine concentration. We have examined some of the misconceptions and misunderstandings of the classification of renal disease and its course, which have followed this evolution. Furthermore, renal plasma flow and tubular function, which in the past were estimated by the clearance of the exogenous aryl amine, para-aminohippurate, are no longer measured. Over the past decade, studies in experimental animals with reduced nephron mass and in patients with reduced renal function have identified small gut-derived, protein-bound uremic retention solutes ("uremic toxins") that are poorly filtered but are secreted into the lumen by organic anion transporters (OATs) in the proximal renal tubule. These are not effectively removed by conventional hemodialysis or peritoneal dialysis. Residual renal function, urine produced in patients with advanced renal failure or undergoing dialysis treatment, may represent, at least in part, secretion of fluid and uremic toxins, such as indoxyl sulfate, mediated by proximal tubule OATs and might serve as a useful survival function. In light of this new evidence of the physiological role of proximal tubule OATs, we suggest that measurement of renal tubular function and renal plasma flow may be of considerable value in understanding and managing chronic kidney disease. Data obtained in normal subjects indicate that renal plasma flow and renal tubular function might be measured by the clearance of the endogenous aryl amine, hippurate.

  13. The rebirth of interest in renal tubular function.

    PubMed

    Lowenstein, Jerome; Grantham, Jared J

    2016-06-01

    The measurement of glomerular filtration rate by the clearance of inulin or creatinine has evolved over the past 50 years into an estimated value based solely on plasma creatinine concentration. We have examined some of the misconceptions and misunderstandings of the classification of renal disease and its course, which have followed this evolution. Furthermore, renal plasma flow and tubular function, which in the past were estimated by the clearance of the exogenous aryl amine, para-aminohippurate, are no longer measured. Over the past decade, studies in experimental animals with reduced nephron mass and in patients with reduced renal function have identified small gut-derived, protein-bound uremic retention solutes ("uremic toxins") that are poorly filtered but are secreted into the lumen by organic anion transporters (OATs) in the proximal renal tubule. These are not effectively removed by conventional hemodialysis or peritoneal dialysis. Residual renal function, urine produced in patients with advanced renal failure or undergoing dialysis treatment, may represent, at least in part, secretion of fluid and uremic toxins, such as indoxyl sulfate, mediated by proximal tubule OATs and might serve as a useful survival function. In light of this new evidence of the physiological role of proximal tubule OATs, we suggest that measurement of renal tubular function and renal plasma flow may be of considerable value in understanding and managing chronic kidney disease. Data obtained in normal subjects indicate that renal plasma flow and renal tubular function might be measured by the clearance of the endogenous aryl amine, hippurate. PMID:26936872

  14. Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor.

    PubMed

    Shi, Daning; Hou, Xiaojuan; Wang, Lei; Gao, Yitian; Wu, Di; Xi, Xinping; Zhou, Mei; Kwok, Hang Fai; Duan, Jinao; Chen, Tianbao; Shaw, Chris

    2016-01-01

    The dermaseptin antimicrobial peptide family contains members of 27-34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5'-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested. PMID:27187467

  15. Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

    PubMed Central

    Shi, Daning; Hou, Xiaojuan; Wang, Lei; Gao, Yitian; Wu, Di; Xi, Xinping; Zhou, Mei; Kwok, Hang Fai; Duan, Jinao; Chen, Tianbao; Shaw, Chris

    2016-01-01

    The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested. PMID:27187467

  16. Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip

    PubMed Central

    Murillo, I.; Martinez-Argudo, I.; Blocker, A. J.

    2016-01-01

    Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB’s role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction. PMID:27277624

  17. Leptin Suppression of Insulin Secretion by the Activation of ATP-Sensitive K+ Channels in Pancreatic β-Cells

    PubMed Central

    Kieffer, Timothy J.; Heller, R. Scott; Leech, Colin A.; Holz, George G.; Habener, Joel F.

    2010-01-01

    In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that β-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting β-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the β-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob β-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob β-cells demonstrated that leptin activated ATP-sensitive potassium channels (KATP) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of KATP. Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the β-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM. PMID:9166685

  18. Activation and inhibition of calcium-dependent histamine secretion by ATP ions applied to rat mast cells.

    PubMed Central

    Cockcroft, S; Gomperts, B D

    1979-01-01

    1. The concentration dependence on ATP of mast cell histamine secretion in the presence of various concentrations of Mg2+ and Ca2+ confirms that the agonist form of ATP is the free form of ATP (ATP(free) not bound to divalent cations, i.e. ATP4-. It induces 50% activation at about 1.2 microM, maximal secretion at about 2.7 microM and 50% self-inhibition at about 4.4 microM. 2. The divalent cations Mg2+ and Ca2+ were used to buffer ATP(tree) in the range 1-8 microM in the presence of much higher concentrations of ATP(total). In addition to its effect as a buffer for ATP, Ca2+ is required for secretion. 3. With ATP(free) at 1 microM, the time-course of histamine secretion is characterized by a delay of about 10 min before secretion commences. With increasing concentration of ATP(free) the delay becomes shorter (less than 5 min with ATP(free) at 2 microM). 4. Secretion commences promptly on addition of Ca2+ to cells which have been pretreated with low concentrations of ATP(free) (less than 2 microM). This observation suggests that the delay normally observed represents the time taken for Ca2+ sensitivity to develop (i.e. probably the time taken for Ca2+ channels to open). 5. Late addition of Ca2+ to cells pretreated with higher concentrations of ATP(free) (greater than 2 microM) results in a reduced amount of histamine secretion compared with that which normally occurs. This reduction (which increases with time of exposure to ATP) and the self-inhibition due to higher concentrations of ATP(free) may be two facets of a common inhibitory mechanism. 6. These results are discussed in the light of other experiments which show that mast cells treated with ATP(free) at self-inhibitory concentrations become permeable to phosphorylated metabolites and nucleotides. PMID:93638

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

    PubMed Central

    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

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

    PubMed

    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

  1. Medullary nephrocalcinosis, distal renal tubular acidosis and polycythaemia in a patient with nephrotic syndrome

    PubMed Central

    2012-01-01

    Background Medullary nephrocalcinosis and distal renal tubular acidosis are closely associated and each can lead to the other. These clinical entities are rare in patients with nephrotic syndrome and polycythaemia is an unusual finding in such patients. We describe the presence of medullary nephrocalcinosis, distal renal tubular acidosis and polycythaemia in a patient with nephrotic syndrome due to minimal change disease. Proposed mechanisms of polycythaemia in patients with nephrotic syndrome and distal renal tubular acidosis include, increased erythropoietin production and secretion of interleukin 8 which in turn stimulate erythropoiesis. Case presentation A 22 year old Sri Lankan Sinhala male with nephrotic syndrome due to minimal change disease was investigated for incidentally detected polycythaemia. Investigations revealed the presence of renal tubular acidosis type I and medullary nephrocalcinosis. Despite extensive investigation, a definite cause for polycythaemia was not found in this patient. Treatment with potassium and bicarbonate supplementation with potassium citrate led to correction of acidosis thereby avoiding the progression of nephrocalcinosis and harmful effects of chronic acidosis. Conclusion The constellation of clinical and biochemical findings in this patient is unique but the pathogenesis of erythrocytosis is not clearly explained. The proposed mechanisms for erythrocytosis in other patients with proteinuria include increased erythropoietin secretion due to renal hypoxia and increased secretion of interleukin 8 from the kidney. This case illustrates that there may exist hitherto unknown connections between tubular and glomerular dysfunction in patients with nephrotic syndrome. PMID:22834973

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

    PubMed

    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.

  3. STAT3 and STAT6 Signaling Pathways Synergize to Promote Cathepsin Secretion from Macrophages via IRE1α Activation.

    PubMed

    Yan, Dongyao; Wang, Hao-Wei; Bowman, Robert L; Joyce, Johanna A

    2016-09-13

    Tumor-associated macrophages play critical roles during tumor progression by promoting angiogenesis, cancer cell proliferation, invasion, and metastasis. Cysteine cathepsin proteases, produced by macrophages and cancer cells, modulate these processes, but it remains unclear how these typically lysosomal enzymes are regulated and secreted within the tumor microenvironment. Here, we identify a STAT3 and STAT6 synergy that potently upregulates cathepsin secretion by macrophages via engagement of an unfolded protein response (UPR) pathway. Whole-genome expression analyses revealed that the TH2 cytokine interleukin (IL)-4 synergizes with IL-6 or IL-10 to activate UPR via STAT6 and STAT3. Pharmacological inhibition of the UPR sensor IRE1α blocks cathepsin secretion and blunts macrophage-mediated cancer cell invasion. Similarly, genetic deletion of STAT3 and STAT6 signaling components impairs tumor development and invasion in vivo. Together, these findings demonstrate that cytokine-activated STAT3 and STAT6 cooperate in macrophages to promote a secretory phenotype that enhances tumor progression in a cathepsin-dependent manner. PMID:27626662

  4. Anticonvulsant and analgesic activities of crude extract and its fractions of the defensive secretion from the Mediterranean sponge, Spongia officinalis

    PubMed Central

    2012-01-01

    This study progresses in the direction of identifying component(s) from the Mediterranean sponge, Spongia officinalis with anticonvulsant and analgesic activities. We investigated the efficacy of crude extract and its semi-purified fractions (F1-F3) of the defensive secretion from Spongia officinalis for their in vivo anticonvulsant activity using the pentylenetetrazole (PTZ) seizure model and analgesic activity using the writhing test in mice. Among the series the crude extract exhibited interesting analgesic activity in a dose dependent manner. Similarly the fraction F2 showed a partial protection of mice from PTZ-induced seizure and interesting analgesic activity in a dose dependent manner. The purification and the determination of chemical structure(s) of compound(s) of this active fraction are under investigation. PMID:22494441

  5. Single-particle to single-particle transformation of an active type organic μ-tubular homo-structure photonic resonator into a passive type hetero-structure resonator.

    PubMed

    Venkataramudu, Uppari; Venkatakrishnarao, Dasari; Chandrasekhar, Naisa; Mohiddon, Mahamad Ahamad; Chandrasekar, Rajadurai

    2016-06-21

    Self-assembled hexagonal organic submicrotubes, upon electronic excitation with an UV laser, display an active type polarized whispering gallery mode (WGM) resonance in the visible (Vis) range (400-600 nm). Due to the photonic cavity effect the tubes show fluorescence (FL) signal intensity 5× greater than the corresponding powder state. Furthermore, the same tubes, which are passive to a visible laser, produce yellow-orange emitting carbonaceous lumps when burnt with an intense laser beam (42 mW) forming a chemically binary heterogeneous structure. The hetero-structure upon excitation with a visible laser at the passive tubular part showed emission in the Vis-Near infrared (NIR) range (500-800 nm) with WGMs thus producing a passive/active type hetero-structure photonic resonator.

  6. A Simple Tubular Reactor Experiment.

    ERIC Educational Resources Information Center

    Hudgins, Robert R.; Cayrol, Bertrand

    1981-01-01

    Using the hydrolysis of crystal violet dye by sodium hydroxide as an example, the theory, apparatus, and procedure for a laboratory demonstration of tubular reactor behavior are described. The reaction presented can occur at room temperature and features a color change to reinforce measured results. (WB)

  7. Tubular cystourethroneostomy after total prostatectomy.

    PubMed

    Melchior, H

    1975-01-01

    After radical prostatectomy cystourethroneostomy is done as a tubular cystourethroplasty. In the last 13 months 14 patients have been operated on in this manner. In 12 patients continence was achieved; 2 patients had a temporary stress incontinence. The stress incontinence could be treated successfully by temporary electrostimulation of the pelvic floor by an anal plug stimulator.

  8. METHOD OF FABRICATING TUBULAR UNITS

    DOEpatents

    Ohlinger, L.A.

    1961-06-20

    A process is described for making a fuel element comprising a tubular jacket and fuel slugs held by the jacket in longitudinally spaced relation to one another. The jacket is lengthened as a result of being drawn down to grip the fuel slugs. As an intentional incident to this operation, the fuel slugs become longitudinally spaced from one another.

  9. Tubular solid oxide fuel cell development program

    SciTech Connect

    1995-08-01

    This paper presents an overview of the Westinghouse Solid Oxide Fuel Cell (SOFC) development activities and current program status. The Westinghouse goal is to develop a cost effective cell that can operate for 50,000 to 100,000 hours. Progress toward this goal will be discussed and test results presented for multiple single cell tests which have now successfully exceeded 56,000 hours of continuous power operation at temperature. Results of development efforts to reduce cost and increase power output of tubular SOFCs are described.

  10. Development of tubular SOFC using metallic substrate

    SciTech Connect

    Nagata, S.; Okuo, T.; Kaga, Y.; Kasuga, Y.; Momma, K.; Tsukamoto, K.; Uchiyama, F.

    1995-12-31

    The tubular SOFCs using porous metallic substrates have been developed. The substrates can act as excellent fuel electrodes of low activation polarizations by baking fine Ni layers on them. These substrates can be compatible with other materials composing SOFCs in life tests including sudden and scheduled interruptions. The cells were fabricated by the combined method of the spray process and the wet process. In the life test at 1,198K, the power density of 0.3W/cm{sup 2} was kept over 3,000 hours at the current density of 0.4A/cm{sup 2}.

  11. Distal Renal Tubular Acidosis and Calcium Nephrolithiasis

    NASA Astrophysics Data System (ADS)

    Moe, Orson W.; Fuster, Daniel G.; Xie, Xiao-Song

    2008-09-01

    Calcium stones are commonly encountered in patients with congenital distal renal tubular acidosis, a disease of renal acidification caused by mutations in either the vacuolar H+-ATPase (B1 or a4 subunit), anion exchanger-1, or carbonic anhydrase II. Based on the existing database, we present two hypotheses. First, heterozygotes with mutations in B1 subunit of H+-ATPase are not normal but may harbor biochemical abnormalities such as renal acidification defects, hypercalciuria, and hypocitraturia which can predispose them to kidney stone formation. Second, we propose at least two mechanisms by which mutant B1 subunit can impair H+-ATPase: defective pump assembly and defective pump activity.

  12. Substrate-Activated Conformational Switch on Chaperones Encodes aTargeting Signal in Type III Secretion

    PubMed Central

    Chen, Li; Ai, Xuanjun; Portaliou, Athina G.; Minetti, Conceicao A.S.A.; Remeta, David P.; Economou, Anastassios; Kalodimos, Charalampos G.

    2013-01-01

    SUMMARY Targeting of type III secretion proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone CesAB to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB, but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB–EspA complex and EscN resulted in severe secretion and infection defects. We further show that the targeting and secretion signals are distinct and the two processes are likely regulated by different mechanisms. PMID:23523349

  13. Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria.

    PubMed

    Turner, Mark S; Waldherr, Florian; Loessner, Martin J; Giffard, Philip M

    2007-01-01

    The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

  14. Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha.

    PubMed

    Agaphonov, Michael O; Sokolov, Sviatoslav S; Romanova, Nina V; Sohn, Jung-Hoon; Kim, So-Young; Kalebina, Tatyana S; Choi, Eui-Sung; Ter-Avanesyan, Michael D

    2005-10-15

    Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal. PMID:16200504

  15. Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows.

    PubMed

    Denis, Michel; Parlane, Natalie A; Lacy-Hulbert, S Jane; Summers, Emma L; Buddle, Bryce M; Wedlock, D Neil

    2006-11-15

    to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.

  16. Power generation characteristics of tubular type SOFC by wet process

    SciTech Connect

    Tajiri, H.; Nakayama, T.; Kuroishi, M.

    1996-12-31

    The development of a practical solid oxide fuel cell requires improvement of a cell performance and a cell manufacturing technology suitable for the mass production. In particular tubular type SOFC is thought to be superior in its reliability because its configuration can avoid the high temperature sealing and reduce the thermal stress resulting from the contact between cells. The authors have fabricated a tubular cell with an air electrode support by a wet processing technique, which is suitable for mass production in improving a power density. To enhance the power output of the module, the Integrated Tubular-Type (ITT) cell has been developed. This paper reports the performance of the single cells with various active anode areas and the bundle with series-connected 9-ITT cells with an active anode area of 840 cm{sup 2}.

  17. Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

    PubMed

    Sala-Rabanal, Monica; Yurtsever, Zeynep; Nichols, Colin G; Brett, Tom J

    2015-03-17

    Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

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

    PubMed

    Cunningham, Katherine A; Bouagnon, Aude D; Barros, Alexandre G; Lin, Lin; Malard, Leandro; Romano-Silva, Marco Aurélio; Ashrafi, Kaveh

    2014-06-01

    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. PMID:24921650

  19. Immunoglobulin G subclasses secreted by human B cells in vitro in response to interleukin-2 and polyclonal activators.

    PubMed Central

    Calvert, J E; Johnstone, R; Duggan-Keen, M F; Bird, P

    1990-01-01

    The IgG subclasses secreted by human B cells in vitro in response to IL-2 have been analysed. B cells were prepared from tonsil, blood and spleen, and cultured with recombinant IL-2 in the presence or absence of two polyclonal activators: Staphylococcus aureus Cowan 1 (SAC) and bacterial lipopolysaccharide (LPS). Secretion of all four subclasses and of IgM was stimulated by IL-2, but the relative amounts varied according to (i) the tissue source of the B cells, and (ii) which polyclonal activator was used. The amount of IgG1 tended to be higher and IgG2 tended to be lower when SAC was the polyclonal activator (compared to LPS). This difference was most marked for tonsil B cells, and it was found that SAC had a negative effect on secretion of IgM and IgG2 in these cultures, whilst synergizing with IL-2 to stimulate the production of IgG1, 3 and 4. When the degree of stimulation of different pairs of isotypes was analysed, several interesting positive correlations emerged. In tonsil B-cell cultures, stimulation of IgM and IgG2 was linked with each other, but not with IgG1, whilst in blood B-cell cultures all isotypes appeared to be stimulated co-ordinately. Stimulation of IgG1 and IgG3 were positively correlated in cultures of B cells from all tissues. The results emphasize that the effects of a single cytokine on immunoglobulin isotype production can be influenced by the source of the B cells, and by other signals delivered to the cells. PMID:2373516

  20. Interaction of Ca(2+)-dependent activator protein for secretion 1 (CAPS1) with septin family proteins in mouse brain.

    PubMed

    Hosono, Mayu; Shinoda, Yo; Hirano, Touko; Ishizaki, Yasuki; Furuichi, Teiichi; Sadakata, Tetsushi

    2016-03-23

    The Ca(2+)-dependent activator protein for secretion 1 (CAPS1) protein plays a regulatory role in the dense-core vesicle exocytosis pathway. To clarify the functions of this protein in the brain, we searched for novel interaction partners of CAPS1 by mass spectrometry. We identified a specific interaction of CAPS1 with septin family proteins. We also demonstrated that the C-terminal region of the CAPS1 protein binds to part of the deduced GTP-binding domain of septin proteins. It is possible that a tertiary complex of septin, CAPS, and syntaxin contributes to dense-core vesicle trafficking and exocytosis in neurons.

  1. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

  2. The Pseudomonas aeruginosa Type VI Secretion PGAP1-like Effector Induces Host Autophagy by Activating Endoplasmic Reticulum Stress.

    PubMed

    Jiang, Feng; Wang, Xia; Wang, Bei; Chen, Lihong; Zhao, Zhendong; Waterfield, Nicholas R; Yang, Guowei; Jin, Qi

    2016-08-01

    Pseudomonas aeruginosa is an opportunistic pathogen that regularly causes nosocomial infections in hospitalized patients. The type VI secretion system (T6SS) is responsible for the secretion of numerous virulence effector proteins that can both interfere with competing microbes and manipulate host cells. Here, we report a detailed investigation of a P. aeruginosa H2-T6SS-dependent phospholipase effector, TplE, which acts as a trans-kingdom toxin. Delivery of TplE to the periplasmic space of rival bacteria leads to growth inhibition. Importantly, TplE, also contains a eukaryotic PGAP1-like domain, which targets the host ER apparatus, ultimately leading to disruption of the ER. TplE activity leads to the activation of the unfolded protein response (UPR) through the IRE1α-XBP1 pathway, enhancing autophagic flux. These findings indicate that this T6SS-delivered phospholipase effector is active against both prokaryotic and eukaryotic cellular targets, highlighting the T6SS as a versatile weapon in the Pseudomonas arsenal. PMID:27477276

  3. The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis.

    PubMed

    Sieben, S; Hertle, R; Gumpert, J; Braun, V

    1998-10-01

    The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.

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

    PubMed

    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.

  5. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  6. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.

    PubMed

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-10-16

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

  7. Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets.

    PubMed

    Takahashi, Rui; Ishihara, Hisamitsu; Tamura, Akira; Yamaguchi, Suguru; Yamada, Takahiro; Takei, Daisuke; Katagiri, Hideki; Endou, Hitoshi; Oka, Yoshitomo

    2006-02-01

    Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.

  8. Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression.

    PubMed Central

    da Silva Xavier, Gabriela; Leclerc, Isabelle; Varadi, Aniko; Tsuboi, Takashi; Moule, S Kelly; Rutter, Guy A

    2003-01-01

    AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet beta-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023-4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 beta-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPK alpha 1.T(172)D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca(2+)] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPK alpha 1.D(157)A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca(2+)], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the beta-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus. PMID:12589707

  9. Chronic activation of central AMPK attenuates glucose-stimulated insulin secretion and exacerbates hepatic insulin resistance in diabetic rats.

    PubMed

    Park, Sunmin; Kim, Da Sol; Kang, Suna; Shin, Bae Keun

    2014-09-01

    We investigated the effects of chronic AMP-activated kinase (AMPK) activation in the hypothalamus on energy and glucose metabolism in 90% pancreatectomized diabetic rats. Diabetic rats fed a high fat diet were divided into 3 groups and intracerebroventricular (ICV) administered with one of the following: 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, AMPK activator; 80 μg/day), AICAR+compound C (AMPK inhibitor; 6.2 μg/day), or an artificial cerebrospinal fluid (control) by means of osmotic pumps for 4 weeks. In the hypothalamus, central AICAR activated the phosphorylation of AMPK whereas adding compound C suppressed the activation. AICAR increased body weight and epididymal and retroperitoneal fat mass by increasing energy intake for the first 2 weeks and decreasing energy expenditure, whereas compound C reversed the AICAR effect on energy metabolism. Indirect calorimetry revealed that ICV-AICAR decreased carbohydrate oxidation, but not fat oxidation, compared to the control. During euglycemic hyperinsulinemic clamp, central AICAR increased hepatic glucose output at hyperinsulinemic states. ICV-AICAR increased expressions of hepatic genes involved in fatty acid synthesis and decreased expression of hepatic genes related to thermogenesis whereas compound C nullified the AICAR effect. Insulin secretion in the first and second phases decreased in AICAR-treated rats at hyperglycemic clamp, but compound C nullified the decrease. However, central AICAR did not alter β-cell mass via its proliferation or apoptosis. In conclusion, chronic hypothalamic AMPK activation impaired energy metabolism and glucose homeostasis by increasing food intake, increasing hepatic glucose output and decreasing insulin secretion in diabetic rats. The impairment of energy and glucose homeostasis by AMPK activation was nullified by an AMPK inhibitor.

  10. Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

    PubMed

    Hendrick, Siobhán M; Mroz, Magdalena S; Greene, Catherine M; Keely, Stephen J; Harvey, Brian J

    2014-09-01

    Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

  11. Open–closed switching of synthetic tubular pores

    PubMed Central

    Kim, Yongju; Kang, Jiheong; Shen, Bowen; Wang, Yanqiu; He, Ying; Lee, Myongsoo

    2015-01-01

    While encouraging progress has been made on switchable nanopores to mimic biological channels and pores, it remains a great challenge to realize long tubular pores with a dynamic open–closed motion. Here we report μm-long, dynamic tubular pores that undergo rapid switching between open and closed states in response to a thermal signal in water. The tubular walls consist of laterally associated primary fibrils stacked from disc-shaped molecules in which the discs readily tilt by means of thermally regulated dehydration of the oligoether chains placed on the wall surfaces. Notably, this pore switching mediates a controlled water-pumping catalytic action for the dehydrative cyclization of adenosine monophosphate to produce metabolically active cyclic adenosine monophosphate. We believe that our work may allow the creation of a variety of dynamic pore structures with complex functions arising from open–closed motion. PMID:26456695

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

    PubMed

    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.

  13. Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K+ channel activity.

    PubMed

    Nomura, Masatoshi; Morinaga, Hidetaka; Zhu, Hai-Lei; Wang, Lixiang; Hasuzawa, Nao; Takayanagi, Ryoichi; Teramoto, Noriyoshi

    2014-07-18

    In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K(+) channels (KATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of KATP channel activity.

  14. Purification of peptides with differential cytolytic activities from the skin secretions of the Central American frog, Lithobates vaillanti (Ranidae).

    PubMed

    Conlon, J Michael; Raza, Haider; Coquet, Laurent; Jouenne, Thierry; Leprince, Jérôme; Vaudry, Hubert; King, Jay D

    2009-08-01

    Peptide-based defenses of ranid frogs from Mexico and Central America have been studied in much less detail than those from North America. Peptides belonging to the brevinin-1 (5 peptides), palustrin-2 (1 peptide), and ranatuerin-2 (3 peptides) families were isolated from norepinephrine-stimulated skin secretions of the Costa Rican frog, Lithobates vaillanti (Ranidae) and characterized structurally. Brevinin-1VLa (FLGAIAGVAAKFLPKVFCFITKKC) and brevinin-1VLc (FLPVIASVAAKVLPK VFCFITKKC) showed particularly high growth-inhibitory potency (MIC < or =3 microM) against a Gram-positive microorganism Staphylococcus aureus and the opportunistic yeast pathogen Candida albicans and potent cytolytic activity (LC(50)< or =8 microM) against both human erythrocytes and HepG2 hepatoma-derived cells. The peptides were also active against a Gram-negative microorganism Escherichia coli (MIC< or =50 microM). Substitutions in brevinin-1VLd (Lys(11) --> Asn) and brevinin-1VLe (Lys(11) --> Ser) that decrease cationicity result in loss of activity against E. coli. Ranatuerin-2VLb (GIMDTIKGAAKDLAGQLLDKLKCKITKC) showed relatively weak antimicrobial activity (MIC> or =75 microM) but selective cytolytic activity against HepG2 tumor cells (LC(50)=30 microM) compared with erythrocytes (LC(50)>200 microM). In addition, a dodecapeptide (RICYAMWIPYPC) were isolated from the secretions that were devoid of antimicrobial activity. This component contains an Ala-Met bond that constitutes the scissile bond in the selective elastase inhibitor, elafin but the peptide did not inhibit pancreatic elastase at concentrations up to 100 microM.

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

    PubMed

    Jacobs, M; Andersen, J B; Kontinen, V; Sarvas, M

    1993-05-01

    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 by the N-terminal pro-sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin-alkaline phosphatase fusion genes in the prsA3 mutant. We found massive degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro-sequence (like subtilisin) and those without (like alpha-amylase).

  16. Spergularia marina Induces Glucagon-Like Peptide-1 Secretion in NCI-H716 Cells Through Bile Acid Receptor Activation

    PubMed Central

    Kim, Kyong; Lee, Yu Mi; Rhyu, Mee-Ra

    2014-01-01

    Abstract Spergularia marina Griseb. (SM) is a halophyte that grows in mud flats. The aerial portions of SM have been eaten as vegetables and traditionally used to prevent chronic diseases in Korea. However, there has been no scientific report that demonstrates the pharmacological effects of SM. Glucagon-like peptide-1 (GLP-1) is important for the maintenance of glucose and energy homeostasis through acting as a signal in peripheral and neural systems. To discover a functional food for regulating glucose and energy homeostasis, we evaluated the effect of an aqueous ethanolic extract (AEE) of SM on GLP-1 release from enteroendocrine NCI-H716 cells. In addition, we explored the Takeda G-protein-coupled receptor 5 (TGR5) agonist activity of AEE-SM in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5. As a result, treatment of NCI-H716 cells with AEE-SM increased GLP-1 secretion and intracellular Ca2+ and cyclic AMP (cAMP) levels in a dose-dependent manner. Transfection of NCI-H716 cells with TGR5-specific small interference RNA inhibited AEE-SM-induced GLP-1 secretion and the increase in Ca2+ and cAMP levels. Moreover, AEE-SM showed that the TGR5 agonist activity in CHO-K1 cells transiently transfected with TGR5. The results suggest that AEE-SM might be a candidate for a functional food to regulate glucose and energy homeostasis. PMID:25260089

  17. Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity

    PubMed Central

    Verdeguer, Francisco; Soustek, Meghan S.; Hatting, Maximilian; Blättler, Sharon M.; McDonald, Devin; Barrow, Joeva J.

    2015-01-01

    Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight. PMID:26503783

  18. Hydathode trichomes actively secreting water from leaves play a key role in the physiology and evolution of root-parasitic rhinanthoid Orobanchaceae

    PubMed Central

    Světlíková, Petra; Hájek, Tomáš; Těšitel, Jakub

    2015-01-01

    Background and Aims Root hemiparasites from the rhinanthoid clade of Orobanchaceae possess metabolically active glandular trichomes that have been suggested to function as hydathode trichomes actively secreting water, a process that may facilitate resource acquisition from the host plant’s root xylem. However, no direct evidence relating the trichomes to water secretion exists, and carbon budgets associated with this energy-demanding process have not been determined. Methods Macro- and microscopic observations of the leaves of hemiparasitic Rhinanthus alectorolophus were conducted and night-time gas exchange was measured. Correlations were examined among the intensity of guttation, respiration and transpiration, and analysis of these correlations allowed the carbon budget of the trichome activity to be quantified. We examined the intensity of guttation, respiration and transpiration, correlations among which indicate active water secretion. Key Results Guttation was observed on the leaves of 50 % of the young, non-flowering plants that were examined, and microscopic observations revealed water secretion from the glandular trichomes present on the abaxial leaf side. Night-time rates of respiration and transpiration and the presence of guttation drops were positively correlated, which is a clear indicator of hydathode trichome activity. Subsequent physiological measurements on older, flowering plants indicated neither intense guttation nor the presence of correlations, which suggests that the peak activity of hydathodes is in the juvenile stage. Conclusions This study provides the first unequivocal evidence for the physiological role of the hydathode trichomes in active water secretion in the rhinanthoid Orobanchaceae. Depending on the concentration of organic elements calculated to be in the host xylem sap, the direct effect of water secretion on carbon balance ranges from close to neutral to positive. However, it is likely to be positive in the xylem-only feeding

  19. Metabolite secretion, Fe(3+)-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869.

    PubMed

    Aguiar, André; Gavioli, Daniela; Ferraz, André

    2014-11-01

    Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m(-)(1)). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m(-)(1)) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m(-)(1)). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe(3+)-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe(3+)-reducing activity, which was restored in the cultures after the first week of biodegradation.

  20. Metabolite secretion, Fe(3+)-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869.

    PubMed

    Aguiar, André; Gavioli, Daniela; Ferraz, André

    2014-11-01

    Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m(-)(1)). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m(-)(1)) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m(-)(1)). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe(3+)-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe(3+)-reducing activity, which was restored in the cultures after the first week of biodegradation. PMID:25442296

  1. Understanding the sequential activation of Type III and Type VI Secretion Systems in Salmonella typhimurium using Boolean modeling

    PubMed Central

    2013-01-01

    Background Three pathogenicity islands, viz. SPI-1 (Salmonella pathogenicity island 1), SPI-2 (Salmonella pathogenicity island 2) and T6SS (Type VI Secretion System), present in the genome of Salmonella typhimurium have been implicated in the virulence of the pathogen. While the regulation of SPI-1 and SPI-2 (both encoding components of the Type III Secretion System - T3SS) are well understood, T6SS regulation is comparatively less studied. Interestingly, inter-connections among the regulatory elements of these three virulence determinants have also been suggested to be essential for successful infection. However, till date, an integrated view of gene regulation involving the regulators of these three secretion systems and their cross-talk is not available. Results In the current study, relevant regulatory information available from literature have been integrated into a single Boolean network, which portrays the dynamics of T3SS (SPI-1 and SPI-2) and T6SS mediated virulence. Some additional regulatory interactions involving a two-component system response regulator YfhA have also been predicted and included in the Boolean network. These predictions are aimed at deciphering the effects of osmolarity on T6SS regulation, an aspect that has been suggested in earlier studies, but the mechanism of which was hitherto unknown. Simulation of the regulatory network was able to recreate in silico the experimentally observed sequential activation of SPI-1, SPI-2 and T6SS. Conclusions The present study integrates relevant gene regulatory data (from literature and our prediction) into a single network, representing the cross-communication between T3SS (SPI-1 and SPI-2) and T6SS. This holistic view of regulatory interactions is expected to improve the current understanding of pathogenesis of S. typhimurium. PMID:24079299

  2. Ernest Henry Starling (1866-1927) on the glomerular and tubular functions of the kidney.

    PubMed

    Fine, Leon G

    2014-01-01

    Around the turn of the 20th century, Ernest Henry Starling (1866-1927) made many fundamental contributions to the understanding of human physiology. With a deep interest in how fluid balance is regulated, he naturally turned to explore the intricacies of kidney function. Early in his career he focused upon the process of glomerular filtration and was able to substantiate the view of Carl Ludwig that this process can be explained entirely upon the basis of hydrostatic and oncotic pressure gradients across the glomerular capillary wall and that the process can be regulated by alterations in the tone of the afferent and efferent arterioles. To explore renal tubular function he employed a heart-lung-kidney model in the dog and was able to infer that certain substances are reabsorbed by the tubules (e.g. sodium chloride) and certain by tubular secretion (e.g. uric acid, indigo carmine dye). By temporarily blocking tubular function using hydrocyanic acid he was able to conclude that secreted substances must be taken up on the peritubular side of the cell and concentrated within the cell to drive the secretory process. Finally, he was able to appreciate that the kidney is an organ which is regulated according to the needs of the organism and that the processes of glomerular filtration, tubular secretion and reabsorption are all subject to regulatory influences, which have evolved to conserve the normal chemical composition of the cells and fluids of the body. PMID:24970544

  3. The activity of candidate virucidal agents, low pH and genital secretions against HIV-1 in vitro.

    PubMed

    O'Connor, T J; Kinchington, D; Kangro, H O; Jeffries, D J

    1995-01-01

    The effect of low pH, normally present in the female genital tract, on HIV viability was examined. HIV is more acid stable than previously reported with no substantial reduction in infectivity occurring until pH levels are reduced below 4.5. The virucidal activity of 3 topical spermicides and chlorhexidine was assessed in vitro using previously established and newly modified assay systems. None of the agents tested had a selectivity index (SI) greater than 5.2. Semen and cervical secretions were assessed for their ability to inhibit HIV-1. While no virucidal effect was found in the latter, seminal fluid was found to have significant activity against HIV-1 and a SI of approximately 50. PMID:7548290

  4. Effect of enzyme secreting bacterial pretreatment on enhancement of aerobic digestion potential of waste activated sludge interceded through EDTA.

    PubMed

    Kavitha, S; Adish Kumar, S; Yogalakshmi, K N; Kaliappan, S; Rajesh Banu, J

    2013-12-01

    In this study, the effect of Ethylene diamine tetra acetic acid (EDTA) on Extracellular polymeric substance (EPS) removal tailed with bacterial enzymatic pretreatment on aerobic digestion of activated sludge was studied. In order to enhance the accessibility of sludge to the enzyme secreting bacteria; the extracellular polymeric substances were removed using EDTA. EDTA efficiently removed the EPS with limited cell lysis and enhanced the sludge enzyme activity at its lower concentration of 0.2 g/g SS. The sludge was then subjected to bacterial pretreatment to enhance the aerobic digestion. In aerobic digestion the best results in terms of Suspended solids (SS) reduction (48.5%) and COD (Chemical oxygen demand) solubilization (47.3%) was obtained in experimental reactor than in control. These results imply that aerobic digestion can be enhanced efficiently through bacterial pretreatment of EPS removed sludge.

  5. The high mobility group box 1 protein of Sciaenops ocellatus is a secreted cytokine that stimulates macrophage activation.

    PubMed

    Zhao, Lu; Hu, Yong-Hua; Sun, Jin-Sheng; Sun, Li

    2011-10-01

    High mobility group box 1 protein (HMGB1) is a chromatin-associated nonhistone protein that is involved in nucleosome formation and transcriptional regulation. In addition, HMGB1 is also known as an extracellular cytokine that triggers inflammation and immune responses. HMGB1-like sequences have been identified in a number of fish species, however, the function of piscine HMGB1 remains uninvestigated. In this study, we reported the identification and analysis of SoHMGB1, an HMGB1 homologue from red drum (Sciaenops ocellatus). SoHMGB1 is 206 residues in length and contains two basic HMG boxes and a highly acidic C-terminal domain. SoHMGB1 shares 71-87% overall sequence identities with the HMGB1 counterparts from human, rat, and several fish species. Quantitative real time RT-PCR analysis showed that constitutive SoHMGB1 expression was detected in various tissues, with the lowest and highest levels found in kidney and muscle respectively. Bacterial challenge upregulated SoHMGB1 expression in head kidney (HK) and HK macrophages and induced extracellular secretion of SoHMGB1 by the activated macrophages. Recombinant SoHMGB1 (rSoHMGB1) purified from yeast exhibited no direct antimicrobial effect but was significantly stimulatory on the proliferation, activation, and bactericidal activity of HK macrophages. Taken together, these results indicate for the first time that a fish HMGB1, SoHMGB1, can function as a secreted cytokine in the event of bacterial infection and promote innate defense through the activation of macrophages. PMID:21527276

  6. Geothrix fermentans Secretes Two Different Redox-Active Compounds To Utilize Electron Acceptors across a Wide Range of Redox Potentials

    PubMed Central

    Mehta-Kolte, Misha G.

    2012-01-01

    The current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial genera Geobacter and Shewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of the Acidobacteria, Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE), G. fermentans required potentials as high as 0.55 V to respire at its maximum rate. In addition, G. fermentans secreted two different soluble redox-active electron shuttles with separate redox potentials (−0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found in G. fermentans supernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals that Geothrix is able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined to Shewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies of Geothrix and Geobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential. PMID:22843516

  7. Tubular aggregates: their association with myalgia.

    PubMed Central

    Niakan, E; Harati, Y; Danon, M J

    1985-01-01

    Three thousand consecutive muscle biopsies were reviewed for the presence of tubular aggregates and their association with clinical symptomatology. Tubular aggregates were detected in 19 patients (0.6%). Twelve of these nineteen patients had severe myalgia, and the most abundant tubular aggregates were found in biopsies of patients with myalgia. Seven patients had only myalgia as their clinical symptomatology with normal physical examination. An additional five patients with tubular aggregates and myalgia had concomitant amyotrophic lateral sclerosis (2) or neuropathy (3). The high incidence of myalgia associated with tubular aggregates in our patients and the fact that tubular aggregates originate from sarcoplasmic reticulum suggest a role played by this structure in the pathogenesis of myalgia. Images PMID:2995591

  8. Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk.

    PubMed

    Mikus, Tomás; Poplstein, Martin; Sedláková, Jirina; Landa, Vladimír; Jeníkova, Gabriela; Trefil, Pavel; Lidický, Jan; Malý, Petr

    2004-10-01

    Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

  9. Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

    PubMed Central

    Lu, Connie; Turley, Stewart; Marionni, Samuel T.; Park, Young-Jun; Lee, Kelly K.; Patrick, Marcella; Shah, Ripal; Sandkvist, Maria; Bush, Matthew F.; Hol, Wim G. J.

    2013-01-01

    SUMMARY The Type II Secretion System (T2SS), a multi-protein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multi-domain T2SS assembly ATPase GspEEpsE had so far not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspEEpsE are obtained when fused to Hcp1 as an assistant hexamer, as shown by native mass spectrometry. The enzymatic activity of the GspEEpsE-Hcp1 fusions is ~20 times higher than that of a GspEEpsE monomer indicating that increasing the local concentration of GspEEpsE by the fusion strategy was successful. Crystal structures of GspEEpsE-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspEEpsE with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspEEpsE-Hcp1 fusions suggest that even further variability in GspEEpsE hexamer architecture is likely. PMID:23954505

  10. Response of plasma prorenin and active renin to chronic and acute alterations of renin secretion in normal humans. Studies using a direct immunoradiometric assay.

    PubMed Central

    Toffelmire, E B; Slater, K; Corvol, P; Menard, J; Schambelan, M

    1989-01-01

    We employed a novel immunoradiometric assay to measure plasma levels of active renin and prorenin in physiologic and pharmacologic studies designed to characterize renin biosynthesis and processing in response to both chronic and acute stimuli of renin secretion in normal human subjects. Stimulation of renin secretion with prolonged dietary sodium restriction or amiloride resulted in marked increases in the plasma levels of prorenin, active renin, and plasma renin activity (PRA); suppression of renin secretion with indomethacin resulted in parallel decreases in prorenin, active renin, and PRA. In contrast, acute stimulation with upright activity or administration of an angiotensin-converting enzyme inhibitor, which increased active renin and PRA from 2- to 15-fold, had no effect on prorenin levels. Based on studies in cultured human juxtaglomerular tumor cells, it has been proposed that prorenin is secreted constitutively whereas active renin is stored in and released from secretory granules through a regulated pathway. Our studies are consistent with such a model: the parallel changes in active renin and prorenin with experimental maneuvers of long duration suggest that both the constitutive and regulated pathways are altered under these conditions. The increase in active renin levels in the absence of a change in prorenin that occurs in response to acute stimuli presumably represents the release of preformed active enzyme that is stored in secretory granules. PMID:2643635

  11. Circulating anti-double-stranded DNA antibody-secreting cells in patients with systemic lupus erythematosus: a novel biomarker for disease activity.

    PubMed

    Hanaoka, H; Okazaki, Y; Satoh, T; Kaneko, Y; Yasuoka, H; Seta, N; Kuwana, M

    2012-10-01

    Antibodies against double-stranded DNA (dsDNA) are widely used to diagnose systemic lupus erythematosus (SLE) and evaluate its activity in patients. This study was undertaken to examine the clinical utility of circulating anti-dsDNA antibody-secreting cells for evaluating SLE patients. Anti-dsDNA antibody-secreting cells quantified using an enzyme-linked immunospot assay were detected in the spleen, bone marrow and peripheral blood from MRL/lpr but not in control BALB/c mice. Circulating anti-dsDNA antibody-secreting cells were detected in 29 (22%) of 130 patients with SLE, but in none of 49 with non-SLE connective-tissue disease or 18 healthy controls. The presence of circulating anti-dsDNA antibody-secreting cells was associated with persistent proteinuria, high SLE disease activity index and systemic lupus activity measures, and a high serum anti-dsDNA antibody titre measured with an enzyme-linked immunosorbent assay. The positive predictive value for active disease was 48% for circulating anti-dsDNA antibody-secreting cells versus 17% for serum anti-dsDNA antibodies. A prospective cohort of patients with circulating anti-dsDNA antibodies and inactive SLE showed that the cumulative disease flare-free rate was significantly lower in patients with than without circulating anti-dsDNA antibody-secreting cells (p < 0.001). Circulating anti-dsDNA antibody-secreting cells are a useful biomarker for assessing disease activity in SLE patients.

  12. Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function

    SciTech Connect

    Bussey, H.; Sacks, W.; Galley, D.; Saville, D.

    1982-04-01

    M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only one M plasmid. Mutants with neutral (K/sup -/), immune (R/sup +/) or suicide (killer (K/sup +/), sensitive (R/sup -/)) phenotypes were examined. All mutants became K/sup -/ R/sup -/ sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.

  13. Renal tubular acidosis type 4 in pregnancy.

    PubMed

    Jakes, Adam Daniel; Baynes, Kevin; Nelson-Piercy, Catherine

    2016-03-17

    We describe the clinical course of renal tubular acidosis (RTA) type 4 in pregnancy, which has not been previously published. Renal tubular acidosis type 4 is a condition associated with increased urinary ammonia secondary to hypoaldosteronism or pseudohypoaldosteronism. Pregnancy may worsen the hyperkalaemia and acidosis of renal tubular acidosis type 4, possibly through an antialdosterone effect. We advise regular monitoring of potassium and pH throughout pregnancy to ensure safe levels are maintained.

  14. Probabilistic secretion of quanta and the synaptosecretosome hypothesis: evoked release at active zones of varicosities, boutons, and endplates.

    PubMed Central

    Bennett, M R; Gibson, W G; Robinson, J

    1997-01-01

    A quantum of transmitter may be released upon the arrival of a nerve impulse if the influx of calcium ions through a nearby voltage-dependent calcium channel is sufficient to activate the vesicle-associated calcium sensor protein that triggers exocytosis. A synaptic vesicle, together with its calcium sensor protein, is often found complexed with the calcium channel in active zones to form what will be called a "synaptosecretosome." In the present work, a stochastic analysis is given of the conditions under which a quantum is released from the synaptosecretosome by a nerve impulse. The theoretical treatment considers the rise of calcium at the synaptosecretosome after the stochastic opening of a calcium channel at some time during the impulse, followed by the stochastic binding of calcium to the vesicle-associated protein and the probability of this leading to exocytosis. This allows determination of the probabilities that an impulse will release 0, 1, 2,... quanta from an active zone, whether this is in a varicosity, a bouton, or a motor endplate. A number of experimental observations of the release of transmitter at the active zones of sympathetic varicosities and boutons as well as somatic motor endplates are described by this analysis. These include the likelihood of the secretion of only one quantum at an active zone of endplates and of more than one quantum at an active zone of a sympathetic varicosity. The fourth-power relationship between the probability of transmitter release at the active zones of sympathetic varicosities and motor endplates and the external calcium concentration is also explained by this approach. So, too, is the fact that the time course of the increased rate of quantal secretion from a somatic active zone after an impulse is invariant with changes in the amount of calcium that enters through its calcium channel, whether due to changes consequent on the actions of autoreceptor agents such as adenosine or to facilitation. The increased

  15. Effects of exposure to male goat hair extracts on luteinizing hormone secretion and neuronal activation in seasonally anestrous ewes.

    PubMed

    Ohara, Hiromi; Mogi, Kazutaka; Ichimaru, Toru; Ohkura, Satoshi; Takeuchi, Yukari; Mori, Yuji; Okamura, Hiroaki

    2014-10-01

    In sheep and goats, exposure of seasonally anestrous females to males or their fleece/hair activates the gonadotropin-releasing hormone (GnRH) pulse generator leading to pulsatile luteinizing hormone (LH) secretion. Pheromones emitted by sexually mature males are thought to play a prominent role in this male effect. In the present study, we first aimed to clarify whether the male goat pheromone is effective in ewes. Seasonally anestrous St. Croix ewes were exposed to hair extracts derived from either intact or castrated (control) male Shiba goats. The male goat-hair extract significantly increased LH secretion compared to the control, suggesting that an interspecies action of the male pheromone occurs between sheep and goats. Using the male goat-hair extract as the pheromone source, we then aimed to clarify the neural pathway involved in the signal transduction of the male pheromone. Ewes were exposed to either the goat-hair extract or the control and sacrificed 2 hr after the exposure. Expression of c-Fos, a marker of neuronal activation, was immunohistochemically examined. The male goat-hair extract significantly increased the c-Fos expression compared to the control in regions of the vomeronasal system, such as the accessory olfactory bulb and medial amygdala, and the arcuate nucleus. The main olfactory bulb did not exhibit any significant increase in the c-Fos expression by the male goat-hair extract. This result suggests that the neural signal of the male pheromone is conveyed to the GnRH pulse generator through the activated regions in ewes.

  16. Biologic Activity of Autologous, Granulocyte-Macrophage Colony Stimulating Factor Secreting Alveolar Soft Parts Sarcoma and Clear Cell Sarcoma Vaccines

    PubMed Central

    Goldberg, John; Fisher, David E.; Demetri, George D.; Neuberg, Donna; Allsop, Stephen A.; Fonseca, Catia; Nakazaki, Yukoh; Nemer, David; Raut, Chandrajit P.; George, Suzanne; Morgan, Jeffrey A.; Wagner, Andrew J.; Freeman, Gordon J.; Ritz, Jerome; Lezcano, Cecilia; Mihm, Martin; Canning, Christine; Hodi, F. Stephen; Dranoff, Glenn

    2015-01-01

    Purpose Alveolar soft parts sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor (GM-CSF). Experimental Design Metastatic tumors from ASPS and CCS patients were resected, processed to single cell suspensions, transduced with a replication defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly times three and then every other week. Results Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from 3 to 13 immunizations. Toxicities were restricted to grade 1–2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell mediated delayed type-hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1) positive CD8+ T cells in association with PD ligand-1 (PD-L1) expressing sarcoma cells. No tumor regressions were observed. Conclusions Vaccination with irradiated, GM-CSF secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1 negative regulatory pathway might intensify immune-mediated tumor destruction. PMID:25805798

  17. Downdraft stove with tubular grating

    SciTech Connect

    Zimmerman, H.G.

    1986-08-26

    This patent describes a downdraft stove, a tubular grating assembly for positioning in a reaction chamber which consists of: a substantially vertically oriented central tube open at its upper end and connected at its lower end to an air inlet opening; a cap supported above the open upper end for protecting the open upper end from entry of matter, the space between the cap and the upper end constituting a primary air inlet nozzle; grating tubes radially distributed around and taking off substantially horizontally from and communicating with the central tube, thereby defining a grating, and thence turning downwardly and being open at their downward ends to thereby constitute secondary air inlets.

  18. Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma.

    PubMed

    Kamath, Arun T; Sheasby, Christopher E; Tough, David F

    2005-01-15

    Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-alphabeta, secreted by dendritic cells (DCs), and IFN-gamma, secreted by NK cells; notably, IFN-gamma-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-gamma by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-gamma, maximal IFN-gamma production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-alphabeta, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-gamma, whereas IFN-alphabeta was shown to be important for induction of T cell bystander activation and NK cell IFN-gamma production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells. PMID:15634897

  19. Lipolanthionine peptides act as inhibitors of TLR2-mediated IL-8 secretion. Synthesis and structure-activity relationships.

    PubMed

    Seyberth, Tobias; Voss, Söhnke; Brock, Roland; Wiesmüller, Karl-Heinz; Jung, Günther

    2006-03-01

    Lipoproteins from gram-positive and -negative bacteria, mycoplasma, and shorter synthetic lipopeptide analogues activate cells of the innate immune system via the Toll-like receptor TLR2/TLR1 or TLR2/TLR6 heterodimers. For this reason, these compounds constitute highly active adjuvants for vaccines either admixed or covalently linked. The lanthionine scaffold has structural similarity with the S-(2,3-dihydroxypropyl)cysteine core structure of the lipopeptides. Therefore, lanthionine-based lipopeptide amides were synthesized and probed for activity as potential TLR2 agonists or antagonists. A collection of analytically defined lipolanthionine peptide amides exhibited an inhibitory effect of the TLR2-mediated IL-8 secretion when applied in high molar excess to the agonistic synthetic lipopeptide Pam3Cys-Ser-(Lys)4-OH. Structure-activity relationships revealed the influence of the chirality of the two alpha-carbon atoms, the chain lengths of the attached fatty acids and fatty amines, and the oxidation level of the sulfur atom on the inhibitory activity of the lipolanthionine peptide amides. PMID:16509590

  20. Novel series of tacrine-tianeptine hybrids: Synthesis, cholinesterase inhibitory activity, S100B secretion and a molecular modeling approach.

    PubMed

    Ceschi, Marco Antonio; da Costa, Jessie Sobieski; Lopes, João Paulo Bizarro; Câmara, Viktor Saraiva; Campo, Leandra Franciscato; Borges, Antonio César de Amorim; Gonçalves, Carlos Alberto Saraiva; de Souza, Daniela Fraga; Konrath, Eduardo Luis; Karl, Ana Luiza Martins; Guedes, Isabella Alvim; Dardenne, Laurent Emmanuel

    2016-10-01

    Tianeptine was linked to various 9-aminoalkylamino-1,2,3,4-tetrahydroacridines using EDC·HCl/HOBt to afford a series of tacrine-tianeptine hybrids. The hybrids were tested for their ability to inhibit AChE and BuChE and IC50 values in the nanomolar concentration scale were obtained. AChE molecular modeling studies of these hybrids indicated that tacrine moiety interacts in the bottom of the gorge with the catalytic active site (CAS) while tianeptine binds to peripheral anionic site (PAS). Furthermore, the compounds 2g and 2e were able to reduce the in vitro basal secretion of S100B, suggesting its therapeutic action in some cases or stages of Alzheimer's disease.

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

    PubMed

    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. PMID:19160674

  2. Novel series of tacrine-tianeptine hybrids: Synthesis, cholinesterase inhibitory activity, S100B secretion and a molecular modeling approach.

    PubMed

    Ceschi, Marco Antonio; da Costa, Jessie Sobieski; Lopes, João Paulo Bizarro; Câmara, Viktor Saraiva; Campo, Leandra Franciscato; Borges, Antonio César de Amorim; Gonçalves, Carlos Alberto Saraiva; de Souza, Daniela Fraga; Konrath, Eduardo Luis; Karl, Ana Luiza Martins; Guedes, Isabella Alvim; Dardenne, Laurent Emmanuel

    2016-10-01

    Tianeptine was linked to various 9-aminoalkylamino-1,2,3,4-tetrahydroacridines using EDC·HCl/HOBt to afford a series of tacrine-tianeptine hybrids. The hybrids were tested for their ability to inhibit AChE and BuChE and IC50 values in the nanomolar concentration scale were obtained. AChE molecular modeling studies of these hybrids indicated that tacrine moiety interacts in the bottom of the gorge with the catalytic active site (CAS) while tianeptine binds to peripheral anionic site (PAS). Furthermore, the compounds 2g and 2e were able to reduce the in vitro basal secretion of S100B, suggesting its therapeutic action in some cases or stages of Alzheimer's disease. PMID:27392529

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

    PubMed

    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.

  4. Nocardia farcinica Activates Human Dendritic Cells and Induces Secretion of Interleukin-23 (IL-23) Rather than IL-12p70

    PubMed Central

    Eisenblätter, Martin; Buchal, Ariane; Gayum, Hermine; Jasny, Edith; Renner Viveros, Pablo; Ulrichs, Timo; Schneider, Thomas; Schumann, Ralf R.; Zweigner, Janine

    2012-01-01

    Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-α) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses. PMID:22988018

  5. The HMG-CoA reductase inhibitor rosuvastatin inhibits plasminogen activator inhibitor-1 expression and secretion in human adipocytes.

    PubMed

    Laumen, Helmut; Skurk, Thomas; Hauner, Hans

    2008-02-01

    Human preadipocytes and adipocytes are known to produce the proatherogenic factor PAI-1 and proinflammatory cytokines, and obesity was found to be state of increased adipose production of these factors. In the present study, we investigated the effect of rosuvastatin on the regulation of PAI-1 gene expression in human adipocytes. Human preadipocytes, adipocytes in primary culture and the SGBS cell line were used as cell models. Cells were transfected using various constructs and promoter activity was measured as luciferase activity. PAI-1 expression was measured by quantitative RT-PCR and ELISA. Rosuvastatin inhibited PAI-1 mRNA expression and secretion of the protein in a concentration-dependent manner. This effect was reversed by isoprenoids. Addition of MEK-inhibitors and NFkappaB inhibitors also reduced PAI-1 expression and PAI-1 promoter luciferase activity. Further experiments revealed that rosuvastatin down-regulated the MEKK-1 mediated activation of the PAI-1 promoter. In conclusion our data suggest that rosuvastatin inhibits PAI-1 expression and release from human adipocytes via a MEKK-1-dependent but not a NFkappaB-dependent mechanism.

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

    PubMed

    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. PMID:27609291

  7. Antibacterial Activity of Recombinant Pig Intestinal Parasite Cecropin P4 Peptide Secreted from Pichia pastoris

    PubMed Central

    Song, Ki-Duk; Lee, Woon-Kyu

    2014-01-01

    Cecropins (Cec) are antibacterial peptides and their expression is induced in a pig intestinal parasite Ascaris suum by bacterial infection. To explore the usefulness of its activity as an antibiotic, CecP4 cDNA was prepared and cloned into the pPICZ B expression vector and followed by the integration into AOX1 locus in Pichia pastoris. The supernatants from cell culture were collected after methanol induction and concentrated for the test of antimicrobial activity. The recombinant P. patoris having CecP4 showed antimicrobial activity when tested against Staphyllococcus aureus in disc diffusion assay. We selected one of the CecP4 clones (CecP4-2) and performed further studies with it. The growth of recombinant P. pastoris was optimized using various concentration of methanol, and it was found that 2% methanol in the culture induced more antibacterial activity, compared to 1% methanol. We extended the test of antimicrobial activity by applying the concentrated supernatant of CecP4 culture to Pseudomonas aeruginosa and E. coli respectively. Recombinant CecP4 also showed antimicrobial activity against both Pseudomona and E. coli, suggesting the broad spectrum of its antimicrobial activity. After improvements for the scale-up, it will be feasible to use recombinant CecP4 for supplementation to the feed to control microbial infections in young animals, such as piglets. PMID:25049952

  8. Activation of microglia by endotoxin suppresses the secretion of glial cell line-derived neurotrophic factor (GDNF) through the action of protein kinase C alpha (PKCalpha) and mitogen-activated protein kinases (MAPKS).

    PubMed

    Matsushita, Yuichi; Nakajima, Kazuyuki; Tohyama, Yoko; Kurihara, Tadashi; Kohsaka, Shinichi

    2008-07-01

    The ability of microglia to produce/secrete glial cell line-derived neurotrophic factor (GDNF) in vitro was examined. Immunoblotting analysis revealed that nonstimulated microglia release limited amounts of GDNF with molecular sizes of 14 and 17 kDa. However, the secreted amounts significantly decreased when the microglia were activated with the endotoxin lipopolysaccharide (LPS). Comparison of the amounts of GDNF in the cells and the conditioned medium between the nonstimulated microglia and LPS-stimulated microglia clarified that the secretion of GDNF, but not its production, is strongly suppressed when the microglia are activated with LPS. The inhibitor experiments suggested that the GDNF secretion is depressed by a signaling cascade associated with protein kinase C alpha (PKCalpha) and/or mitogen-activated protein kinases (MAPKs). As expected from the above results, a PKC activator suppressed the secretion of GDNF in nonstimulated microglia. Taken together, these results demonstrated that microglia have the ability to produce and secrete GDNF in vitro, and that the secretion is suppressed by stimulation with endotoxin, probably due to a signaling mechanism involving PKCalpha and/or MAPKs.

  9. Anatomicohistological characteristics of the tubular genital organs of the female collared peccary (Tayassu tajacu) from North-eastern Amazon.

    PubMed

    Mayor, P; Jori, F; López-Béjar, M

    2004-04-01

    The present study examines anatomical and histological characteristics of tubular genital organs and its relationships with the reproductive state of 24 wild adult collared peccary (Tayassu tajacu) females. The tunica mucosa of the uterine tube presents a pseudostratified, intermittently ciliated columnar epithelium. The epithelial secretory cells of pregnant females and females in the luteal phase of the oestrous cycle became taller than the ciliated cells and showed abundant apical secretory blebs, whereas secretory cells of females in the follicular phase showed abundant mucous secretory activity (periodic acid-Schiff positive cells). The uterus is composed of two narrow and convoluted uterine horns, separated by the velum uteri, a small uterine body and a long and muscular cervix. The endometrial lining of both uterine horns and body is a monostratified, columnar ciliated epithelium. Pregnant females and females in luteal phase showed a more developed hyperplasia of the endometrial simple tubular glands than females in the follicular phase. The cervix presents interdigitated rows of mucosal prominences that project into the lumen, structures similar to pulvini cervicali, occluding the cervical canal. In pregnant females, the endocervical canal was filled by a viscous cervical secretion. Females in follicular phase presented a thicker vaginal epithelium than pregnant females and females in luteal phase. The present study suggests that the collared peccary female showed different histological features of the uterine tubes, uterus and vagina in accordance with the reproductive state of the females.

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

    SciTech Connect

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

    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.

  11. Identification and characterization of a novel secreted glycosidase with multiple glycosidase activities in Streptococcus intermedius.

    PubMed

    Imaki, Hidenori; Tomoyasu, Toshifumi; Yamamoto, Naoki; Taue, Chiharu; Masuda, Sachiko; Takao, Ayuko; Maeda, Nobuko; Tabata, Atsushi; Whiley, Robert A; Nagamune, Hideaki

    2014-08-01

    Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal

  12. Tubular inverse opal scaffolds for biomimetic vessels

    NASA Astrophysics Data System (ADS)

    Zhao, Ze; Wang, Jie; Lu, Jie; Yu, Yunru; Fu, Fanfan; Wang, Huan; Liu, Yuxiao; Zhao, Yuanjin; Gu, Zhongze

    2016-07-01

    There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially oriented elliptical pattern microstructures on their surfaces. It is demonstrated that these tailored tubular scaffolds can effectively make endothelial cells to form an integrated hollow tubular structure on their inner surface and induce smooth muscle cells to form a circumferential orientation on their outer surface. These features of our tubular scaffolds make them highly promising for the construction of biomimetic blood vessels.There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially

  13. FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line.

    PubMed

    Hamaguchi, M; Morishita, Y; Takahashi, I; Ogura, M; Takamatsu, J; Saito, H

    1991-01-01

    We studied the effect of fibrinogen degradation products D, E, and D-dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)-mol/L fragment D, E, and D-dimer, whereas a significant increase in IL-1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation. PMID:1845845

  14. Schistosoma mansoni Hemozoin Modulates Alternative Activation of Macrophages via Specific Suppression of Retnla Expression and Secretion

    PubMed Central

    Truscott, Martha; Evans, D. Andrew; Gunn, Matt

    2013-01-01

    The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis. PMID:23090958

  15. Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG

    PubMed Central

    Claes, Ingmar J. J.; Schoofs, Geert; Regulski, Krzysztof; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Rolain, Thomas; Hols, Pascal; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Vanderleyden, Jos; De Keersmaecker, Sigrid C. J.; Lebeer, Sarah

    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 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. PMID:22359601

  16. A 25 kDa polypeptide is the ligand for p185neu and is secreted by activated macrophages.

    PubMed

    Tarakhovsky, A; Zaichuk, T; Prassolov, V; Butenko, Z A

    1991-12-01

    Medium conditioned by mouse peritoneal macrophages, activated by muramyl dipeptide (MDP), was used as a possible source of p185neu-specific ligand. MDP-activated macrophage-conditioned medium (MDP-CM) was shown to induce p185neu down-regulation in NEU-expressing NIH3T3 cells in a dose-dependent and temperature-sensitive manner. To exclude the possibility of an indirect action of proteins/metabolites present in MDP-CM on p185neu turnover, a ligand-trapping approach was used. Secreted NEU protein possessing only the extracellular domain but lacking transmembrane and protein kinase domains was expressed in HeLa cells and then purified from conditioned medium, using affinity chromatography on WGA-Sepharose. Co-incubation of the truncated, soluble NEU protein preparation with MDP-CM abolished MDP-CM-induced p185neu down-regulation and reduced self-phosphorylation. It is concluded that a putative p185neu-specific ligand is produced by macrophages activated by MDP. Using MDP-CM, the presence of a 25 kDa polypeptide distinct from EGF, PDGF, FGF, IGF, TGF-alpha and TGF-beta and TNF-alpha, could be demonstrated by decorating a Western blot with soluble NEU and anti-NEU antibodies. Thus, a 25 kDa (non-reduced) p185neu ligand has been described.

  17. Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with MS detection

    PubMed Central

    Temporini, C.; Pochetti, G.; Fracchiolla, G.; Piemontese, L.; Montanari, R.; Moaddel, R.; Laghezza, A.; Altieri, F.; Cervoni, L.; Ubiali, D.; Prada, E.; Loiodice, F.; Massolini, G.; Calleri, E.

    2013-01-01

    The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening towards PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of Frontal Affinity Chromatography coupled to Mass Spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments towards new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes. PMID:23466198

  18. Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88- and Syk kinase-dependent pathways.

    PubMed

    Li, Bing; Cramer, Daniel; Wagner, Stephanie; Hansen, Richard; King, Chelsea; Kakar, Shelly; Ding, Chuanlin; Yan, Jun

    2007-08-01

    The therapeutic benefits of fungal beta-glucans have been demonstrated as immuno-stimulating agents. In this study, we aimed to explore the mechanisms used by yeast beta-glucan-rich particles to activate murine resident macrophages for cytokine secretion. We demonstrated that resident macrophages were effectively activated by whole yeast beta-glucan particles (WGPs), such as with the upregulation of co-stimulatory molecules and the secretion of cytokines. The binding ability of WGPs and the levels of cytokine secretion in resident macrophages were significantly inhibited by soluble yeast beta-glucan but not by blockade of zymosan glucan receptor dectin-1. In addition, WGP-stimulated cytokine secretion was partially dependent on the MyD-88 pathway but was not significantly affected in CR3-deficient (CR3(-/-)) mice. Furthermore, we showed that Syk kinase was recruited upon WGP stimulation and was required for the production of cytokines. Taken together, these observations suggest that beta-glucan recognition is necessary but not sufficient to induce inflammatory response on resident macrophages. In addition, beta-glucan particles may use differential mechanisms for cytokine secretion in resident macrophages that may modulate both innate and adaptive immunity.

  19. Yeast Glucan Particles Activate Murine Resident Macrophages to Secrete Proinflammatory Cytokines Via MyD88- and Syk Kinase-dependent Pathways1

    PubMed Central

    Li, Bing; Cramer, Daniel; Wagner, Stephanie; Hansen, Richard; King, Chelsea; Kakar, Shelly; Ding, Chuanlin; Yan, Jun

    2007-01-01

    The therapeutic benefits of fungal β-glucans have been demonstrated as immuno-stimulating agents. In this study, we aimed to explore the mechanisms used by yeast β-glucan-rich particles to activate murine resident macrophages for cytokine secretion. We demonstrated that resident macrophages were effectively activated by whole yeast β-glucan particles (WGPs), such as with the up-regulation of co-stimulatory molecules and the secretion of cytokines. The binding ability of WGPs and the levels of cytokine secretion in resident macrophages were significantly inhibited by soluble yeast β-glucan but not by blockade of zymosan glucan receptor dectin-1. In addition, WGP-stimulated cytokine secretion was partially dependent on the MyD-88 pathway but was not significantly affected in CR3-deficient (CR3−/−) mice. Furthermore, we showed that Syk kinase was recruited upon WGP stimulation and was required for the production of cytokines. Taken together, these observations suggest that β-glucan recognition is necessary but not sufficient to induce inflammatory response on resident macrophages. In addition, β-glucan particles may use differential mechanisms for cytokine secretion in resident macrophages that may modulate both innate and adaptive immunity. PMID:17572156

  20. Inhibition of IL-1beta and TNF-alpha secretion from resting and activated human immunocytes by the homeopathic medication Traumeel S.

    PubMed

    Porozov, Svetlana; Cahalon, Liora; Weiser, Michael; Branski, David; Lider, Ofer; Oberbaum, Menachem

    2004-06-01

    Traumeel S (Traumeel), a mixture of highly diluted (10(-1)-10(-9)) extracts from medicinal plants and minerals is widely used in humans to relieve trauma, inflammation and degenerative processes. However, little is known about its possible effects on the behavior of immune cells. The effects of Traumeel were examined in vitro on the ability of resting and PHA-, PMA- or TNF-alpha-activated human T cells, monocytes, and gut epithelial cells to secrete the prototypic pro-inflammatory mediators IL-1beta, TNF-alpha and IL-8 over a period of 24-72 h. Traumeel inhibited the secretion of all three agents in resting, as well as activated immune cells. IL-beta secretion was reduced by up to 70% in both resting and activated cells; TNF-alpha secretion was reduced by up to 65 and 54%, respectively, and IL-8 secretion was reduced by 50% in both resting and activated cells (P < 0.01 for all cells). Interestingly, the effect appeared to be inversely dose-related; maximal inhibition (usually 30-60% inhibition; P < 0.01) was seen with dilutions of 10(-3)-10(-6) of the Traumeel stock material. This finding suggests that Traumeel does not inhibit immune cells functions by exerting a toxic effect. Indeed, Traumeel did not affect T cell and monocyte proliferation. Although additional studies are needed to clarify the mode of action of Traumeel and to demonstrate causative relationship between the inhibition of cytokine/chemokine secretion in cell culture and the reported clinical effects of the preparation, our in vitro results offer a mechanism for the anti-inflammatory effects of Traumeel observed in clinical use. PMID:15330450

  1. Secrets of the Marsh. An Activity Resource Book For and By Children. Second Edition.

    ERIC Educational Resources Information Center

    Euler, Aline, Ed.

    This publication contains a collection of student-produced activities about wildlife. Children's views of the varying life forms that they encountered in field walks to a marsh are expressed through the games, puzzles, stories, and pictures that they created. Samples of the students' work are organized into separate topic areas. These include: (1)…

  2. Neural stem cells secrete factors facilitating brain regeneration upon constitutive Raf-Erk activation

    PubMed Central

    Rhee, Yong-Hee; Yi, Sang-Hoon; Kim, Joo Yeon; Chang, Mi-Yoon; Jo, A-Young; Kim, Jinyoung; Park, Chang-Hwan; Cho, Je-Yoel; Choi, Young-Jin; Sun, Woong; Lee, Sang-Hun

    2016-01-01

    The intracellular Raf-Erk signaling pathway is activated during neural stem cell (NSC) proliferation, and neuronal and astrocytic differentiation. A key question is how this signal can evoke multiple and even opposing NSC behaviors. We show here, using a constitutively active Raf (ca-Raf), that Raf-Erk activation in NSCs induces neuronal differentiation in a cell-autonomous manner. By contrast, it causes NSC proliferation and the formation of astrocytes in an extrinsic autocrine/paracrine manner. Thus, treatment of NSCs with medium (CM) conditioned in ca-Raf-transduced NSCs (Raf-CM; RCM) became activated to form proliferating astrocytes resembling radial glial cells (RGCs) or adult-type NSCs. Infusion of Raf-CM into injured mouse brains caused expansion of the NSC population in the subventricular zone, followed by the formation of new neurons that migrated to the damaged site. Our study shows an example how molecular mechanisms dissecting NSC behaviors can be utilized to develop regenerative therapies in brain disorders. PMID:27554447

  3. Neural stem cells secrete factors facilitating brain regeneration upon constitutive Raf-Erk activation.

    PubMed

    Rhee, Yong-Hee; Yi, Sang-Hoon; Kim, Joo Yeon; Chang, Mi-Yoon; Jo, A-Young; Kim, Jinyoung; Park, Chang-Hwan; Cho, Je-Yoel; Choi, Young-Jin; Sun, Woong; Lee, Sang-Hun

    2016-01-01

    The intracellular Raf-Erk signaling pathway is activated during neural stem cell (NSC) proliferation, and neuronal and astrocytic differentiation. A key question is how this signal can evoke multiple and even opposing NSC behaviors. We show here, using a constitutively active Raf (ca-Raf), that Raf-Erk activation in NSCs induces neuronal differentiation in a cell-autonomous manner. By contrast, it causes NSC proliferation and the formation of astrocytes in an extrinsic autocrine/paracrine manner. Thus, treatment of NSCs with medium (CM) conditioned in ca-Raf-transduced NSCs (Raf-CM; RCM) became activated to form proliferating astrocytes resembling radial glial cells (RGCs) or adult-type NSCs. Infusion of Raf-CM into injured mouse brains caused expansion of the NSC population in the subventricular zone, followed by the formation of new neurons that migrated to the damaged site. Our study shows an example how molecular mechanisms dissecting NSC behaviors can be utilized to develop regenerative therapies in brain disorders. PMID:27554447

  4. [In vitro evaluation of antacid activity in gastric acid secretion in static and dynamic systems].

    PubMed

    Vatier, J; Malikova-Sekera, E; Vitre, M T; Mignon, M

    1992-01-01

    A valid in vitro evaluation of antacid capacity should consider: 1) the intragastric pH-range; 2) the antacid mechanism; 3) the dependence of antacid activity from intraluminal flux variations; 4) the interaction between proteins and antacids. Pharmacologically, a static method allows 1) to quantify H+ binding sites at different pH-end points of the titration: pH 3.0, 2.0 and 1.0 and 2) to characterize the antacid mechanism, neutralizing activity and/or buffering capacity. In dynamic conditions, using the "artificial stomach-duodenum" model the antacid-induced resistance to acidification was measured, the antacid mechanisms were characterized in regard to intraluminal gastroduodenal flux variations and the incidence of antacid activity on duodenal pH was evaluated. These procedures were applied to antacid evaluation of proteins, as natural antacids, and of drugs containing aluminium salts alone or combined with magnesium salts. Pharmacologically, antacid drugs exhibited a greater amount of H+ binding sites when titration end-point was pH 1.0 than pH 3.0 corresponding to the development of neutralizing activity and/or buffering capacity. In dynamic conditions, the drugs, like proteins, induced a potent resistance to acidification related to gastric emptying fluxes. Antacid effect was supported by neutralizing activity and/or by buffering capacity. It was prolonged by removal of H+ ions since lagtimes for recovering initial pH were longer than antacid total emptying, the dilution of intragastric content by H+ impoverished secretory flux contributing thus to prevent gastric acidification. At duodenal site, proteins and aluminium-containing antacids induced the same duodenal pH as controls, without antacids, while magnesium-containing antacids increased it. PMID:1412150

  5. Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

    PubMed Central

    Micaroni, Massimo; Stanley, Amanda C.; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X. F.; Lim, Jet P.; Marsh, Brad J.; Storrie, Brian; Gleeson, Paul A.; Stow, Jennifer L.

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages. PMID:23437303

  6. Emerin expression in tubular aggregates.

    PubMed

    Manta, Panagiota; Terzis, Gerasimos; Papadimitriou, Constantinos; Kontou, Chrysanthi; Vassilopoulos, Demetris

    2004-06-01

    Emerin is an inner nuclear membrane protein that is mutated or not expressed in patients with X-linked Emery-Dreifuss muscular dystrophy (X-EDMD/EMD). Cytoplasmic localization of emerin in cultured cells or tissues has been reported, although this remains a controversial issue. Tubular aggregates (TAs) are pathological structures seen in the sarcoplasm of human skeletal muscle fibers in various disorders. The TAs derive from the sarcoplasmic reticulum (SR) and represent, probably, an adaptive response of the SR to various insults to the muscle fibers. In the present study, we present immunohistochemical evidence of emerin expression in TAs. Muscle biopsies with tubular aggregates from four male, unrelated patients were studied. The percentage of muscle fibers containing TAs varied between 5 and 20%. Routine histochemistry revealed intense reaction of TAs with NADH-TR, AMPDA, and NSE, but not with COX, SDH, myosin ATPase (pH 9.4, 4.3, 4.6), PAS, and Oil red O staining. Immunohistochemical study revealed strong immunostaining of TAs with antibodies against emerin and 7 SERCA2-ATPase. Immunostaining of TAs was also seen with antibodies against heat shock protein and dysferlin, but not with antibodies to lamin A, dystrophin, adhalin, beta, gamma, delta sarcoglycans, and merosin. These results suggest that emerin, an inner nuclear membrane protein, is present at the TAs. The interpretation and significance of this finding is discussed in relation to experimental data suggesting that normal emerin localization at the inner nuclear membrane depends on lamin A and mutations in the N-terminal domain of emerin cause mislocalization of the protein to the sarcoplasmic membranes.

  7. Geochemical characterization of tubular alteration features in subseafloor basalt glass

    NASA Astrophysics Data System (ADS)

    Knowles, Emily; Staudigel, Hubert; Templeton, Alexis

    2013-07-01

    There are numerous indications that subseafloor basalts may currently host a huge quantity of active microbial cells and contain biosignatures of ancient life in the form of physical and chemical basalt glass alteration. Unfortunately, technological challenges prevent us from observing the formation and mineralization of these alteration features in situ, or reproducing tubular basalt alteration processes in the laboratory. Therefore, comprehensive analysis of the physical and chemical traces retained in mineralized tubules is currently the best approach for deciphering a record of glass alteration. We have used a number of high-resolution spectroscopic and microscopic methods to probe the geochemical and mineralogical characteristics of tubular alteration features in basalt glasses obtained from a suite of subseafloor drill cores that covers a range of different collection locations and ages. By combining three different synchrotron-based X-ray measurements - X-ray fluorescence microprobe mapping, XANES spectroscopy, and μ-XRD - with focused ion beam milling and transmission electron microscopy, we have spatially resolved the major and trace element distributions, as well as the oxidation state of Fe, determined the coordination chemistry of Fe, Mn and Ti at the micron-scale, and constrained the secondary minerals within these features. The tubular alteration features are characterized by strong losses of Fe2+, Mn2+, and Ca2+ compared to fresh glass, oxidation of the residual Fe, and the accumulation of Ti and Cu. The predominant phases infilling the alteration regions are Fe3+-bearing silicates dominated by 2:1 clays, with secondary Fe- and Ti-oxides, and a partially oxidized Mn-silicate phase. These geochemical patterns observed within the tubular alteration features are comparable across a diverse suite of samples formed over the past 5-100 Ma, which shows that the microscale mineralization processes are common and consistent throughout the ocean basins and

  8. Role of Nitric Oxide in the Regulation of Renin and Vasopressin Secretion

    NASA Technical Reports Server (NTRS)

    Reid, Ian A.

    1994-01-01

    Research during recent years has established nitric oxide as a unique signaling molecule that plays important roles in the regulation of the cardiovascular, nervous, immune, and other systems. Nitric oxide has also been implicated in the control of the secretion of hormones by the pancreas, hypothalamus, and anterior pituitary gland, and evidence is accumulating that it contributes to the regulation of the secretion of renin and vasopressin, hormones that play key roles in the control of sodium and water balance. Several lines of evidence have implicated nitric oxide in the control of renin secretion. The enzyme nitric oxide synthase is present in vascular and tubular elements of the kidney, particularly in cells of the macula densa, a structure that plays an important role in the control of renin secretion. Guanylyl cyclase, a major target for nitric oxide, is also present in the kidney. Drugs that inhibit nitric oxide synthesis generally suppress renin release in vivo and in vitro, suggesting a stimulatory role for the L-arginine/nitric oxide pathway in the control of renin secretion. Under some conditions, however, blockade of nitric oxide synthesis increases renin secretion. Recent studies indicate that nitric oxide not only contributes to the regulation of basal renin secretion, but also participates in the renin secretory responses to activation of the renal baroreceptor, macula densa, and beta adrenoceptor mechanisms that regulate renin secretion. Histochemical and immunocytochemical studies have revealed the presence of nitric oxide synthase in the supraoptic and paraventricular nuclei of the hypothalamus and in the posterior pituitary gland. Colocalization of nitric oxide synthase and vasopressin has been demonstrated in some hypothalamic neurons. Nitric oxide synthase activity in the hypothalamus and pituitary is increased by maneuvers known to stimulate vasopressin secretion, including salt loading and dehydration, Administration of L-arginine and nitric

  9. Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis

    PubMed Central

    Federico, Giuseppina; Meister, Michael; Mathow, Daniel; Heine, Gunnar H.; Moldenhauer, Gerhard; Popovic, Zoran V.; Nordström, Viola; Kopp-Schneider, Annette; Hielscher, Thomas; Nelson, Peter J.; Schaefer, Franz; Porubsky, Stefan; Fliser, Danilo; Arnold, Bernd; Gröne, Hermann-Josef

    2016-01-01

    Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia–derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis. PMID:27699213

  10. Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

    PubMed Central

    Benito-Martin, Alberto; Ucero, Alvaro Conrado; Zubiri, Irene; Posada-Ayala, Maria; Fernandez-Fernandez, Beatriz; Cannata-Ortiz, Pablo; Sanchez-Nino, Maria Dolores; Ruiz-Ortega, Marta; Egido, Jesus; Alvarez-Llamas, Gloria; Ortiz, Alberto

    2013-01-01

    Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine. PMID:24058411

  11. Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis

    PubMed Central

    Federico, Giuseppina; Meister, Michael; Mathow, Daniel; Heine, Gunnar H.; Moldenhauer, Gerhard; Popovic, Zoran V.; Nordström, Viola; Kopp-Schneider, Annette; Hielscher, Thomas; Nelson, Peter J.; Schaefer, Franz; Porubsky, Stefan; Fliser, Danilo; Arnold, Bernd; Gröne, Hermann-Josef

    2016-01-01

    Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia–derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.

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

    SciTech Connect

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

    2013-09-10

    We announce the discovery of a {approx}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{sup -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.

  13. Secretly Eccentric: The Giant Planet and Activity Cycle of GJ 328

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

    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.

  14. Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

    PubMed Central

    Piumi, Francois; Valli, Richard; Aramburu, Juan Carro; Ferreira, Patricia; Faulds, Craig B.; Record, Eric

    2016-01-01

    Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus. PMID:26873317

  15. Sleep Deprivation Aggravates Median Nerve Injury-Induced Neuropathic Pain and Enhances Microglial Activation by Suppressing Melatonin Secretion

    PubMed Central

    Huang, Chun-Ta; Chiang, Rayleigh Ping-Ying; Chen, Chih-Li; Tsai, Yi-Ju

    2014-01-01

    deprivation aggravates median nerve injury-induced neuropathic pain and enhances microglial activation by suppressing melatonin secretion. SLEEP 2014;37(9):1513-1523. PMID:25142572

  16. Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets.

    PubMed

    Berdan, Charles A; Erion, Karel A; Burritt, Nathan E; Corkey, Barbara E; Deeney, Jude T

    2016-01-01

    Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

  17. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step. PMID:27510653

  18. Effects of free amino acids on cytokine secretion and proliferative activity of feline T cells in an in vitro study using the cell line MYA-1.

    PubMed

    Paßlack, Nadine; Doherr, Marcus G; Zentek, Jürgen

    2016-10-01

    In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step.

  19. Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling

    PubMed Central

    Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M.; Maddocks, Kami; Yu, Lianbo; Byrd, John C.

    2015-01-01

    Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile. PMID:25833959

  20. Active and secreted IgA-coated bacterial fractions from the human gut reveal an under-represented microbiota core

    PubMed Central

    D'Auria, Giuseppe; Peris-Bondia, Francesc; Džunková, Mária; Mira, Alex; Collado, Maria Carmen; Latorre, Amparo; Moya, Andrés

    2013-01-01

    Host-associated microbiota varies in distribution depending on the body area inhabited. Gut microbes are known to interact with the human immune system, maintaining gut homoeostasis. Thus, we studied whether secreted-IgA (S-IgA) coat specific microbial taxa without inducing strong immune responses. To do so, we fractionated gut microbiota by flow cytometry. We found that active and S-IgA-coated bacterial fractions were characterized by a higher diversity than those observed in raw faecal suspensions. A long-tail effect was observed in family distribution, revealing that rare bacteria represent up to 20% of total diversity. While Firmicutes was the most abundant phylum, the majority of its sequences were not assigned at the genus level. Finally, the single-cell-based approach enabled us to focus on active and S-IgA-coated bacteria. Thus, we revealed a microbiota core common to the healthy volunteers participating in the study. Interestingly, this core was composed mainly of low frequency taxa (e.g. Sphingomonadaceae). PMID:24343271

  1. Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

    PubMed Central

    Egashira, Mahiro; Bai, Rulan; Nomura, Nana; Nomura, Shintaro; Hirota, Yasushi; Sakurai, Toshihiro; Imakawa, Kazuhiko

    2012-01-01

    Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity. PMID:23152823

  2. Tubular inverse opal scaffolds for biomimetic vessels.

    PubMed

    Zhao, Ze; Wang, Jie; Lu, Jie; Yu, Yunru; Fu, Fanfan; Wang, Huan; Liu, Yuxiao; Zhao, Yuanjin; Gu, Zhongze

    2016-07-14

    There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially oriented elliptical pattern microstructures on their surfaces. It is demonstrated that these tailored tubular scaffolds can effectively make endothelial cells to form an integrated hollow tubular structure on their inner surface and induce smooth muscle cells to form a circumferential orientation on their outer surface. These features of our tubular scaffolds make them highly promising for the construction of biomimetic blood vessels. PMID:27241065

  3. Tubular inverse opal scaffolds for biomimetic vessels.

    PubMed

    Zhao, Ze; Wang, Jie; Lu, Jie; Yu, Yunru; Fu, Fanfan; Wang, Huan; Liu, Yuxiao; Zhao, Yuanjin; Gu, Zhongze

    2016-07-14

    There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially oriented elliptical pattern microstructures on their surfaces. It is demonstrated that these tailored tubular scaffolds can effectively make endothelial cells to form an integrated hollow tubular structure on their inner surface and induce smooth muscle cells to form a circumferential orientation on their outer surface. These features of our tubular scaffolds make them highly promising for the construction of biomimetic blood vessels.

  4. Tubular up-regulation of clusterin mRNA in murine lupus-like nephritis.

    PubMed Central

    Moll, S.; Menoud, P. A.; French, L.; Sappino, A. P.; Pastore, Y.; Schifferli, J. A.; Izui, S.

    1998-01-01

    Clusterin, a widely distributed glycoprotein, is detected in most tissues and in numerous physiological fluids. In the kidney, this protein is constitutively expressed in tubular epithelial cells, and its expression is enhanced following tubular injuries. In addition, clusterin has been detected in glomerular immune deposits of glomerulonephritis. The present study was designed to define the sites of clusterin mRNA accumulation in murine lupus-like nephritis in comparison with murine tubulopathies. In lupus-like nephritis, a significant increase of clusterin mRNA abundance was demonstrated. This up-regulation was localized exclusively in tubular epithelial cells exhibiting tubulointerstitial alterations, whereas no clusterin mRNA was detectable in diseased glomeruli, excluding an active synthesis of clusterin by glomerular cells. A similar tubular increase of clusterin mRNA abundance was observed in myeloma-like cast nephropathy induced by IgG3 monoclonal cryoglobulins and even in the absence of any detectable histological alterations in a model of septic shock induced by the injection of bacterial lipopolysaccharides. Our results suggest that tubular epithelial cells are the only sites of clusterin mRNA accumulation during the course of lupus-like nephritis and that the tubular up-regulation of clusterin gene expression may reflect the cellular response to various types of tubular injuries. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 7 PMID:9546356

  5. Tubular up-regulation of clusterin mRNA in murine lupus-like nephritis.

    PubMed

    Moll, S; Menoud, P A; French, L; Sappino, A P; Pastore, Y; Schifferli, J A; Izui, S

    1998-04-01

    Clusterin, a widely distributed glycoprotein, is detected in most tissues and in numerous physiological fluids. In the kidney, this protein is constitutively expressed in tubular epithelial cells, and its expression is enhanced following tubular injuries. In addition, clusterin has been detected in glomerular immune deposits of glomerulonephritis. The present study was designed to define the sites of clusterin mRNA accumulation in murine lupus-like nephritis in comparison with murine tubulopathies. In lupus-like nephritis, a significant increase of clusterin mRNA abundance was demonstrated. This up-regulation was localized exclusively in tubular epithelial cells exhibiting tubulointerstitial alterations, whereas no clusterin mRNA was detectable in diseased glomeruli, excluding an active synthesis of clusterin by glomerular cells. A similar tubular increase of clusterin mRNA abundance was observed in myeloma-like cast nephropathy induced by IgG3 monoclonal cryoglobulins and even in the absence of any detectable histological alterations in a model of septic shock induced by the injection of bacterial lipopolysaccharides. Our results suggest that tubular epithelial cells are the only sites of clusterin mRNA accumulation during the course of lupus-like nephritis and that the tubular up-regulation of clusterin gene expression may reflect the cellular response to various types of tubular injuries.

  6. The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-κB early during infection.

    PubMed

    Pallett, Mitchell A; Berger, Cedric N; Pearson, Jaclyn S; Hartland, Elizabeth L; Frankel, Gad

    2014-11-01

    The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the host inflammatory response during infection. Previously, it has been reported that EPEC, in a T3SS-dependent manner, induces an early proinflammatory response through activation of NF-κB via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cζ (PKCζ). However, the activation of NF-κB during infection has not yet been attributed to an effector. At later time points postinfection, NF-κB signaling is inhibited through the translocation of multiple effectors, including NleE and NleC. Here we report that the highly conserved non-LEE (locus of enterocyte effacement)-encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic expression. Moreover, NleF induces the nuclear translocation of NF-κB p65 and the expression of interleukin 8 (IL-8) following ectopic expression and during EPEC infection. Furthermore, the proinflammatory activity and localization of NleF were dependent on the C-terminal amino acids LQCG. While the C-terminal domain of NleF has previously been shown to be essential for interaction with caspase-4, caspase-8, and caspase-9, the proinflammatory activity of NleF was independent of interaction with caspase-4, -8, or -9. In conclusion, EPEC, through the T3SS-dependent translocation of NleF, induces a proinflammatory response in an NF-κB-dependent manner in the early stages of infection.

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

    PubMed

    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. PMID:22765374

  8. The Type III Secretion Effector NleF of Enteropathogenic Escherichia coli Activates NF-κB Early during Infection

    PubMed Central

    Pallett, Mitchell A.; Berger, Cedric N.; Pearson, Jaclyn S.; Hartland, Elizabeth L.

    2014-01-01

    The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the host inflammatory response during infection. Previously, it has been reported that EPEC, in a T3SS-dependent manner, induces an early proinflammatory response through activation of NF-κB via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cζ (PKCζ). However, the activation of NF-κB during infection has not yet been attributed to an effector. At later time points postinfection, NF-κB signaling is inhibited through the translocation of multiple effectors, including NleE and NleC. Here we report that the highly conserved non-LEE (locus of enterocyte effacement)-encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic expression. Moreover, NleF induces the nuclear translocation of NF-κB p65 and the expression of interleukin 8 (IL-8) following ectopic expression and during EPEC infection. Furthermore, the proinflammatory activity and localization of NleF were dependent on the C-terminal amino acids LQCG. While the C-terminal domain of NleF has previously been shown to be essential for interaction with caspase-4, caspase-8, and caspase-9, the proinflammatory activity of NleF was independent of interaction with caspase-4, -8, or -9. In conclusion, EPEC, through the T3SS-dependent translocation of NleF, induces a proinflammatory response in an NF-κB-dependent manner in the early stages of infection. PMID:25183730

  9. High Systemic Levels of the Cytokine-Inducing HMGB1 Isoform Secreted in Severe Macrophage Activation Syndrome

    PubMed Central

    Palmblad, Karin; Schierbeck, Hanna; Sundberg, Erik; Horne, Anna-Carin; Harris, Helena Erlandsson; Henter, Jan-Inge; Antoine, Daniel J; Andersson, Ulf

    2014-01-01

    Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS. PMID:25247290

  10. Activated extracellular signal-regulated kinases are necessary and sufficient to initiate tubulogenesis in renal tubular MDCK strain I cell cysts.

    PubMed

    Hellman, Nathan E; Greco, Andres J; Rogers, Katherine K; Kanchagar, Chitra; Balkovetz, Daniel F; Lipschutz, Joshua H

    2005-10-01

    A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. Most cyst and tubule formation studies that utilized MDCK cells have been performed with MDCK strain II cells. MDCK strain II cells form hollow cysts in a three-dimensional collagen matrix over 10 days and tubulate in response to hepatocyte growth factor, which increases levels of active (phosphorylated) ERK1/2. In this study, we demonstrate that MDCK strain I cells also form cysts when grown in a collagen matrix; however, MDCK strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK strain I and strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. MDCK strain I cell cysts can spontaneously initiate tubulogenesis mainly because of relatively higher levels of active ERK in MDCK strain I, compared with strain II, cells. The presence of either of two distinct inhibitors of ERK activation (UO126 and PD09059) prevents tubulogenesis from occurring spontaneously in MDCK strain I cell cysts and, in response to hepatocyte growth factor, in strain II cell cysts. The difference between MDCK strain I and strain II cell lines is likely explained by differing embryological origins, with strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK and spontaneously tubulate in our in vitro collagen gel system, with tubulogenesis inhibited by UO126 and PD09059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells destined to form the collecting tubules.

  11. Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.

    PubMed

    Silanikove, Nissim; Shapiro, Fira; Shamay, Avi; Leitner, Gabriel

    2005-05-01

    The aims of this study were to test whether xanthine oxidase, lactoperoxidase, and NO are components of the innate immune system of mammary secretion during active involution in dairy cows, and whether the innate immune system is activated by casein hydrolysates. Our laboratory has shown recently that infusion of CNH into mammary glands induced involution and was associated with earlier increases in the concentrations of components of the innate immune system. Intact casein is inactive and served as control. Half of the glands of 8 Holstein cows scheduled for dry off (approximately 60 days before parturition) were injected for 3 days with a single dose of casein hydrolyzates and the contralateral glands with a single dose of intact casein with the same concentration. Involution elicited marked increases in xanthine oxidase and lactoperoxidase activities, and accumulation of urate and nitrate. NO and H(2)O(2) were constantly produced in the mammary gland secretion. Nitrite formed either by autooxidation of NO or by conversion of nitrate to nitrite by xanthine oxidase was converted into the powerful nitric dioxide radical by lactoperoxidase and H(2)O(2) that is derived from the metabolism of xanthine oxidase. Nitric dioxide is most likely responsible for the formation of nitrosothiols on thiol-bearing groups, which allows an extended NO presence in mammary secretion. Nitrite is effectively converted to nitrate, which accumulated in the range of approximately 25 microM -1 mM from the start of the experiment to the complete involution of glands. The mammary secretion in all glands was bactericidal and bacteriostatic during established involution, and this appeared sooner and more acutely in glands treated with casein hydrolyzates, within 8 to 24 h. It is concluded that xanthine oxidase, lactoperoxidase, and NO are components of the mammary innate immune system that form bactericidal and bacteriostatic activities in mammary secretions. The innate immune system play a

  12. Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.

    PubMed

    Silanikove, Nissim; Shapiro, Fira; Shamay, Avi; Leitner, Gabriel

    2005-05-01

    The aims of this study were to test whether xanthine oxidase, lactoperoxidase, and NO are components of the innate immune system of mammary secretion during active involution in dairy cows, and whether the innate immune system is activated by casein hydrolysates. Our laboratory has shown recently that infusion of CNH into mammary glands induced involution and was associated with earlier increases in the concentrations of components of the innate immune system. Intact casein is inactive and served as control. Half of the glands of 8 Holstein cows scheduled for dry off (approximately 60 days before parturition) were injected for 3 days with a single dose of casein hydrolyzates and the contralateral glands with a single dose of intact casein with the same concentration. Involution elicited marked increases in xanthine oxidase and lactoperoxidase activities, and accumulation of urate and nitrate. NO and H(2)O(2) were constantly produced in the mammary gland secretion. Nitrite formed either by autooxidation of NO or by conversion of nitrate to nitrite by xanthine oxidase was converted into the powerful nitric dioxide radical by lactoperoxidase and H(2)O(2) that is derived from the metabolism of xanthine oxidase. Nitric dioxide is most likely responsible for the formation of nitrosothiols on thiol-bearing groups, which allows an extended NO presence in mammary secretion. Nitrite is effectively converted to nitrate, which accumulated in the range of approximately 25 microM -1 mM from the start of the experiment to the complete involution of glands. The mammary secretion in all glands was bactericidal and bacteriostatic during established involution, and this appeared sooner and more acutely in glands treated with casein hydrolyzates, within 8 to 24 h. It is concluded that xanthine oxidase, lactoperoxidase, and NO are components of the mammary innate immune system that form bactericidal and bacteriostatic activities in mammary secretions. The innate immune system play a

  13. Active sodium transport and fluid secretion in the gall-bladder epithelium of Necturus.

    PubMed Central

    Giraldez, F

    1984-01-01

    Intracellular Na, K and Cl activities (acNa, acK and acCl) and membrane potentials were measured in Necturus gall-bladder epithelium using double-barrelled ion-sensitive micro-electrodes. Mucosal membrane potential was about -55 mV and the mean control activities were acNa = 14.7 mM, acK = 91.6 mM and acCl = 20.3 mM. Replacing mucosal Na by K caused a fall in acNa that followed an exponential time course. The rate of change in acNa was linearly related to acNa above a certain value (congruent to 3 mM). acK and acCl both increased in K Ringer solution. From the change in all three ions the cell was estimated to swell at an initial rate of 0.13% s-1. From the initial rate of change in acNa, a net cell efflux of Na of 405 pmol cm-2 s-1 was calculated. Replacement of Na by Tris or choline led to a similar result. The transepithelial Na transport rate was for this group of animals 346 pmol cm-2 s-1. Ouabain (10(-3) M) produced an increase in acNa and acCl, whereas acK decreased. The cells were estimated to swell at an initial rate of 0.06% s-1. The initial Na influx after Na-pump inhibition was calculated to be 162 pmol cm-2 s-1. The parallel measure of the transepithelial rate of transport of Na gave a value of 189 pmol cm-2 s-1. Ouabain inhibited the decrease in acNa after replacement of Na by K by about 80%. A fast depolarization, ranging from 2 to 7 mV, occurred after the perfusion with ouabain. Em then slowly decreased from about 53 to 32 mV in 1 h. It is concluded that (a) the major fraction of the transepithelial transport of Na is transcellular and mediated by the Na pump, (b) the pumping rate is linearly dependent on internal Na within a certain range and (c) the Na pump is electrogenic under normal circumstances. PMID:6716291

  14. A theoretical study of factors influencing calcium-secretion coupling in a presynaptic active zone model.

    PubMed

    Gil, Amparo; González-Vélez, Virginia; Segura, Javier; Gutiérrez, Luis Miguel

    2014-10-01

    A theoretical analysis of some of the relevant factors influencing the calcium time course and the strength and timing of release probabilities of vesicles evoked by an action potential in a calyx-type active zone is presented in this paper. In particular, our study focus on the comparison of cooperative vs non-cooperative calcium binding by the release site and the effect of the number of Ca(2+) binding sites on the calcium sensitivity for release. Regarding the comparison of cooperative and non-cooperative kinetic schemes, our simulations show that quite different results are obtained when considering one or another: a reduction in the release probability of more than a 50% is obtained when considering the cooperative kinetic scheme. Also, a delay in the average time for release appears when using this model for the calcium sensor. Our study also shows that a non-cooperative kinetic binding scheme gives rise to a well defined average calcium level for release assuming that the same kinetic constants are considered for all the sites. Our results also suggest that the central value of the calcium sensitivity for release depends on the number of binding sites N and the dissociation constant KD with a scaling law depending on NKD.

  15. Secretion of multi-protein migratory complex induced by Toxoplasma gondii infection in macrophages involves the uPA/uPAR activation system.

    PubMed

    Schuindt, Sara Hellen Santos; Oliveira, Bruno Cabral de Lima; Pimentel, Pollyana Maria de Oliveira; Resende, Thatiane Lacerda; Retamal, Cláudio A; DaMatta, Renato A; Seipel, Daniele; Arnholdt, Andrea Cristina Vetö

    2012-05-25

    Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular mechanisms or outcomes of the subversion of the host cell are largely unknown. Recently our group established that metalloproteinases are involved in migration of infected macrophages. Herein, we evaluated the recruitment of host invasive machinery components in T. gondii infected murine macrophages. We showed by immunoprecipitation assays that MMP-9, CD44 TIMP-1 and uPAR were secreted as a multi-protein complex by infected macrophages. Zymographic analysis revealed that MMP-9 was present in its pro- and active form. Moreover, inhibition of uPA/uPAR pathway by PAI-1 decreased secretion of MMP-9 active forms, as well those associated to uPAR and TIMP-1, but not to CD44. Data presented here suggest that MMP-9 is secreted as a multiprotein complex by T. gondii infected macrophages, similar to that observed in metastatic cells. We further speculate that uPA/uPAR system is involved in the expression/secretion of complexes containing active MMP-9 forms.

  16. Cdk5 inhibitory peptide (CIP) inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

    PubMed

    Zheng, Ya-Li; Li, Congyu; Hu, Ya-Fang; Cao, Li; Wang, Hui; Li, Bo; Lu, Xiao-Hua; Bao, Li; Luo, Hong-Yan; Shukla, Varsha; Amin, Niranjana D; Pant, Harish C

    2013-01-01

    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. PMID:24039692

  17. Activation of PI3K-Akt-GSK3{beta} pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

    SciTech Connect

    Gong Rujun . E-mail: rgong@Brown.edu; Rifai, Abdalla; Dworkin, Lance D.

    2005-04-29

    Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-{alpha}-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-{alpha}-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3{beta} or an uninhibitable mutant GSK3{beta}, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3{beta}) in HKC. Overexpression of wild type GSK3{beta} did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3{beta} abolished HGF inhibition of basal and TNF-{alpha} stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3{beta} are required for HGF-induced suppression of RANTES in HKC.

  18. Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2.

    PubMed

    Kirby, Elizabeth D; Muroy, Sandra E; Sun, Wayne G; Covarrubias, David; Leong, Megan J; Barchas, Laurel A; Kaufer, Daniela

    2013-04-16

    Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain. DOI:http://dx.doi.org/10.7554/eLife.00362.001.

  19. Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity.

    PubMed

    Mechkarska, Milena; Ojo, Opeolu O; Meetani, Mohammed A; Coquet, Laurent; Jouenne, Thierry; Abdel-Wahab, Yasser H A; Flatt, Peter R; King, Jay D; Conlon, J Michael

    2011-02-01

    Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30nM, with a maximum response (236% of basal rate, P<0.001) at a concentration of 3μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P<0.001 at 3μM) but the peptide was cytotoxic at this concentration. PMID:21087647

  20. The secret struggle of the active girl: a qualitative synthesis of interpersonal factors that influence physical activity in adolescent girls.

    PubMed

    Standiford, Anne

    2013-10-01

    The author conducted a systematic review of 19 international, multidisciplinary, qualitative studies of interpersonal factors that influence physical activity in adolescent girls. Themes were deductively generated based on reported findings, and were organized according to frequency of occurrence. Themes were further organized according to a theoretical model to illustrate how interpersonal, perceptual, and situational influences affect physical activity in adolescent girls. The three most frequently discovered themes follow: (a) ability comparison and competition; (b) family, peer, and teacher influence; and (c) appearance concerns. It is important to consider the influence of gender role conflict on physical activity.

  1. The secret struggle of the active girl: a qualitative synthesis of interpersonal factors that influence physical activity in adolescent girls.

    PubMed

    Standiford, Anne

    2013-10-01

    The author conducted a systematic review of 19 international, multidisciplinary, qualitative studies of interpersonal factors that influence physical activity in adolescent girls. Themes were deductively generated based on reported findings, and were organized according to frequency of occurrence. Themes were further organized according to a theoretical model to illustrate how interpersonal, perceptual, and situational influences affect physical activity in adolescent girls. The three most frequently discovered themes follow: (a) ability comparison and competition; (b) family, peer, and teacher influence; and (c) appearance concerns. It is important to consider the influence of gender role conflict on physical activity. PMID:23790150

  2. Tubular cell phenotype in HIV-associated nephropathy: role of phospholipid lysophosphatidic acid.

    PubMed

    Ayasolla, Kamesh R; Rai, Partab; Rahimipour, Shai; Hussain, Mohammad; Malhotra, Ashwani; Singhal, Pravin C

    2015-08-01

    Collapsing glomerulopathy and microcysts are characteristic histological features of HIV-associated nephropathy (HIVAN). We have previously reported the role of epithelial mesenchymal transition (EMT) in the development of glomerular and tubular cell phenotypes in HIVAN. Since persistent tubular cell activation of NFκB has been reported in HIVAN, we now hypothesize that HIV may be contributing to tubular cell phenotype via lysophosphatidic acid (LPA) mediated downstream signaling. Interestingly, LPA and its receptors have also been implicated in the tubular interstitial cell fibrosis (TIF) and cyst formation in autosomal dominant polycystic kidney disease (PKD). Primary human proximal tubular cells (HRPTCs) were transduced with either empty vector (EV/HRPTCs), HIV (HIV/HRPTCs) or treated with LPA (LPA/HRPTC). Immunoelectrophoresis of HIV/HRPTCs and LPA/HRPTCs displayed enhanced expression of pro-fibrotic markers: a) fibronectin (2.25 fold), b) connective tissue growth factor (CTGF; 4.8 fold), c) α-smooth muscle actin (α-SMA; 12 fold), and d) collagen I (5.7 fold). HIV enhanced tubular cell phosphorylation of ILK-1, FAK, PI3K, Akt, ERKs and P38 MAPK. HIV increased tubular cell transcriptional binding activity of NF-κB; whereas, a LPA biosynthesis inhibitor (AACOCF3), a DAG kinase inhibitor, a LPA receptor blocker (Ki16425), a NF-κB inhibitor (PDTC) and NFκB-siRNA not only displayed downregulation of a NFκB activity but also showed attenuated expression of profibrotic/EMT genes in HIV milieu. These findings suggest that LPA could be contributing to HIV-induced tubular cell phenotype via NFκB activation in HIVAN. PMID:26079546

  3. Angiotensin II and renal tubular ion transport.

    PubMed

    Valles, Patricia; Wysocki, Jan; Batlle, Daniel

    2005-08-29

    Angiotensin II, a potent vasoconstrictor, also participates in the regulation of renal sodium and water excretion, not only via a myriad of effects on renal hemodynamics, glomerular filtration rate, and regulation of aldosterone secretion, but also via direct effects on renal tubule transport. In addition, angiotensin II stimulates H+ secretion and HCO3- reabsorption in both proximal and distal tubules and regulates H+-ATPase activity in intercalated cells of the collecting tubule. Different results regarding the effect of angiotensin II on bicarbonate reabsorption and proton secretion have been reported at the functional level, depending on the angiotensin II concentration and tubule segment studied. It is likely that interstitial angiotensin II is more important in regulating hemodynamic and transport functions than circulating angiotensin II. In proximal tubules, stimulation of bicarbonate reabsorption, Na+/H+-exchange, and Na+/HCO3- cotransport has been found using low concentrations (<10(-9) M), while inhibition of bicarbonate reabsorption has been documented using concentrations higher than 10(-8) M. Evidence for the regulation of H+-ATPase activity in vivo and in vitro by trafficking/exocytosis has been provided. An additional level of H+-ATPase regulation via protein synthesis may be important as well. Recently, we have shown that both aldosterone and angiotensin II provide such a mechanism of regulation in vivo at the level of the medullary collecting tubule. Interestingly, in this part of the nephron, the effects of aldosterone and angiotensin II are not sodium dependent, whereas in the cortical collecting duct, both aldosterone and angiotensin II, by contrast, affect H+ secretion by sodium-dependent mechanisms.

  4. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  5. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  6. Actions of Agonists and Antagonists of the ghrelin/GHS-R Pathway on GH Secretion, Appetite, and cFos Activity.

    PubMed

    Hassouna, Rim; Labarthe, Alexandra; Zizzari, Philippe; Videau, Catherine; Culler, Michael; Epelbaum, Jacques; Tolle, Virginie

    2013-01-01

    The stimulatory effects of ghrelin, a 28-AA acylated peptide originally isolated from stomach, on growth hormone (GH) secretion and feeding are exclusively mediated through the growth hormone secretagogue 1a receptor (GHS-R1a), the only ghrelin receptor described so far. Several GHS-R1a agonists and antagonists have been developed to treat metabolic or nutritional disorders but their mechanisms of action in the central nervous system remain poorly understood. In the present study, we compared the activity of BIM-28163, a GHS-R1a antagonist, and of several agonists, including native ghrelin and the potent synthetic agonist, BIM-28131, to modulate food intake, GH secretion, and cFos activity in arcuate nucleus (ArcN), nucleus tractus solitarius (NTS), and area postrema (AP) in wild-type and NPY-GFP mice. BIM-28131 was as effective as ghrelin in stimulating GH secretion, but more active than ghrelin in inducing feeding. It stimulated cFos activity similarly to ghrelin in the NTS and AP but was more powerful in the ArcN, suggesting that the super-agonist activity of BIM-28131 is mostly mediated in the ArcN. BIM-28163 antagonized ghrelin-induced GH secretion but not ghrelin-induced food consumption and cFos activation, rather it stimulated food intake and cFos activity without affecting GH secretion. The level of cFos activation was dependent on the region considered: BIM-28163 was as active as ghrelin in the NTS, but less active in the ArcN and AP. All compounds also induced cFos immunoreactivity in ArcN NPY neurons but BIM-28131 was the most active. In conclusion, these data demonstrate that two peptide analogs of ghrelin, BIM-28163, and BIM-28131, are powerful stimulators of appetite in mice, acting through pathways and key brain regions involved in the control of appetite that are only partially superimposable from those activated by ghrelin. A better understanding of the molecular pathways activated by these compounds could be useful in devising future therapeutic

  7. Actions of Agonists and Antagonists of the ghrelin/GHS-R Pathway on GH Secretion, Appetite, and cFos Activity

    PubMed Central

    Hassouna, Rim; Labarthe, Alexandra; Zizzari, Philippe; Videau, Catherine; Culler, Michael; Epelbaum, Jacques; Tolle, Virginie

    2012-01-01

    The stimulatory effects of ghrelin, a 28-AA acylated peptide originally isolated from stomach, on growth hormone (GH) secretion and feeding are exclusively mediated through the growth hormone secretagogue 1a receptor (GHS-R1a), the only ghrelin receptor described so far. Several GHS-R1a agonists and antagonists have been developed to treat metabolic or nutritional disorders but their mechanisms of action in the central nervous system remain poorly understood. In the present study, we compared the activity of BIM-28163, a GHS-R1a antagonist, and of several agonists, including native ghrelin and the potent synthetic agonist, BIM-28131, to modulate food intake, GH secretion, and cFos activity in arcuate nucleus (ArcN), nucleus tractus solitarius (NTS), and area postrema (AP) in wild-type and NPY-GFP mice. BIM-28131 was as effective as ghrelin in stimulating GH secretion, but more active than ghrelin in inducing feeding. It stimulated cFos activity similarly to ghrelin in the NTS and AP but was more powerful in the ArcN, suggesting that the super-agonist activity of BIM-28131 is mostly mediated in the ArcN. BIM-28163 antagonized ghrelin-induced GH secretion but not ghrelin-induced food consumption and cFos activation, rather it stimulated food intake and cFos activity without affecting GH secretion. The level of cFos activation was dependent on the region considered: BIM-28163 was as active as ghrelin in the NTS, but less active in the ArcN and AP. All compounds also induced cFos immunoreactivity in ArcN NPY neurons but BIM-28131 was the most active. In conclusion, these data demonstrate that two peptide analogs of ghrelin, BIM-28163, and BIM-28131, are powerful stimulators of appetite in mice, acting through pathways and key brain regions involved in the control of appetite that are only partially superimposable from those activated by ghrelin. A better understanding of the molecular pathways activated by these compounds could be useful in devising future therapeutic

  8. Treatment of well tubulars with gelatin

    SciTech Connect

    Lowther, F.E.

    1992-08-04

    This patent describes a method for treating a tubular in a well. It comprises: passing a mass of gelatin downward through the tubular; and passing the mass of gelating, upward in the well tubular toward the surface. This patent also describes a method of treating tubulars in a cased well having at least one string of tubing therein. It comprises positioning a mass in the annulus formed between the casing and the at least one string of tubing; and passing the mass downward in the annulus and in contact with both the inner wall of the casing and the outer wall of the tubing to deposit a protective layer on each of the walls.

  9. METHOD AND APPARATUS FOR FABRICATING TUBULAR UNITS

    DOEpatents

    Haldeman, G.W.

    1959-02-24

    A method and apparatus are described for fabricating tubular assemblies such as clad fuel elements for nuclear reactors. According to this method, a plurality of relatively short cylindrical slug-shaped members are inserted in an outer protective tubular jacket, and the assembly is passed through a reducing die to draw the outer tubular member into tight contact with the slug members, the slugs being automatically spaced with respect to each other and helium being inserted during the drawing operation to fill the spaces. The apparatus includes a pusher rod which functions to space the slugelements equidistantly by pushing on them in the direction of drawing but traveling at a slower rate than that of the tubular member.

  10. An open tubular ion chromatograph.

    PubMed

    Yang, Bingcheng; Zhang, Min; Kanyanee, Tinakorn; Stamos, Brian N; Dasgupta, Purnendu K

    2014-12-01

    We describe an open tubular ion chromatograph (OTIC) that uses anion exchange latex coated 5 μm radius silica and 9.8 μm radius poly(methyl methacrylate) tubes and automated time/pressure based hydrodynamic injection for pL-nL scale injections. It is routinely possible to generate 50,000 plates or more (up to 150,000 plates/m, columns between 0.3 and 0.8 m have been used), and as such, fast separations are possible, comparable to or in some cases better than the current practice of IC. With an optimized admittance detector, nonsuppressed detection permits LODs of submicromolar to double digit micromolar for a variety of analytes. However, large volume injections are possible and can significantly improve on this. A variety of eluents, the use of organic modifiers, and variations of eluent pH can be used to tailor a given separation. The approach is discussed in the context of extraterrestrial exploration, especially Mars, where the existence of large amounts of perchlorate in the soil needs to be confirmed. These columns can survive drying and freezing, and small footprint, low power consumption, and simplicity make OTIC a good candidate for such a mission. PMID:25394230

  11. Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity

    PubMed Central

    Ertunc, Meric Erikci; Sikkeland, Jørgen; Fenaroli, Federico; Griffiths, Gareth; Daniels, Mathew P.; Cao, Haiming; Saatcioglu, Fahri; Hotamisligil, Gökhan S.

    2015-01-01

    Adipocyte fatty acid binding protein 4, aP2, contributes to the pathogenesis of several common diseases including type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Although the biological functions of aP2 have classically been attributed to its intracellular action, recent studies demonstrated that aP2 acts as an adipokine to regulate systemic metabolism. However, the mechanism and regulation of aP2 secretion remain unknown. Here, we demonstrate a specific role for lipase activity in aP2 secretion from adipocytes in vitro and ex vivo. Our results show that chemical inhibition of lipase activity, genetic deficiency of adipose triglyceride lipase and, to a lesser extent, hormone-sensitive lipase blocked aP2 secretion from adipocytes. Increased lipolysis and lipid availability also contributed to aP2 release as determined in perilipin1-deficient adipose tissue explants ex vivo and upon treatment with lipids in vivo and in vitro. In addition, we identify a nonclassical route for aP2 secretion in exosome-like vesicles and show that aP2 is recruited to this pathway upon stimulation of lipolysis. Given the effect of circulating aP2 on glucose metabolism, these data support that targeting aP2 or the lipolysis-dependent secretory pathway may present novel mechanistic and translational opportunities in metabolic disease. PMID:25535287

  12. YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

    PubMed Central

    Amer, Ayad A. A.; Gurung, Jyoti M.; Costa, Tiago R. D.; Ruuth, Kristina; Zavialov, Anton V.; Forsberg, Åke; Francis, Matthew S.

    2016-01-01

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity. PMID:27446813

  13. Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity.

    PubMed

    Lymperopoulos, Anastasios; Rengo, Giuseppe; Zincarelli, Carmela; Soltys, Stephen; Koch, Walter J

    2008-02-01

    We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

  14. DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

    PubMed Central

    Cascales, Eric; Atmakuri, Krishnamohan; Sarkar, Mayukh K.

    2013-01-01

    The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface. PMID:23564169

  15. DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system.

    PubMed

    Cascales, Eric; Atmakuri, Krishnamohan; Sarkar, Mayukh K; Christie, Peter J

    2013-06-01

    The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface. PMID:23564169

  16. Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells.

    PubMed

    Kim, Jung-Hee; Kim, Hye-Rim; Lee, Bo-Ram; Choi, Eun-Sook; In, Su-Il; Kim, Eunjoo

    2015-01-01

    Lead sulfide (PbS) quantum dots (QDs) have been applied in the biomedical area because they offer an excellent platform for theragnostic applications. In order to comprehensively evaluate the biocompatibility of PbS QDs in human cells, we analyzed the exosomes secreted from cells because exosomes are released during cellular stress to convey signals to other cells and serve as a reservoir of enriched biomarkers. PbS QDs were synthesized and coated with 3-mercaptopropionic acid (MPA) to allow the particles to disperse in water. Exosomes were isolated from HEK293 cells treated with PbS-MPA at concentrations of 0 µg/mL, 5 µg/mL, and 50 µg/mL, and the exosomal expression levels of miRNAs and proteins were analyzed. As a result, five miRNAs and two proteins were proposed as specific exosomal biomarkers for the exposure of HEK293 cells to PbS-MPA. Based on the pathway analysis, the molecular signature of the exosomes suggested that PbS-MPA QDs had carcinogenic activity. The comet assay and expression of molecular markers, such as p53, interleukin (IL)-8, and C-X-C motif chemokine 5, indicated that DNA damage occurred in HEK293 cells following PbS-MPA exposure, which supported the carcinogenic activity of the particles. In addition, there was obvious intensification of miRNA expression signals in the exosomes compared with that of the parent cells, which suggested that exosomal biomarkers could be detected more sensitively than those of whole cellular extracts.

  17. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

    PubMed Central

    Duchez, Anne-Claire; Boudreau, Luc H.; Naika, Gajendra S.; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E.; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J.; Poubelle, Patrice E.; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. PMID:26106157

  18. Acetylcholinesterase secreted by Anisakis simplex larvae (Nematoda: Anisakidae) parasitizing herring, Clupea harengus: an inverse relationship of enzyme activity in the host-parasite system.

    PubMed

    Podolska, Magdalena; Nadolna, Katarzyna

    2014-06-01

    Acetylcholinesterase (AChE) is a key enzyme involved in nerve impulse transmission in both vertebrates and invertebrates. In addition to neuromuscular AChE, many parasitic nematodes synthesize AChE in secretory glands and release the enzyme into their external environment. In this study, we evaluate the activities of both somatic and secreted AChE from larvae (L3) of the parasitic nematode Anisakis simplex, and compare these to the AChE activity in its host, herring, Clupea harengus. A. simplex larvae were obtained from a herring sampled in three areas of the southern Baltic. Enzyme kinetics were determined for excretory/secretory (E/S) products and somatic extracts of larvae as well as for herring muscle tissue. The results reveal that mean AChE activity is approximately fourfold higher in E/S products and eightfold higher in somatic extracts of post-secretory A. simplex larvae than in host muscle tissue. The level of AChE activity in nematodes is inversely related to the enzyme activity in their hosts, i.e. reduced AChE activity in herring was accompanied by increased enzyme activity in its parasites. The physiological function of AChE secreted by parasitic nematodes has been widely discussed in the literature, and numerous roles for this form of enzyme have been suggested. The results of our investigation indicate that AChE secretion by A. simplex larvae may constitute an adaptive mechanism that promotes survival under adverse environmental conditions. Larvae probably increase secretion of AChE in response to a direct and/or indirect effect of neurotoxic compounds. This is the first report of such a phenomenon in A. simplex.

  19. Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally.

    PubMed

    Campbell-Valois, F-X; Pontier, Stéphanie M

    2016-01-01

    Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed. PMID:27014638

  20. Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally

    PubMed Central

    Campbell-Valois, F.-X.; Pontier, Stéphanie M.

    2016-01-01

    Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed. PMID:27014638

  1. Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates.

    PubMed

    Perpetuo, Elen A; Lebrun, Ivo; Juliano, Luis; Juliano, Maria Aparecida; Sakauchi, Maria Aparecida; Prado, Sally M A

    2007-01-01

    Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates. PMID:17849290

  2. 78 FR 37584 - U.S. Steel Tubular Products, Inc., Mckeesport Tubular Operations Division, Subsidiary of United...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-21

    ... Employment and Training Administration U.S. Steel Tubular Products, Inc., Mckeesport Tubular Operations Division, Subsidiary of United States Steel Corporation, Mckeesport, Pennsylvania; Notice of Amended... workers of U.S. Steel Tubular Products, McKeesport Tubular Operations Division, a subsidiary of...

  3. Activation of FoxM1 Revitalizes the Replicative Potential of Aged β-Cells in Male Mice and Enhances Insulin Secretion

    PubMed Central

    Golson, Maria L.; Dunn, Jennifer C.; Maulis, Matthew F.; Dadi, Prasanna K.; Osipovich, Anna B.; Magnuson, Mark A.; Jacobson, David A.

    2015-01-01

    Type 2 diabetes incidence increases with age, while β-cell replication declines. The transcription factor FoxM1 is required for β-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged β-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase β-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged β-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced β-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in β-cell mass, suggesting that the loss of FoxM1 is detrimental to β-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in β-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases β-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes. PMID:26251404

  4. Secretion of monocyte chemotactic activity by cultured rat aortic smooth muscle cells in response to PDGF is due predominantly to the induction of JE/MCP-1.

    PubMed Central

    Poon, M.; Hsu, W. C.; Bogdanov, V. Y.; Taubman, M. B.

    1996-01-01

    Inflammation is a critical feature of atherosclerosis and is characterized in part by the migration of circulating monocytes to the atherosclerotic plaque. These monocytes, together with macrophages, are a source of cytokines, growth factors, proteases, and procoagulants, which contribute to the progression of the atherosclerosis lesion. This study employed a modified Boyden chamber to examine the secretion of monocyte chemotactic activity by cultured rat aortic vascular smooth muscle cells in response to growth factors and cytokines. The induction of monocyte chemotactic activity showed a surprising specificity for platelet-derived growth factor-BB. This activity was blocked by actinomycin D and cycloheximide and thus required de novo transcription and protein synthesis. The ability to stimulate monocyte migration appeared to be solely due to the secretion of the monocyte chemoattractant protein JE/MCP-1 and was completely blocked by antisense oligonucleotides and antibodies to JE/MCP-1. The induction of chemotactic activity was also blocked by dexamethasone, an inhibitor of JE mRNA accumulation. This study suggests that the secretion of monocyte chemotactic activity by vascular smooth muscle cells is a highly regulatable and specific event and underscores the importance of JE/MCP-1 in the inflammatory response of the vessel wall. Images Figure 5 PMID:8686755

  5. Salivary Gland Secretion.

    ERIC Educational Resources Information Center

    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)

  6. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    PubMed Central

    Gupta, Sachin; Termini, James M.; Issac, Biju; Guirado, Elizabeth; Stone, Geoffrey W.

    2016-01-01

    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. PMID:26849062

  7. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

    PubMed

    Gupta, Sachin; Termini, James M; Issac, Biju; Guirado, Elizabeth; Stone, Geoffrey W

    2016-01-01

    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. PMID:26849062

  8. Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

    PubMed

    Matoba, Yuko; Nonaka, Naoko; Takagi, Yoshitoki; Imamura, Eisaku; Narukawa, Masayuki; Nakamachi, Tomoya; Shioda, Seiji; Banks, William A; Nakamura, Masanori

    2016-09-01

    Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc. PMID:27339371

  9. Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum

    PubMed Central

    Lagha, Amel Ben; Grenier, Daniel

    2016-01-01

    Fusobacterium nucleatum has been associated with both periodontal disease and inflammatory bowel disease. This Gram-negative bacterium possesses a high inflammatory potential that may contribute to the disease process. We hypothesized that green and black tea polyphenols attenuate the inflammatory response of monocytes/macrophages mediated by F. nucleatum. We first showed that the tea extracts, EGCG and theaflavins reduce the NF-κB activation induced by F. nucleatum in monocytes. Since NF-κB is a key regulator of genes coding for inflammatory mediators, we tested the effects of tea polyphenols on secretion of IL-1β, IL-6, TNF-α, and CXCL8 by macrophages. A pre-treatment of macrophages with the tea extracts, EGCG, or theaflavins prior to a stimulation with F. nucleatum significantly inhibited the secretion of all four cytokines and reduced the secretion of MMP-3 and MMP-9, two tissue destructive enzymes. TREM-1 expressed by macrophages is a cell-surface receptor involved in the propagation of the inflammatory response to bacterial challenges. Interestingly, tea polyphenols inhibited the secretion/shedding of soluble TREM-1 induced by a stimulation of macrophages with F. nucleatum. The anti-inflammatory properties of tea polyphenols identified in the present study suggested that they may be promising agents for the prevention and/or treatment of periodontal disease and inflammatory bowel disease. PMID:27694921

  10. Group V Secretory Phospholipase A2 Is Involved in Tubular Integrity and Sodium Handling in the Kidney

    PubMed Central

    Moraes-Santos, Felipe; Landgraf, Sharon Schilling; Silva, Leandro Souza; Sirtoli, Gabriela Modenesi; Zamith-Miranda, Daniel; Takiya, Christina Maeda; Pinheiro, Ana Acacia Sá; Diaz, Bruno Lourenço; Caruso-Neves, Celso

    2016-01-01

    Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5−/−) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5−/− mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5−/− mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5−/− mice. The increased FENa+ observed in Pla2g5−/− mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5−/− mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity. PMID:26820468

  11. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Veyo, S.E.

    1995-08-01

    The development of a viable fuel cell driven electrical power generation system involves not only the development of cell and stack technology, but also the development of the overall system concept, the strategy for control, and the ancillary subsystems. The design requirements used to guide system development must reflect a customer focus in order to evolve a commercial product. In order to obtain useful customer feedback, Westinghouse has practiced the deployment with customers of fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units have served to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  12. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Ray, E.R.; Veyo, S.E.

    1995-12-31

    This reports on a solid oxide fuel cell demonstration program in which utilities are provided fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units serve to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  13. Toward high-torque electrostatic tubular motors

    NASA Astrophysics Data System (ADS)

    Helin, Philippe; Bourbon, Gilles; Minotti, Patrice; Fujita, Hiroyuki

    1999-10-01

    A new generation of electrostatic micro-motors is investigated using cooperation of arrayed direct-drive actuators. Electrostatic scratch-drive actuators (SDA), which combine active frictional contact mechanisms with electrostatic actuation, are particularly analyzed. Active polysilicon sheets of 2*3 mm2 that integrate up to several thousands of electrostatic scratch drive actuators are fabricated by silicon surface micro-machining process. Each elementary actuator provides its contribution according to the driving force superposition principle, with internal forces as high as 105uN are available from this sheet. According to their natural flexibility, active polysilicon sheets can be coated onto large surfaces. A new generation of self-assembled tubular electrostatic micromotors is developed using this concept. A prototype of a cylindrical micromotor, whose external diameter and length are 1 mm and 2 mm, respectively, has been realized through the insertion of a flexible active polysilicon sheet at the rotor/motor- frame interface. After final assembling, the sheet has to be jammed onto the chassis, in order to allow the rotor to be moved with respect to the motor frame. Thus, the sheet must be in close contact with both the rotor and the motor frame, whatever the gap, which separates the two macroscopic parts. The problem related to the micro/macro world interfacing is solved during the design of sheet in allowing an out-of- plane motion of SDA in order to provide a self gap compensation, whatever both the thermal expansion effects and the macroscopic machining tolerances. The expected driving characteristics show the interest of both cooperative arrayed microactuators and direct drive frictional mechanisms.

  14. Listeriolysin O-dependent bacterial entry into the cytoplasm is required for calpain activation and interleukin-1 alpha secretion in macrophages infected with Listeria monocytogenes.

    PubMed

    Dewamitta, Sita R; Nomura, Takamasa; Kawamura, Ikuo; Hara, Hideki; Tsuchiya, Kohsuke; Kurenuma, Takeshi; Shen, Yanna; Daim, Sylvia; Yamamoto, Takeshi; Qu, Huixin; Sakai, Shunsuke; Xu, Yanting; Mitsuyama, Masao

    2010-05-01

    Listeriolysin O (LLO), an hly-encoded cytolysin of Listeria monocytogenes, plays an essential role in the entry of L. monocytogenes into the host cell cytoplasm. L. monocytogenes-infected macrophages produce various proinflammatory cytokines, including interleukin-1 alpha (IL-1 alpha), that contribute to the host immune response. In this study, we have examined IL-1 alpha production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Delta hly). Expression of IL-1 alpha mRNA and accumulation of pro-IL-1 alpha in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1 alpha from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Delta hly mutant. A recovery of the ability to induce IL-1 alpha secretion was shown in a mutant strain complemented with the hly gene. The Toll-like receptor (TLR)/MyD88 signaling pathway was exclusively required for the expression of pro-IL-1 alpha, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1 alpha was abolished by addition of calcium chelators, and only LLO-producing L. monocytogenes strains were able to induce elevation of the intracellular calcium level in infected macrophages. A calcium-dependent protease, calpain, was implicated in the maturation and secretion of IL-1 alpha induced by LLO-producing L. monocytogenes strains based on the effect of calpain inhibitor. Functional activation of calpain was detected in macrophages infected with LLO-producing L. monocytogenes strains but not with a mutant strain lacking LLO. These results clearly indicated that LLO-mediated cytoplasmic entry of bacteria could induce the activation of intracellular calcium signaling, which is essential for maturation and secretion of IL-1 alpha in macrophages during L. monocytogenes infection through activation of a calcium-dependent calpain protease. In

  15. β-Adrenergic activation of electrogenic K+ and Cl− secretion in guinea pig distal colonic epithelium proceeds via separate cAMP signaling pathways

    PubMed Central

    Halm, Susan T.; Zhang, Jin

    2010-01-01

    Adrenergic stimulation of isolated guinea pig distal colonic mucosa produced transient Cl− and sustained K+ secretion. Transient short-circuit current (Isc) depended on β2-adrenergic receptors (β2-AdrR), and sustained Isc relies on a β1-AdrR/β2-AdrR complex. Epinephrine (epi) increased cAMP content with a biphasic time course similar to changes in epi-activated Isc (epiIsc). Inhibition of transmembrane adenylyl cyclases (tmACs) reduced peak epiIsc and cAMP to near zero without decreasing sustained epiIsc, consistent with cAMP from tmAC signaling for only Cl− secretion. Inhibition of soluble adenylyl cyclase (sAC) reduced sustained epiIsc and cAMP to near zero without decreasing peak epiIsc or cAMP, consistent with cAMP from sAC signaling for K+ secretion. Sensitivity to phosphodiesterase (PDE) inhibitors and peptide YY (PYY) stimulation further supported separate signaling for the two components. PDE3 or PDE4 inhibitors enhanced peak epiIsc but not sustained epiIsc, consistent with these PDEs as part of the β2-AdrR signaling domain. PYY suppressed peak epiIsc in a pertussis toxin (PTx)-sensitive manner, supporting Gαi-dependent inhibition of tmACs producing cAMP for Cl− secretion. Since PYY or PTx did not alter sustained epiIsc, signaling for K+ secretion occurred via a Gαi-independent mechanism. Presence of multiple sAC variants in colonic epithelial cells was supported by domain-specific antibodies. Responses to specific activators and inhibitors suggested that protein kinase A was not involved in activating peak or sustained components of epiIsc, but the cAMP-dependent guanine nucleotide exchange factor, Epac, may contribute. Thus β-adrenergic activation of electrogenic Cl− and K+ secretion, respectively, required tmAC- and sAC-dependent signaling pathways. PMID:20413718

  16. Melatonin receptor activation regulates GnRH gene expression and secretion in GT1-7 GnRH neurons. Signal transduction mechanisms.

    PubMed

    Roy, Deboleena; Belsham, Denise D

    2002-01-01

    Melatonin plays a significant role in the control of the hypothalamic-pituitary-gonadal axis. Using the GT1-7 cell line, an in vitro model of GnRH-secreting neurons of the hypothalamus, we examined the potential signal transduction pathways activated by melatonin directly at the level of the GT1-7 neuron. We found that melatonin inhibits forskolin-stimulated adenosine 3'-, 5'-cyclic monophosphate accumulation in GT1-7 cells through an inhibitory G protein. Melatonin induced protein kinase C activity by 1.65-fold over basal levels, increased the phosphorylation of extracellular signal-regulated kinase 1 and 2 proteins, and activated c-fos and junB mRNA expression in GT1-7 cells. Using the protein kinase A inhibitor H-89, the protein kinase C inhibitor bisindolylmaleimide, and the mitogen-activated protein kinase kinase inhibitor PD98059, we found that the melatonin-mediated cyclical regulation of GnRH mRNA expression may involve the protein kinase C and the extracellular signal-regulated kinase 1 and 2 pathways, but not the protein kinase A pathway. We found that melatonin suppresses GnRH secretion by approximately 45% in the GT1-7 neurons. However, in the presence of the inhibitors H-89, bisindolylmaleimide, and PD98059 melatonin was unable to suppress GnRH secretion. These results provide insights into the potential signal transduction mechanisms involved in the control of GnRH gene expression and secretion by melatonin.

  17. Salmonella-secreted Virulence Factors

    SciTech Connect

    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.

  18. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase.

    PubMed

    da Silva Xavier, G; Varadi, A; Ainscow, E K; Rutter, G A

    2000-11-17

    Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta

  19. Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets.

    PubMed

    Neumüller, Olga; Hoffmeister, Meike; Babica, Jan; Prelle, Carola; Gegenbauer, Kristina; Smolenski, Albert P

    2009-08-13

    The small guanine-nucleotide-binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O-permeabilized platelets stimulated with calcium or guanosine 5'-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1. PMID:19528539

  20. Screening for Inhibition of Vibrio cholerae VipA-VipB Interaction Identifies Small-Molecule Compounds Active against Type VI Secretion

    PubMed Central

    Sun, Kun; Bröms, Jeanette; Lavander, Moa; Gurram, Bharat Kumar; Enquist, Per-Anders; Andersson, C. David; Elofsson, Mikael

    2014-01-01

    The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A1 activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance. PMID:24798289

  1. Some aspects of proximal tubular sodium chloride reabsorption in Necturus kidney.

    PubMed

    Whittembury, G; Diezi, F; Diezi, J; Spring, K; Giebisch, G

    1975-05-01

    Some aspects of proximal tubular sodium chloride reabsorption in Necturus kidney. Renal tubular reabsorption of fluid and sodium was measured by clearance methods in the doubly perfused Necturus kidney in which the bicarbonate concentration was varied between 0 and 60 mEq/liter. The effects of Damox (2.2 times 10-3M), ocubain (10-5M) and ethacrynic acid (10-4M) and of acidosis were also investigated. In addition to clearance experiments, stationary microperfusion experiments were carried out on promimal tubules to measure volume flow and steady-state sodium and chloride concentration differences across the tubular epithelium. In some experiments, the transepithelial electrical potential difference was also measured using an axial electrode system. The following results were obtained: 1) Bicarbonate is not essential to the operation of renal tubular fluid and sodium transport. 2) Total renal and proximal tubular fluid and sodium transport are partially inhibited by Diamox, ouabian and ethacrynic acid. 3) The proximal tubule maintains a significant transepithelial sodium and chloride concentration difference and a significant electrical potential difference (lumen-negative) in the presence of a poorly permeant nonelectrolyte. The direction and magnitude of the electrical polarization fully accounts for the observed chloride concentration difference. The data support the thesis that sodium chloride transport accross the proximal tubular epithelium takes place by active sodium transport and electically coupled passive chloride reabsorption. Important species differences with respect to mammalian transport mechanisms are discussed.

  2. Salicylate-induced proximal tubular dysfunction.

    PubMed

    Tsimihodimos, Vasilis; Psychogios, Nikolaos; Kakaidi, Varvara; Bairaktari, Eleni; Elisaf, Moses

    2007-09-01

    We describe the case of a 17-year-old girl who was admitted to our clinic for drug poisoning. Twelve hours after the ingestion of 25 tablets of aspirin (12.5 g of acetylsalicylic acid), the patient had a generalized proximal tubular dysfunction characterized by glucosuria (in the face of normal serum glucose levels), proteinuria, and uric acid wasting. Further characterization of the tubular dysfunction using high-resolution proton nuclear magnetic resonance spectroscopy of the urine showed a pattern consistent with proximal tubular injury. An important characteristic of the salicylate-induced proximal tubular dysfunction in our patient was its rapid reversibility. A trend toward normalization of fractional excretion values of electrolytes was observed 2 days after ingestion. Determination of serum and urine metabolites and spectroscopy of urine 15 days later showed no evidence of tubular dysfunction. The mechanisms potentially implicated in the pathogenesis of salicylate-induced Fanconi syndrome are discussed and a brief review of the relevant literature is provided. PMID:17720526

  3. Immunoregulation by macrophages II. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and interleukin I

    SciTech Connect

    Hopper, K.E.; Cahill, J.M.

    1983-06-01

    Macrophage subpopulations having bactericidal or tumoricidal activities and secreting interleukin I (IL1) or prostaglandin E (PGE) were identified through primary or secondary infection with Salmonella enteritidis and separated by sedimentation velocity. Bactericidal activity was measured by (3H)-thymidine release from Listeria monocytogenes and tumoricidal activity by 51Cr-release from C-4 fibrosarcoma or P815 mastocytoma cells. Macrophages with bactericidal activity were distinguished from those with tumoricidal activity a) during secondary infection when cytolytic activity occurred only at days 1-4 post injection and bactericidal activity remained high throughout and b) after sedimentation velocity separation. Cytolysis was consistently greatest among adherent cells of low sedimentation velocity, whereas cells with bactericidal activity increased in size during the infection. Tumour cytostasis (inhibition and promotion of (3H)-thymidine uptake) differed from cytolysis in that the former was more prolonged during infection and was also detected among large cells. Secretion of immunoregulatory molecules PGE and IL1 occurred maximally among different macrophage subpopulations separated by sedimentation velocity and depending on the type of stimulus used in vitro. There was an inverse correlation between IL1 production and PGE production after stimulation with C3-zymosan or lipopolysaccharide (LPS). The development of immunity during infection may therefore be dependent upon the relative proportions of effector and regulatory macrophage subpopulations and the selective effects of environmental stimuli on these functions.

  4. Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona).

    PubMed

    George, Jancy M; Smita, Matthew; Kadalmani, Balamuthu; Girija, Ramankutty; Oommen, Oommen V; Akbarsha, Mohammad A

    2004-12-01

    Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes.

  5. Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona).

    PubMed

    George, Jancy M; Smita, Matthew; Kadalmani, Balamuthu; Girija, Ramankutty; Oommen, Oommen V; Akbarsha, Mohammad A

    2004-12-01

    Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes. PMID:15487004

  6. VPAC1 receptors regulate intestinal secretion and muscle contractility by activating cholinergic neurons in guinea pig jejunum.

    PubMed

    Fung, Candice; Unterweger, Petra; Parry, Laura J; Bornstein, Joel C; Foong, Jaime P P

    2014-05-01

    In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin(+) excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY(+) secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 μM), PG97-269 (VPAC1 antagonist; 1 μM), and hyoscine (10 μM), but not by hexamethonium (200 μM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl(-) secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.

  7. Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

    SciTech Connect

    Suzuki, Shigeki; Kulkarni, Ashok B.

    2010-07-30

    Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.

  8. Activation and negative selection of functionally distinct subsets of antibody-secreting cells by influenza hemagglutinin as a viral and a neo-self antigen

    PubMed Central

    1996-01-01

    We have compared transgenic mice that express the influenza virus PR8 hemagglutinin (PR8 HA) as a membrane-bound neo-self antigen (HA104 mice) with nontransgenic (non-Tg) mice for their ability to generate HA- specific B cell responses after primary immunization with PR8 virus. HA- specific, IgM-secreting B cells were induced with similar frequencies in HA104 and non-Tg mice. In addition, a B cell clonotype (C4) that is characteristic of anti-HA immune responses of BALB/c mice was identified among HA-specific IgM hybridomas from HA104 mice. A subset of HA-specific, IgG-secreting B cells that arises rapidly after primary virus immunization in non-Tg mice, however, was substantially reduced in HA104 mice. Likewise, a B cell clonotype (C12) that dominates HA- specific IgG hybridomas generated after primary immunization of non-Tg mice was present at greatly reduced frequencies among hybridomas from HA104 mice. Because HA-specific, IgG-secreting B cells were generated by HA104 mice in response to a mutant HA containing an amino acid interchange in a B cell antigenic site, we conclude that these PR8 HA- specific, IgG-secreting B cells are negatively selected in HA104 mice as a result of their specificity for the neo-self PR8 HA. The findings demonstrate that HA-specific B cells that display distinct phenotypic potentials in non-Tg mice also differ in their susceptibility to negative selection from the primary B cell repertoire of HA104 mice: a subset of B cells that undergo rapid differentiation to become HA- specific IgG antibody-secreting cells (ASC) after activation in non-Tg mice is negatively selected in HA104 mice. By contrast, a subset that gives rise to HA-specific, IgM-secreting ASC persists in the primary repertoire of HA104 mice and can be activated by virus immunization. PMID:8551216

  9. A novel TMEM16A splice variant lacking the dimerization domain contributes to calcium-activated chloride secretion in human sweat gland epithelial cells.

    PubMed

    Ertongur-Fauth, Torsten; Hochheimer, Andreas; Buescher, Joerg Martin; Rapprich, Stefan; Krohn, Michael

    2014-11-01

    Sweating is an important physiological process to regulate body temperature in humans, and various disorders are associated with dysregulated sweat formation. Primary sweat secretion in human eccrine sweat glands involves Ca(2+) -activated Cl(-) channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia; however, their molecular identity in sweat glands remained elusive. Here, we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(acΔe3) lacking the TMEM16A-dimerization domain. Chloride flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca(2+) -dependent Cl(-) secretion in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(acΔe3) is forming a functional CaCC, with basal and Ca(2+) -activated Cl(-) permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl(-) secretion providing opportunities to develop sweat gland-specific therapeutics for treatment of sweating disorders.

  10. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

    PubMed Central

    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. PMID:27148251

  11. Mycobacterium tuberculosis RNA Expression Patterns in Sputum Bacteria Indicate Secreted Esx Factors Contributing to Growth are Highly Expressed in Active Disease

    PubMed Central

    Bukka, Archana; Price, Christopher T. D.; Kernodle, Douglas S.; Graham, James E.

    2012-01-01

    To identify factors contributing to the ability of tubercle bacilli to grow in the lung during active infection, we analyzed RNA expression patterns in bacteria present in patient sputum. Prominent among bacterial transcripts identified were those encoding secreted peptides of the Esat-6 subfamily that includes EsxK and EsxL (Rv1197 and Rv1198). H37Rv esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered growth phase kinetics in typical laboratory batch broth cultures. These growth defects, including the reduced intracellular growth of an ΔesxKL mutant in primary human macrophages, were reversed by either low multiplicity co-infection or co-culture with wild-type bacteria, demonstrating the ability of the secreted factors to rescue isogenic mutants. Complementing either only esxL or esxI alone (Rv1198 or Rv1037c) also reduced observed growth defects, indicating these genes encode factors capable of contributing to growth. Our studies indicate that the Mycobacterium tuberculosis Mtb9.9 family secreted factors EsxL and EsxI can act in trans to modulate growth of intracellular bacteria, and are highly expressed during active human lung infection. PMID:22291682

  12. C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

    PubMed

    Fujiwara, Miho; Kato, Shintaro; Niwa, Yuki; Suzuki, Takehiro; Tsuchiya, Miyu; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2016-08-01

    R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling.

  13. Tubular solid oxide fuel cell current collector

    DOEpatents

    Bischoff, Brian L.; Sutton, Theodore G.; Armstrong, Timothy R.

    2010-07-20

    An internal current collector for use inside a tubular solid oxide fuel cell (TSOFC) electrode comprises a tubular coil spring disposed concentrically within a TSOFC electrode and in firm uniform tangential electrical contact with the electrode inner surface. The current collector maximizes the contact area between the current collector and the electrode. The current collector is made of a metal that is electrically conductive and able to survive under the operational conditions of the fuel cell, i.e., the cathode in air, and the anode in fuel such as hydrogen, CO, CO.sub.2, H.sub.2O or H.sub.2S.

  14. Tubular membrane bioreactors for biotechnological processes.

    PubMed

    Wolff, Christoph; Beutel, Sascha; Scheper, Thomas

    2013-02-01

    This article is an overview of bioreactors using tubular membranes such as hollow fibers or ceramic capillaries for cultivation processes. This diverse group of bioreactor is described here in regard to the membrane materials used, operational modes, and configurations. The typical advantages of this kind of system such as environments with low shear stress together with high cell densities and also disadvantages like poor oxygen supply are summed up. As the usage of tubular membrane bioreactors is not restricted to a certain organism, a brief overview of various applications covering nearly all types of cells from prokaryotic to eukaryotic cells is also given here. PMID:23224587

  15. Pyruvate kinase, muscle isoform 2 promotes proliferation and insulin secretion of pancreatic β-cells via activating Wnt/CTNNB1 signaling

    PubMed Central

    Wang, Suijun; Yang, Zhen; Gao, Ying; Li, Quanzhong; Su, Yong; Wang, Yanfang; Zhang, Yun; Man, Hua; Liu, Hongxia

    2015-01-01

    Failure of pancreatic β-cells is closely associated with type 2 diabetes mellitus (T2DM), an intractable disease affecting numerous patients. Pyruvate kinase, muscle isoform 2 (PKM2) is a potential modulator of insulin secretion in β-cells. This study aims at revealing roles and possible mechanisms of PKM2 in pancreatic β-cells. Mouse pancreatic β-cell line NIT-1 was used for high glucose treatment and PKM2 overexpression by its specific expression vector. Cell proliferation by Thiazolyl blue assay, cell apoptosis by annexin V-fluorescein isothiocyanate/prodium iodide staining and insulin secretion assay by ELISA were performed in each group. The mRNA and protein levels of related factors were analyzed by real-time quantitative PCR and western blot. Results showed that Pkm2 was inhibited under high glucose conditions compared to the untreated cells (P < 0.01). Its overexpression significantly suppressed NIT-1 cell apoptosis (P < 0.01), and induced cell proliferation (P < 0.05) and insulin secretion (P < 0.05). Related factors showed consistent mRNA expression changes. Protein levels of β-catenin (CTNNB1), insulin receptor substrate 1 (IRS1) and IRS2 were all promoted by PKM2 overexpression (P < 0.01), indicating the activated Wnt/CTNNB1 signaling. These results indicated the inductive roles of PKM2 in pancreatic β-cell NIT-1, including promoting cell proliferation and insulin secretion, and inhibiting cell apoptosis, which might be achieved via activating the Wnt/CTNNB1 signaling and downstream factors. This study offers basic information on the role and mechanism of PKM2 in pancreatic β-cells, and lays the foundation for using PKM2 as a potential therapeutic target in T2DM. PMID:26823761

  16. Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2

    PubMed Central

    DuMont, Ashley L.; Karaba, Sara M.

    2015-01-01

    Stenotrophomonas maltophilia is an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported that S. maltophilia strain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps- and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of the S. maltophilia Xps type II secretion system and its StmPr1 and StmPr2 substrates. PMID:26169274

  17. Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-κB activation and interleukin-8 secretion in human bronchial epithelial cells

    PubMed Central

    2004-01-01

    LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1–LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription–PCR and Western blotting revealed the presence and expression of LPP-1–3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2–3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IκB (inhibitory κB) and translocation of NF-κB (nuclear factor-κB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IκB, NF-κB activation or IL-8 gene expression. Our results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation. PMID:15461590

  18. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

    PubMed Central

    Owen, M R; Corstorphine, C C; Zammit, V A

    1997-01-01

    The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed. PMID:9173878

  19. Suppressed Microglial E Prostanoid Receptor 1 Signaling Selectively Reduces TNFα and IL-6 Secretion from Toll-like Receptor 3 Activation

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Keene, C. Dirk; Breyer, Richard M.; Montine, Thomas J.

    2010-01-01

    Activation of innate immunity via Toll-like receptors (TLRs) is associated with neurodegenerative diseases, and some effectors, like tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), directly contribute to neurodegeneration. We tested the hypothesis that prostaglandin (PG) E2 receptor subtype 1 (EP1) was necessary for induction of microglial cytokines following activation of innate immunity. Primary murine microglia had cytokine secretion by activators of TLR3 > TLR9 >TLR4 > TLR2. TLR3 activation induced early expression of cyclooxygenase 2 (COX2) and delayed expression of membranous PGE synthase and secretion of PGE2. Non-selective and COX2-selective inhibitors blocked TLR3 induction of TNFα and IL-6. Moreover, of the eight out of twenty cytokines and chemokines induced by TLR3 activation, only TNFα and IL-6 were significantly dependent on EP1 signaling as determined using microglia from mice homozygous deficient for EP1 gene or wild type (WT) microglia co-incubated with an EP1 antagonist. These results were confirmed by blocking intracellular Ca2+ release with 2-aminoethoxy-diphenyl borate (2-APB) or Xestospongin C (XC), inhibitors of IP3 receptors. Our results show that suppression of microglial EP1 signaling achieves much of the desired effect of COX inhibitors by selectively blocking TLR3-induced microglial secretion of two major effectors of paracrine neuron damage. In combination with the ability of EP1 suppression to ameliorate excitotoxicity, these data point to blockade of EP1 as an attractive candidate therapeutic for neurodegenerative diseases. PMID:21319223

  20. Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

    PubMed Central

    Bodemann, René Roberto; Rahfeld, Peter; Stock, Magdalena; Kunert, Maritta; Wielsch, Natalie; Groth, Marco; Frick, Sindy; Boland, Wilhelm; Burse, Antje

    2012-01-01

    Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants. PMID:22874750

  1. Engineered woven gauntlets to improve the performance of lead/acid tubular plates

    NASA Astrophysics Data System (ADS)

    Terzaghi, G.

    Several research studies have recently demonstrated that adequate compression of positive active material is one of the key determinants of the life of lead/acid batteries. The superior life displayed by batteries with tubular plates, as opposed to those with flat pasted plates, is related to the ability of the gauntlet to retain the active material around each conductive spine of the tubular plate. Woven multi-tubular gauntlets with engineered fabric structure offer higher resistance to chemical oxidation, better energy utilization due to enhanced elastic compression of the active material, and longer trouble-free battery life. Test results are presented to demonstrate that the choice of the gauntlet affects both the performance and life of the cells.

  2. Cytomorphology of tubular adenoma breast--a case report.

    PubMed

    Ravindra, Savithri; Suguna, B V

    2006-04-01

    Tubular adenoma a 'pure adenoma' is a benign neoplasm of breast presenting clinically like fibroadenoma. We report cytological and histological features of tubular adenoma in a 24 year old female with brief review of literature.

  3. Factors affecting proximal tubular reabsorption during development

    SciTech Connect

    Kaskel, F.J.; Kumar, A.M.; Lockhart, E.A.; Evan, A.; Spitzer, A.

    1987-01-01

    Studies performed in several animal species have demonstrated that glomerulotubular balance is maintained throughout development despite the many changes that occur in the factors known to control it. In an attempt to understand the nature of this phenomenon the authors quantified the magnitude and described the profile of these changes in guinea pigs. The changes in physical forces were assessed from measurements of hydrostatic and oncotic pressures, whereas those in the permeability characteristics of the proximal tubule epithelium were estimated from permanence to radioactivity-labelled macromolecules of graded radii, histologic measurements of the intercellular channels, and measurements of end-proximal ratio of tubular fluid-to-plasma osmolality (TF/P/sub osm/). Between 1 and 50 days of age the net pressure for reabsorption increased from 15.0 to 30.9 mmHg with the major change occurring during the first 2-3 wk of postnatal life. The urinary recovery of (/sup 3/H)inulin, (/sup 14/C)sucrose, and (/sup 14/C)creatinine, injected in the early segment of proximal tubules did not vary with age. The urinary recovery of (/sup 14/C)mannitol increased from 92% at birth to 100% at 49 days of age. The length of the zonulae occludens and the width of the intercellular channels did not change during this period. The findings support the hypothesis that during early postnatal life glomerulotubular balance is made possible by a high permeability of the proximal tubule, which compensates for the low net reabsorptive pressure. As the animal matures and the proximal tubule epithelium becomes tighter, for glomerulotubular balance to be maintained, an increase in the number of intercellular channels and in the active transport of sodium need to be postulated.

  4. Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

    PubMed

    Nicholls, Linda I; Ainscow, Edward K; Rutter, Guy A

    2002-03-01

    Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.

  5. High IFN-γ Release and Impaired Capacity of Multi-Cytokine Secretion in IGRA Supernatants Are Associated with Active Tuberculosis

    PubMed Central

    Pisoni, Amandine; Bendriss, Sophie; Marin, Grégory; Peries, Marianne; Bolloré, Karine; Terru, Dominique; Godreuil, Sylvain; Bourdin, Arnaud; Van de Perre, Philippe; Tuaillon, Edouard

    2016-01-01

    Interferon gamma (IFN-γ) release assays (IGRAs) detect Mycobacterium tuberculosis (Mtb) infection regardless of the active (ATB) or latent (LTBI) forms of tuberculosis (TB). In this study, Mtb-specific T cell response against region of deletion 1 (RD1) antigens were explored by a microbead multiplex assay performed in T-SPOT TB assay (T-SPOT) supernatants from 35 patients with ATB and 115 patients with LTBI. T-SPOT is positive when over 7 IFN-γ secreting cells (SC)/250 000 peripheral blood mononuclear cells (PBMC) are enumerated. However, over 100 IFN-γ SC /250 000 PBMC were more frequently observed in the ATB group compared to the LTBI group. By contrast, lower cytokine concentrations and lower cytokine productions relative to IFN-γ secretion were observed for IL 4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α when compared to LTBI. Thus, high IFN-γ release and low cytokine secretions in relation with IFN-γ production appeared as signatures of ATB, corroborating that multicytokine Mtb-specific response against RD1 antigens reflects host capacity to contain TB reactivation. In this way, testing cytokine profile in IGRA supernatants would be helpful to improve ATB screening strategy including immunologic tests. PMID:27603919

  6. Carbonyl stress-induced 5-hydroxytriptamine secretion from RIN-14B, rat pancreatic islet tumor cells, via the activation of transient receptor potential ankyrin 1.

    PubMed

    Suzawa, Sayaka; Takahashi, Kenji; Shimada, Takahisa; Ohta, Toshio

    2016-07-01

    Methylglyoxal (MG), a highly reactive dicarbonyl substance, is known as an endogenous carbonyl stress-inducing substance related to various disease states. Irritable bowel syndrome (IBS) is one of the most frequently encountered gastrointestinal disorders and MG is considered to be its causal substance. An increased serum 5-hydroxytryptamine (5-HT) level is related to IBS symptoms and the majority of 5-HT originates from enterochromaffin (EC) cells in the intestine. Here we examine the mechanisms of MG-induced 5-HT secretion using RIN-14B cells derived from a rat pancreatic islet tumor since these cells are used as a model for EC cells. MG increased the intracellular Ca(2+) concentration ([Ca(2+)]i) and 5-HT secretion, both of which were inhibited by the removal of extracellular Ca(2+) and specific transient receptor potential ankyrin 1 (TRPA1) antagonists. MG elicited an inward current under voltage-clamped conditions. Prior application of MG evoked reciprocal suppression of subsequent [Ca(2+)]i responses to allylisothiocyanate, a TRPA1 agonist, and vice versa. Glyoxal, an analog of MG, also evoked [Ca(2+)]i and secretory responses but its potency was much lower than that of MG. The present results suggest that MG promotes 5-HT secretion through the activation of TRPA1 in RIN-14B cells. These results may indicate that TRPA1 is a promising target for the treatment of IBS and that the RIN-14B cell line is a useful model for investigation of IBS. PMID:27423812

  7. High IFN-γ Release and Impaired Capacity of Multi-Cytokine Secretion in IGRA Supernatants Are Associated with Active Tuberculosis.

    PubMed

    Carrère-Kremer, Séverine; Rubbo, Pierre-Alain; Pisoni, Amandine; Bendriss, Sophie; Marin, Grégory; Peries, Marianne; Bolloré, Karine; Terru, Dominique; Godreuil, Sylvain; Bourdin, Arnaud; Van de Perre, Philippe; Tuaillon, Edouard

    2016-01-01

    Interferon gamma (IFN-γ) release assays (IGRAs) detect Mycobacterium tuberculosis (Mtb) infection regardless of the active (ATB) or latent (LTBI) forms of tuberculosis (TB). In this study, Mtb-specific T cell response against region of deletion 1 (RD1) antigens were explored by a microbead multiplex assay performed in T-SPOT TB assay (T-SPOT) supernatants from 35 patients with ATB and 115 patients with LTBI. T-SPOT is positive when over 7 IFN-γ secreting cells (SC)/250 000 peripheral blood mononuclear cells (PBMC) are enumerated. However, over 100 IFN-γ SC /250 000 PBMC were more frequently observed in the ATB group compared to the LTBI group. By contrast, lower cytokine concentrations and lower cytokine productions relative to IFN-γ secretion were observed for IL 4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α when compared to LTBI. Thus, high IFN-γ release and low cytokine secretions in relation with IFN-γ production appeared as signatures of ATB, corroborating that multicytokine Mtb-specific response against RD1 antigens reflects host capacity to contain TB reactivation. In this way, testing cytokine profile in IGRA supernatants would be helpful to improve ATB screening strategy including immunologic tests. PMID:27603919

  8. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    SciTech Connect

    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.

  9. Drill pipes and casings utilizing multi-conduit tubulars

    SciTech Connect

    Curlett, H.B.

    1989-01-24

    A seal adapted for use with a multi-conduit well tubular, or the like, is described which consists of: a plate with fluid passages, each passage corresponding to an opening of a conduit of the multiconduit tubular, and a groove on the plate around each passage; and elastomer means partially embeddable into each groove for sealing each conduit of a tubular to a corresponding conduit of another similar tubular.

  10. Infanticide secrets

    PubMed Central

    Barr, Jennieffer A.; Beck, Cheryl T.

    2008-01-01

    ABSTRACT OBJECTIVE To explore thoughts of infanticide that did not lead to the act among mothers with postpartum depression. DESIGN A phenomenologic hermeneutic study in which women were invited to share their experiences of having thoughts of infanticide. SETTING Community setting in a large metropolitan city, Brisbane, Australia. PARTICIPANTS Fifteen women who had been diagnosed as clinically depressed with postpartum onset whose babies were 12 months of age or younger. METHOD Audiotaped, in-depth interviews were transcribed verbatim. Thematic analysis commenced immediately after the first interview, and data collection continued until saturation was achieved. A questioning approach that reflected hermeneutics was facilitated by use of journals by the researchers. MAIN FINDINGS Six themes emerged from the data: imagined acts of infanticide, the experience of horror, distorted sense of responsibility, consuming negativity, keeping secrets, and managing the crisis. CONCLUSION Women who experienced nonpsychotic depression preferred not to disclose their thoughts of infanticide to health professionals, including trusted general practitioners or psychiatrists. These women were more likely to mention their suicidal thoughts than their infanticidal thoughts in order to obtain health care. General practitioners and other health professionals should directly ask about whether a woman has been experiencing thoughts of harming herself or her baby, regardless of the reason why she has presented. PMID:19074717

  11. N-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity

    PubMed Central

    TSUCHIYA, MIYU; NIWA, YUKI; SIMIZU, SIRO

    2016-01-01

    N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. In vitro treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect. PMID:27123103

  12. Tubular Membrane Plant-Growth Unit

    NASA Technical Reports Server (NTRS)

    Dreschel, Thomas W.

    1992-01-01

    Hydroponic system controls nutrient solution for growing crops in space. Pump draws nutrient solution along inside of tubular membrane in pipe from reservoir, maintaining negative pressure in pipe. Roots of plants in slot extract nutrient through membrane within pipe. Crop plants such as wheat, rice, lettuce, tomatoes, soybeans, and beans grown successfully with system.

  13. Boron--epoxy tubular structure members

    NASA Technical Reports Server (NTRS)

    Shakespeare, W. B. J.; Nelson, P. T.; Lindkvist, E. C.

    1973-01-01

    Composite materials fabricate thin-walled tubular members which have same load-carrying capabilities as aluminum, titanium, or other metals, but are lighter. Interface between stepped end fitting and tube lends itself to attachments by primary as well as secondary bonding. Interlaminar shear and hoop stress buildup in attachment at end fitting is avoided.

  14. Tubular structured hierarchical mesoporous titania material derived from natural cellulosic substances and application as photocatalyst for degradation of methylene blue

    SciTech Connect

    Huang, Haiqing; Liu, Xiaoyan; Huang, Jianguo

    2011-11-15

    Graphical abstract: Bio-inspired, tubular structured hierarchical mesoporous titania material with high photocatalytic activity under UV light was fabricated employing natural cellulosic substance (cotton) as hard template and cetyltrimethylammonium bromide (CTAB) surfactant as soft template using a one-pot sol-gel method. Highlights: {yields} Tubular structured mesoporous titania material was fabricated by sol-gel method. {yields} The titania material faithfully recorded the hierarchical structure of the template substrate (cotton). {yields} The titania material exhibited high photocatalytic activity in decomposition of methylene blue. -- Abstract: Bio-inspired, tubular structured hierarchical mesoporous titania material was designed and fabricated employing natural cellulosic substance (cotton) as hard template and cetyltrimethylammonium bromide (CTAB) surfactant as soft template by one-pot sol-gel method. The tubular structured hierarchical mesoporous titania material processes large specific surface area (40.23 m{sup 2}/g) and shows high photocatalytic activity in the photodegradation of methylene blue under UV light irradiation.

  15. AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

    SciTech Connect

    Jaguin, Marie; Fardel, Olivier; Lecureur, Valérie

    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.

  16. Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

    PubMed

    Conlon, J M; Sonnevend, A; Patel, M; Davidson, C; Nielsen, P F; Pál, T; Rollins-Smith, L A

    2003-11-01

    The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.

  17. Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

    PubMed Central

    Yun, Bo; Azad, Mohammad A. K.; Nowell, Cameron J.; Nation, Roger L.; Thompson, Philip E.; Roberts, Kade D.

    2015-01-01

    Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. PMID:26392495

  18. 78 FR 14361 - U.S. Steel Tubular Products, Inc., Mckeesport Tubular Operations Division, Subsidiary of United...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-05

    ... Employment and Training Administration U.S. Steel Tubular Products, Inc., Mckeesport Tubular Operations Division, Subsidiary of United States Steel Corporation, Mckeesport, PA; Notice of Initiation of...) filed on December 20, 2012 on behalf of workers of U.S. Steel Tubular Products, McKeesport...

  19. Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion

    PubMed Central

    2013-01-01

    Background As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. Results Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. Conclusions Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for

  20. The wax glands and wax secretion of Matsucoccus matsumurae at different development stages.

    PubMed

    Xie, Yingping; Tian, Fen; Liu, Weimin; Zhang, Yanfeng; Xue, Jiaoliang; Zhao, Youyou; Wu, Jun

    2014-05-01

    In this paper, the wax secretions and wax glands of Matsucoccus matsumurae (Kuwana) at different instars were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The first and second instar nymphs were found to secrete wax filaments via the wax glands located in the atrium of the abdominal spiracles, which have a center open and a series of outer ring pores. The wax gland of the abdominal spiracle possesses a large central wax reservoir and several wax-secreting cells. Third-instar male nymphs secreted long and translucent wax filaments from monolocular, biolocular, trilocular and quadrilocular pores to form twine into cocoons. The adult male secreted long and straight wax filaments in bundles from a group of 18-19 wax-secreting tubular ducts on the abdominal segment VII. Each tube duct contained five or six wax pores. The adult female has dorsal cicatrices distributed in rows, many biolocular tubular ducts and multilocular disc pores with 8-12 loculi secreting wax filaments that form the egg sac, and a rare type wax pores with 10 loculi secreting 10 straight, hollow wax filaments. The ultrastructure and cytological characteristics of the wax glands include wax-secreting cells with a large nucleus, multiple mitochondria and several rough endoplasmic reticulum. The functions of the wax glands and wax secretions are discussed.

  1. Inhibitory effects of C-type natriuretic peptide on the differentiation of cardiac fibroblasts, and secretion of monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1.

    PubMed

    Li, Zhi-Qiang; Liu, Ying-Long; Li, Gang; Li, Bin; Liu, Yang; Li, Xiao-Feng; Liu, Ai-Jun

    2015-01-01

    The present study aimed to investigate the effect of C-type natriuretic peptide (CNP) on the function of cardiac fibroblasts (CFs). Western blotting was used to investigate the expression of myofibroblast marker proteins: α-smooth muscle actin (α-SMA), extra domain-A fibronectin, collagen I and collagen III, and the activity of extracellular signal-regulated kinase 1/2 (ERK1/2). Immunofluorescence was used to examine the morphological changes; a transwell assay was used to analyze migration, and reverse transcription-quantitative polymerase chain reaction and ELISA were employed to determine the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). The results demonstrated that CNP significantly reduced the protein expression of α-SMA, fibronectin, collagen I and collagen III, and suppressed the migratory ability of CFs. Additionally, the mRNA and protein expression of MCP-1 and PAI-1 was inhibited under the CNP treatment; and this effect was mediated by the inhibition of the ERK1/2 activity. In conclusion, CNP inhibited cardiac fibroblast differentiation and migration, and reduced the secretion of MCP-1 and PAI-1, which demonstrates novel mechanisms to explain the antifibrotic effect of CNP.

  2. Role of NF-κB activation in matrix metalloproteinase 9, vascular endothelial growth factor and interleukin 8 expression and secretion in human breast cancer cells.

    PubMed

    Li, Caijuan; Guo, Sufen; Shi, Tiemei

    2013-04-01

    The aims of this study were to assess the effects and potential mechanisms of parthenolide on the expression of vascular endothelial growth factor (VEGF), interleukin 8 (IL-8) and matrix metalloproteinase 9 (MMP-9) in human breast cancer cell line MDA-MB-231. After incubation with different concentrations of parthenolide for 24 h, MDA-MB-231 cells were collected, and the expressions of VEGF, IL-8 and MMP-9 were measured by real-time PCR and Western blot. The secretions of VEGF, IL-8 and MMP-9 in culture supernatant of MDA-MB-231 cells were then measured with ELISA assays. The NF-κB DNA-binding activity of breast cancer cells treated with parthenolide was analyzed using electrophoretic mobility assays. The real-time PCR and Western blot data showed that the expressions of VEGF, IL-8 and MMP-9 were significantly inhibited by parthenolide at both transcription level and protein level in MDA-MB-231 cells. ELISA results also confirmed these effects at a secretion level. The electrophoretic mobility assay results demonstrated that parthenolide can inhibit NF-κB DNA-binding activity of the breast cancer cells. Hence, the expression of VEGF, IL-8 and MMP-9 may be suppressed by parthenolide through the inhibition of NF-κB DNA-binding activity in MDA-MB-231 cells.

  3. Pleiotrophin promotes microglia proliferation and secretion of neurotrophic factors by activating extracellular signal-regulated kinase 1/2 pathway.

    PubMed

    Miao, Jiayin; Ding, Minghui; Zhang, Aiwu; Xiao, Zijian; Qi, Weiwei; Luo, Ning; Di, Wei; Tao, Yuqian; Fang, Yannan

    2012-12-01

    Pleiotrophin (PTN) is an effective neuroprotective factor and its expression is strikingly increased in microglia after ischemia/reperfusion injury. However, whether PTN could provide neurotrophic support to neurons by regulating microglia function is not clear. In this study, we demonstrated that the expression of PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. PTN promoted the proliferation of microglia by enhancing the G1 to S phase transition. PTN also stimulated the secretion of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) in microglia, but did not upregulate the expression of proinflammatory factors such as TNF-α, IL-1β and iNOS. Mechanistically, we found that PTN increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in microglia in both concentration-dependent and time-dependent manners. In addition, ERK1/2 inhibitor U0126 abolished the proliferation and G1 to S phase transition of microglia stimulated by PTN, and inhibited the production of BDNF, CNTF and NGF induced by PTN. In conclusion, our results demonstrated that PTN-ERK1/2 pathway plays important role in regulating microglia growth and secretion of neurotrophic factors. These findings provide new insight into the neuroprotective role of PTN and suggest that PTN is a new target for therapeutic intervention of stroke.

  4. Low-cost synthesis and utilization in mini-tubular electrodes of nano PbO 2

    NASA Astrophysics Data System (ADS)

    Bervas, M.; Perrin, M.; Geniès, S.; Mattera, F.

    The aim of this work was to explore the possibility of using nano PbO 2 in commercial type tubular lead-acid battery positive electrodes. In that prospect, nanosized αPbO 2 and βPbO 2 have been synthesized at room temperature by a low-cost solution technique involving the chemical oxidation of lead nitrate with ammonium persulfate in concentrated alkaline medium. Mini-tubular electrodes were then prepared using the same type of gauntlet as the one used in commercial tubular electrodes. We show that the capacity and the cycling stability of the tubular electrode highly depend on the sulfuric acid concentration in the electrolyte and on the lead dioxide surface area. A percentage of utilization higher than 45% of the active mass with an excellent cycling stability was obtained when our nano βPbO 2 was used as active material in a diluted H 2SO 4 electrolyte.

  5. CD46 activation regulates miR-150-mediated control of GLUT1 expression and cytokine secretion in human CD4+ T cells

    PubMed Central

    King, Ben C.; Esguerra, Jonathan L. S.; Golec, Ewelina; Eliasson, Lena; Kemper, Claudia; Blom, Anna M.

    2015-01-01

    CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4+ T cells with CD3 and CD46 also leads to the differentiation of a ‘switched’ Th1 population, which shuts down IFN-γ secretion and upregulates IL-10, and is believed to be important for negative feedback regulation of the Th1 response. We show here that CD46 costimulation leads to amplified microRNA expression changes in human CD4+ T cells, with associated increases in activation more potent than that mediated by the ‘classic’ costimulator CD28. Blockade of cell-surface CD46 inhibited CD28-mediated costimulation, identifying autocrine CD46 signaling as downstream of CD28. We also identify a downregulation of microRNA-150 in CD46-costimulated T cells, and identify the glucose transporter-1 (GLUT1) encoding transcript SLC2A1 as a target of microRNA-150 regulation, connecting microRNA-150 with modulation of glucose uptake. We also investigated microRNA expression profiles of CD46-induced ‘switched’ IL-10-secreting Th1 T cells and found increased expression of microRNA-150, compared to IFN-γ-secreting Th1 cells. Knockdown of microRNA-150 led to a reduction in IL-10 but not IFN-γ. CD46 therefore controls both Th1 activation and regulation via a miRNA-150-dependent mechanism. PMID:26746193

  6. Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

    PubMed

    Matus, Carola E; Ehrenfeld, Pamela; Pavicic, Francisca; González, Carlos B; Concha, Miguel; Bhoola, Kanti D; Burgos, Rafael A; Figueroa, Carlos D

    2016-09-01

    The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

  7. Characterisation of the morphogenetic course and secretion of two different types of mucoid material by granulated metrial gland/lymphokine-activated killer cells.

    PubMed

    Dyugovskaya, L; Berkutski, T; Ginsburg, H

    1995-12-01

    Lymphocytes from mesenteric lymph nodes of ordinary and nude mice were grown in microtitre wells on embryonic mesenchymal-fibroblast monolayers. Human recombinant interleukin-2 (80 units ml-1) was added. Clones of lymphokine-activated killer (LAK) cells developed. The incidence of clone-forming cells was 52-136 per 10(5) cells in lymph nodes from nude mice and 4.2-8.3 per 10(5) cells in lymph nodes from ordinary mice. On a limited number of fibroblast monolayers propagated in culture, the maturing LAK cells were induced to synthesise and secrete 2 types of flowing mucoid material. After methanol fixation and Alcian blue/periodic acid-Schiff (PAS) (at pH 1) staining, the first type of material was distinctively stained turquoise, indicating a highly sulphated proteoglycan, chondroitin sulphate; the second type of material, a macromolecular neutral polysaccharide, was not stained and appeared to have been dissolved. Glycogen was stained deep brilliant purple. After treatment with PAS alone, the chondroitin sulphate was not stained and appeared as a bright area, the neutral polysaccharide mass being stained deep red. This polysaccharide material was characteristically secreted as droplets or 'streamlets' emerging from the cell surface and extending through the first type of material to coalesce with the already accumulated main extracellular mucoid layer spreading between the cells. Clones of secretory LAK cells were obtained from gravid and nongravid mouse uteri as well as from tracheal explants. Change of medium or passage with fresh medium to a new inducing batch of monolayer, at the blastoid-large granular lymphocyte stage (on d 3 to 7), was critical for high reproducibility of secretion. The course of differentiation was found ultimately to be dependent on the embryonic mesenchymal monolayer, suggesting induction by a morphogenetic signal. A correlation can be drawn between the secretory activity and the morphological profile at maturation of highly distinctive

  8. Sympathetic nerve activity in normal and cystic follicles from isolated bovine ovary: local effect of beta-adrenergic stimulation on steroid secretion

    PubMed Central

    2011-01-01

    Cystic ovarian disease (COD) is an important cause of abnormal estrous behavior and infertility in dairy cows. COD is mainly observed in high-yielding dairy cows during the first months post-partum, a period of high stress. We have previously reported that, in lower mammals, stress induces a cystic condition similar to the polycystic ovary syndrome in humans and that stress is a definitive component in the human pathology. To know if COD in cows is also associated with high sympathetic activity, we studied isolated small antral (5mm), preovulatory (10mm) and cystic follicles (25mm). Cystic follicles which present an area 600 fold greater compared with preovulatory follicles has only 10 times less concentration of NE as compared with small antral and preovulatory follicles but they had 10 times more NE in follicular fluid, suggesting a high efflux of neurotransmitter from the cyst wall. This suggestion was reinforced by the high basal release of recently taken-up 3H-NE found in cystic follicles. While lower levels of beta-adrenergic receptor were found in cystic follicles, there was a heightened response to the beta-adrenergic agonist isoproterenol and to hCG, as measured by testosterone secretion. There was however an unexpected capacity of the ovary in vitro to produce cortisol and to secrete it in response to hCG but not to isoproterenol. These data suggest that, during COD, the bovine ovary is under high sympathetic nerve activity that in addition to an increased response to hCG in cortisol secretion could participate in COD development. PMID:21575217

  9. Hepatocyte nuclear factor 1β controls nephron tubular development.

    PubMed

    Massa, Filippo; Garbay, Serge; Bouvier, Raymonde; Sugitani, Yoshinobu; Noda, Tetsuo; Gubler, Marie-Claire; Heidet, Laurence; Pontoglio, Marco; Fischer, Evelyne

    2013-02-01

    Nephron morphogenesis is a complex process that generates blood-filtration units (glomeruli) connected to extremely long and patterned tubular structures. Hepatocyte nuclear factor 1β (HNF1β) is a divergent homeobox transcription factor that is expressed in kidney from the first steps of nephrogenesis. Mutations in HNF1B (OMIM #137920) are frequently found in patients with developmental renal pathologies, the mechanisms of which have not been completely elucidated. Here we show that inactivation of Hnf1b in the murine metanephric mesenchyme leads to a drastic tubular defect characterized by the absence of proximal, distal and Henle's loop segments. Nephrons were eventually characterized by glomeruli, with a dilated urinary space, directly connected to collecting ducts via a primitive and short tubule. In the absence of HNF1β early nephron precursors gave rise to deformed S-shaped bodies characterized by the absence of the typical bulge of epithelial cells at the bend between the mid and lower segments. The lack of this bulge eventually led to the absence of proximal tubules and Henle's loops. The expression of several genes, including Irx1, Osr2 and Pou3f3, was downregulated in the S-shaped bodies. We also observed decreased expression of Dll1 and the consequent defective activation of Notch in the prospective tubular compartment of comma- and S-shaped bodies. Our results reveal a novel hierarchical relationship between HNF1β and key genes involved in renal development. In addition, these studies define a novel structural and functional component of S-shaped bodies at the origin of tubule formation.

  10. Secret key generation via a modified quantum secret sharing protocol

    NASA Astrophysics Data System (ADS)

    Smith, A. M.; Evans, P. G.; Lawrie, B.; Legré, M.; Lougovski, P.; Ray, W.; Williams, B. P.; Qi, B.; Grice, W. P.

    2015-05-01

    We present and experimentally show a novel protocol for distributing secret information between two and only two parties in a N-party single-qubit Quantum Secret Sharing (QSS) system. We demonstrate this new algorithm with N = 3 active parties over ~6km of telecom. fiber. Our experimental device is based on the Clavis2 Quantum Key Distribution (QKD) system built by ID Quantique but is generalizable to any implementation. We show that any two out of the N parties can build secret keys based on partial information from each other and with collaboration from the remaining N - 2 parties. This algorithm allows for the creation of two-party secret keys were standard QSS does not and significantly reduces the number of resources needed to implement QKD on a highly connected network such as the electrical grid.

  11. Secret Key Generation via a Modified Quantum Secret Sharing Protocol

    SciTech Connect

    Smith IV, Amos M; Evans, Philip G; Lawrie, Benjamin J; Legre, Matthieu; Lougovski, Pavel; Ray, William R; Williams, Brian P; Qi, Bing; Grice, Warren P

    2015-01-01

    We present and experimentally show a novel protocol for distributing secret information between two and only two parties in a N-party single-qubit Quantum Secret Sharing (QSS) system. We demonstrate this new algorithm with N = 3 active parties over 6km of telecom. ber. Our experimental device is based on the Clavis2 Quantum Key Distribution (QKD) system built by ID Quantique but is generalizable to any implementation. We show that any two out of the N parties can build secret keys based on partial information from each other and with collaboration from the remaining N > 2 parties. This algorithm allows for the creation of two-party secret keys were standard QSS does not and signicantly reduces the number of resources needed to implement QKD on a highly connected network such as the electrical grid.

  12. Response of human renal tubular cells to cyclosporine and sirolimus: A toxicogenomic study

    SciTech Connect

    Pallet, Nicolas Rabant, Marion; Xu-Dubois, Yi-Chun; LeCorre, Delphine; Mucchielli, Marie-Helene; Imbeaud, Sandrine; Agier, Nicolas; Thervet, Eric; Legendre, Christophe; Beaune, Philippe; Anglicheau, Dany

    2008-06-01

    The molecular mechanisms involved in the potentially nephrotoxic response of tubular cells to immunosuppressive drugs remain poorly understood. Transcriptional profiles of human proximal tubular cells exposed to cyclosporine A (CsA), sirolimus (SRL) or their combination, were established using oligonucleotide microarrays. Hierarchical clustering of genes implicated in fibrotic processes showed a clear distinction between expression profiles with CsA and CsA + SRL treatments on the one hand and SRL treatment on the other. Functional analysis found that CsA and CsA + SRL treatments preferentially alter biological processes located at the cell membrane, such as ion transport or signal transduction, whereas SRL modifies biological processes within the nucleus and related to transcriptional activity. Genome wide expression analysis suggested that CsA may induce an endoplasmic reticulum (ER) stress in tubular cells in vitro. Moreover we found that CsA exposure in vivo is associated with the upregulation of the ER stress marker BIP in kidney transplant biopsies. In conclusion, this toxicogenomic study highlights the molecular interaction networks that may contribute to the tubular response to CsA and SRL. These results may also offer a new working hypothesis for future research in the field of CsA nephrotoxicity. Further studies are needed to evaluate if ER stress detection in tubular cells in human biopsies can predict CsA nephrotoxicity.

  13. Losartan attenuates renal interstitial fibrosis and tubular cell apoptosis in a rat model of obstructive nephropathy.

    PubMed

    He, Ping; Li, Detian; Zhang, Beiru

    2014-08-01

    Ureteral obstruction leads to renal injury and progresses to irreversible renal fibrosis, with tubular cell atrophy and apoptosis. There is conflicting evidence concerning whether losartan (an angiotensin II type I receptor antagonist) mitigates renal interstitial fibrosis and renal tubular epithelial cell apoptosis following unilateral ureteral obstruction (UUO) in animal models. The aim of this study was to investigate the effect and mechanism of losartan on renal tubular cell apoptosis and renal fibrosis in a rat model of UUO. The rats were subjected to UUO by ureteral ligation and were treated with dimethyl sulfoxide (control) or losartan. The controls underwent sham surgery. The renal tissues were collected 3, 5, 7 and 14 days after surgery for measurement of various indicators of renal fibrosis. UUO increased the expression levels of α‑smooth muscle actin and collagen I, and the extent of renal tubular fibrosis and apoptosis in a time‑dependent manner. Losartan treatment partially attenuated these responses. Progression of renal interstitial fibrosis was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3) and altered the expression levels of two apoptosis‑related proteins (Bax and Bcl2). Losartan treatment also partially attenuated these responses. The results indicated that losartan attenuated renal fibrosis and renal tubular cell apoptosis in a rat model of UUO. This effect appeared to be mediated by partial blockage of STAT3 phosphorylation.

  14. Epithelial-mesenchymal transition (EMT) of renal tubular cells in canine glomerulonephritis.

    PubMed

    Aresu, Luca; Rastaldi, Maria Pia; Scanziani, Eugenio; Baily, James; Radaelli, Enrico; Pregel, Paola; Valenza, Federico

    2007-11-01

    Tubulo-interstitial fibrosis in dogs may result from primary injury to the interstitium or develop secondary to other renal diseases. As in human renal pathology, tubular epithelial cells (TEC) are believed to actively participate in the mechanisms of renal fibrosis. In this study, we examined the changes in the tubular epithelial component in two specific canine diseases. Immunohistochemistry showed the expression of the epithelial marker cytokeratin, the smooth muscle marker alpha-SMA, the mesenchymal marker vimentin and PCNA in 20 dogs with membranous glomerulonephritis and membrano-proliferative glomerulonephritis. Results showed that the loss of the epithelial marker in TEC was directly correlated to the grade of tubulo-interstitial disease present and independent of the type of glomerulonephritis. Varying degrees of vimentin positivity were detected in tubular epithelium in areas of inflammation, and low numbers of scattered alpha-SMA-positive cells were also observed. Immunohistochemistry showed that epithelial tubular cells lose their cytokeratin staining characteristics and transdifferentiate into cells exhibiting key mesenchymal immunophenotypic feature of vimentin-positive staining in both diseases investigated. The integrity of the tubular basement membrane is likely to be fundamental in maintaining the epithelial phenotype of TEC. Animal models provide opportunities for investigating the pathogenesis of renal fibrosis in humans.

  15. Sec-mediated secretion by Coxiella burnetii

    PubMed Central

    2013-01-01

    Background Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication. Results To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. Conclusions C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed. PMID:24093460

  16. Hydrokinetic activity of secretion and secretin analogues, modified in the N-terminal sequence, and of vasoactive intestinal peptide in the dog pancreas.

    PubMed

    Lehnert, P; Forell, M M; Jaeger, E; Moroder, L; Wünsch, E

    1981-01-01

    In the dog pancreas in vivo, the biological activity of secretin and vasoactive intestinal peptide was compared to that of secretin analogues modified in their N-terminal hexapeptide and to X-secretion (alpha, beta-Asp3-secretin) and Y-secretin (a conversion product of X-secretin consisting of about 15% secretin and 85% beta-Asp3-secretin). Replacement of Asp3 by glutamic acid reduced secretin activity markedly. Replacement by neutral amino acids abolished the activity nearly completely. alpha, beta-Asp3-secretin and beta-Asp3-secretin appeared to be ineffective. The results indicate that the free beta-carboxy group of the side chain of the Asp3 residue of the secretin molecule is of decisive importance for hydrokinetic action.

  17. Murine Macrophages Secrete Interferon γ upon Combined Stimulation with Interleukin (IL)-12 and IL-18: A Novel Pathway of Autocrine Macrophage Activation

    PubMed Central

    Munder, Markus; Mallo, Moisés; Eichmann, Klaus; Modolell, Manuel

    1998-01-01

    Interferon (IFN)-γ, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow– derived macrophages (BMMΦ) secrete large amounts of IFN-γ upon appropriate stimulation. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-γ mRNA transcripts, the combined stimulation of BMMΦ with both cytokines leads to the efficient production of IFN-γ protein. The macrophage-derived IFN-γ is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-γ but also a potent IFN-γ–producing cell. PMID:9625771

  18. Hydrokinetic activity of secretion and secretin analogues, modified in the N-terminal sequence, and of vasoactive intestinal peptide in the dog pancreas.

    PubMed

    Lehnert, P; Forell, M M; Jaeger, E; Moroder, L; Wünsch, E

    1981-01-01

    In the dog pancreas in vivo, the biological activity of secretin and vasoactive intestinal peptide was compared to that of secretin analogues modified in their N-terminal hexapeptide and to X-secretion (alpha, beta-Asp3-secretin) and Y-secretin (a conversion product of X-secretin consisting of about 15% secretin and 85% beta-Asp3-secretin). Replacement of Asp3 by glutamic acid reduced secretin activity markedly. Replacement by neutral amino acids abolished the activity nearly completely. alpha, beta-Asp3-secretin and beta-Asp3-secretin appeared to be ineffective. The results indicate that the free beta-carboxy group of the side chain of the Asp3 residue of the secretin molecule is of decisive importance for hydrokinetic action. PMID:7274610

  19. Nano-Tubular Cellulose for Bioprocess Technology Development

    PubMed Central

    Koutinas, Athanasios A.; Sypsas, Vasilios; Kandylis, Panagiotis; Michelis, Andreas; Bekatorou, Argyro; Kourkoutas, Yiannis; Kordulis, Christos; Lycourghiotis, Alexis; Banat, Ibrahim M.; Nigam, Poonam; Marchant, Roger; Giannouli, Myrsini; Yianoulis, Panagiotis

    2012-01-01

    Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in “cold pasteurization” processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc. PMID:22496794

  20. Nano-tubular cellulose for bioprocess technology development.

    PubMed

    Koutinas, Athanasios A; Sypsas, Vasilios; Kandylis, Panagiotis; Michelis, Andreas; Bekatorou, Argyro; Kourkoutas, Yiannis; Kordulis, Christos; Lycourghiotis, Alexis; Banat, Ibrahim M; Nigam, Poonam; Marchant, Roger; Giannouli, Myrsini; Yianoulis, Panagiotis

    2012-01-01

    Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in "cold pasteurization" processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc.

  1. Tubular lap joints for wind turbine applications

    SciTech Connect

    Reedy, E.D. Jr.; Guess, T.R.

    1990-01-01

    A combined analytical/experimental study of the strength of thick- walled, adhesively bonded PMMA-to-aluminum and E-glass/epoxy composite-to-aluminum tubular lap joints under axial load has been conducted. Test results include strength and failure mode data. Moreover, strain gages placed along the length of the outer tubular adherend characterize load transfer from one adherend to the other. The strain gage data indicate that load transfer is nonuniform and that the relatively compliant PMMA has the shorter load transfer length. Strains determined by a finite element analysis of the tested joints are in excellent agreement with those measured. Calculated bond stresses are highest in the region of observed failure, and extensive bond yielding is predicted in the E- glass/epoxy composite-to-aluminum joint prior to joint failure. 4 refs., 13 figs., 1 tab.

  2. Tubular Heart Pumping Mechanisms in Ciona Intestinalis

    NASA Astrophysics Data System (ADS)

    Battista, Nicholas; Miller, Laura

    2015-11-01

    In vertebrate embryogenesis, the first organ to form is the heart, beginning as a primitive heart tube. However, many invertebrates have tubular hearts from infancy through adulthood. Heart tubes have been described as peristaltic and impedance pumps. Impedance pumping assumes a single actuation point of contraction, while traditional peristalsis assumes a traveling wave of actuation. In addition to differences in flow, this inherently implies differences in the conduction system. It is possible to transition from pumping mechanism to the other with a change in the diffusivity of the action potential. In this work we consider the coupling between the fluid dynamics and electrophysiology of both mechanisms, within a basal chordate, the tunicate. Using CFD with a neuro-mechanical model of tubular pumping, we discuss implications of the both mechanisms. Furthermore, we discuss the implications of the pumping mechanism on evolution and development.

  3. Pattern Selection in Growing Tubular Tissues

    NASA Astrophysics Data System (ADS)

    Ciarletta, P.; Balbi, V.; Kuhl, E.

    2014-12-01

    Tubular organs display a wide variety of surface morphologies including circumferential and longitudinal folds, square and hexagonal undulations, and finger-type protrusions. Surface morphology is closely correlated to tissue function and serves as a clinical indicator for physiological and pathological conditions, but the regulators of surface morphology remain poorly understood. Here, we explore the role of geometry and elasticity on the formation of surface patterns. We establish morphological phase diagrams for patterns selection and show that increasing the thickness or stiffness ratio between the outer and inner tubular layers induces a gradual transition from circumferential to longitudinal folding. Our results suggest that physical forces act as regulators during organogenesis and give rise to the characteristic circular folds in the esophagus, the longitudinal folds in the valves of Kerckring, the surface networks in villi, and the crypts in the large intestine.

  4. Optical analysis of solar energy tubular absorbers.

    PubMed

    Saltiel, C; Sokolov, M

    1982-11-15

    The energy absorbed by a solar energy tubular receiver element for a single incident ray is derived. Two types of receiver elements were analyzed: (1) an inner tube with an absorbing coating surrounded by a semitransparent cover tube, and (2) a semitransparent inner tube filled with an absorbing fluid surrounded by a semitransparent cover tube. The formation of ray cascades in the semitransparent tubes is considered. A numerical simulation to investigate the influence of the angle of incidence, sizing, thickness, and coefficient of extinction of the tubes was performed. A comparison was made between receiver elements with and without cover tubes. Ray tracing analyses in which rays were followed within the tubular receiver element as well as throughout the rest of the collector were performed for parabolic and circular trough concentrating collectors.

  5. Pointlike Inclusion Interactions in Tubular Membranes

    NASA Astrophysics Data System (ADS)

    Vahid, Afshin; Idema, Timon

    2016-09-01

    Membrane tubes and tubular networks are ubiquitous in living cells. Inclusions like proteins are vital for both the stability and the dynamics of such networks. These inclusions interact via the curvature deformations they impose on the membrane. We analytically study the resulting membrane mediated interactions in strongly curved tubular membranes. We model inclusions as constraints coupled to the curvature tensor of the membrane tube. First, as special test cases, we analyze the interaction between ring- and rod-shaped inclusions. Using Monte Carlo simulations, we further show how pointlike inclusions interact to form linear aggregates. To minimize the curvature energy of the membrane, inclusions self-assemble into either line- or ringlike patterns. Our results show that the global curvature of the membrane strongly affects the interactions between proteins embedded in it, and can lead to the spontaneous formation of biologically relevant structures.

  6. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates a biologically active peptide, prepro-TRH-(160-169), which regulates TRH-induced thyrotropin secretion

    SciTech Connect

    Bulant, M.; Vaudry, H. ); Roussel, J.P.; Astier, H. ); Nicolas, P. )

    1990-06-01

    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 {sup 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.

  7. p38MAPK activation is involved in androgen-independent proliferation of human prostate cancer cells by regulating IL-6 secretion

    SciTech Connect

    Shida, Yohei; Igawa, Tsukasa . E-mail: tigawa@net.nagasaki-u.ac.jp; Hakariya, Tomoaki; Sakai, Hideki; Kanetake, Hiroshi

    2007-02-16

    Increased levels of serum interleukin-6 (IL-6) are frequently observed in patients with advanced, hormone-refractory prostate cancer. However, the precise mechanism of IL-6 regulation is still largely unknown. Since prostate cancer gradually progresses to an androgen-independent state despite the stress caused by various therapeutic agents, we hypothesized the stress-activated protein kinases (SAPKs) involvement in androgen-independent growth or IL-6 secretion of prostate cancer cells. Using PC-3 and DU145 human prostate cancer cells, we analyzed the role of SAPKs in IL-6 mediated cell growth and found that the p38MAPK and JNK are involved in androgen-independent cancer cell growth. Furthermore, IL-6 secretion by PC-3 and DU145 cells was significantly suppressed by SAPKs inhibitor, especially by p38MAPK inhibitor SB203580, but not by JNK inhibitor SP600125 nor by MEK inhibitor, PD98059. These results raised the possibility that the IL-6 mediated androgen-independent proliferation of PC-3 and DU145 cells is regulated at least partly via SAPKs signaling pathway especially through p38MAPK activation.

  8. Catechin secretion and phytotoxicity

    PubMed Central

    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

  9. A novel ranacyclin-like peptide with anti-platelet activity identified from skin secretions of the frog Amolops loloensis.

    PubMed

    Hao, Xue; Tang, Xiaopeng; Luo, Lei; Wang, Yuming; Lai, Ren; Lu, Qiumin

    2016-01-15

    Albeit many bioactive peptides have been reported from amphibian skins, no anti-platelet peptide has been identified till to date. Here, an anti-platelet peptide, namely Zongdian platelet inhibitor (ZDPI), with the molecular weight of 1798.6 Da, was purified and characterized from skin secretions of the frog, Amolops loloensis. The amino acid sequence of ZDPI was determined as FRGCWLKNYSPRGCL-NH2 by combination methods of Edman degradation, mass spectrometry analysis and carboxypeptidase Y treatment revealing that it is composed of 15 amino acid residues with two cysteines formed an intra-molecular disulfide bridge and C-terminal amidation. cDNA encoding ZDPI precursor was cloned from skin cDNA library of A. loloensis. The precursor is composed of 63 amino acid (aa) residues including the predicted signal peptide (22 aa), an acidic spacer peptide (19 aa), and mature ZDPI. BLAST search indicates that ZDPI belongs to antimicrobial peptide family of ranacyclin, peptide leucine arginine or odorranain. It was found to inhibit ADP-induced platelet aggregation in a dose-dependent manner. At the concentration of 32 μg/ml, ZDPI completely inhibited platelet aggregation induced by ADP. To the best of our knowledge, this is the first report about an anti-platelet peptide from amphibian skin secretions. Considering its strong inhibitory ability on platelets and simple structure, ZDPI might be an excellent candidate or template to develop anti-thrombosis agent. In addition, the discovery of anti-platelet peptide in the frog skin increases biological function spectrum of amphibian skin peptides.

  10. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  11. Exercise-induced cramp, myoglobinuria, and tubular aggregates in phosphoglycerate mutase deficiency.

    PubMed

    Oh, Shin J; Park, Kyung-Seok; Ryan, Hewitt F; Danon, Moris J; Lu, Jiesheng; Naini, Ali B; DiMauro, Salvatore

    2006-11-01

    We report two patients in whom phosphoglycerate mutase (PGAM) deficiency was associated with the triad of exercise-induced cramps, recurrent myoglobinuria, and tubular aggregates in the muscle biopsy. Serum creatine kinase (CK) levels were elevated between attacks of myoglobinuria. Forearm ischemic exercise tests produced subnormal increases of venous lactate. Muscle biopsies showed subsarcolemmal tubular aggregates in type 2 fibers. Muscle PGAM activities were markedly decreased (3% of the normal mean) and molecular genetic studies showed that both patients were homozygous for a described missense mutation (W78X). A review of 15 cases with tubular aggregates in the muscle biopsies from our laboratory and 15 cases with PGAM deficiency described in the literature showed that this clinicopathological triad is highly suggestive of PGAM deficiency. PMID:16881065

  12. Self-Cleaning Tubular-Membrane Module

    NASA Technical Reports Server (NTRS)

    Sarbolouki, M. N.

    1983-01-01

    Tubular membranes made self-cleaning with aid of flow reversing valve. Sponge balls scrub membrane surfaces as they travel inside membrane tubes. A four-way flow-reversal valve automatically reverses flow in tubes at preset intervals so sponge balls reciprocate along tubes. Baskets at ends of tubes prevent sponges from escaping. Automatic cleaning feature added to existing membrane processing equipment with minimal modifications.

  13. Latch ring for connecting tubular member

    SciTech Connect

    Milberger, L.J.

    1991-06-04

    This patent describes a device for releasably locking an inner member well bore of a tubular outer member, comprising a combination of a grooved inner member profile formed on the exterior of the inner member; a grooved outer member profile formed in the bore of the outer member; a split ring carried by the inner member the ring having a grooved outer profile on its exterior mates with the outer member profile; and the inner member being axially movable.