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Sample records for beta cell secretory

  1. The Fas pathway is involved in pancreatic beta cell secretory function

    DEFF Research Database (Denmark)

    Schumann, Desiree M; Maedler, Kathrin; Franklin, Isobel

    2007-01-01

    Pancreatic beta cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in beta cell apoptosis or proliferation, depending...... on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates beta cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a beta cell-specific transcription factor...... regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient beta cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased beta cell mass. Up-regulation of FLIP enhanced NF-kappaB activity via NF...

  2. Evaluation of beta-cell secretory capacity using glucagon-like peptide 1

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    Vilsbøll, Tina; Nielsen, Mette Toft; Krarup, T

    2000-01-01

    Beta-cell secretory capacity is often evaluated with a glucagon test or a meal test. However, glucagon-like peptide 1 (GLP-1) is the most insulinotropic hormone known, and the effect is preserved in type 2 diabetic patients.......Beta-cell secretory capacity is often evaluated with a glucagon test or a meal test. However, glucagon-like peptide 1 (GLP-1) is the most insulinotropic hormone known, and the effect is preserved in type 2 diabetic patients....

  3. Analysis of the effect of diabetes type 2 duration on beta cell secretory function and insulin resistance

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    Popović Ljiljana

    2006-01-01

    Full Text Available Diabetes type 2 is a chronic metabolic disorder. Pathogenesis of diabetes type 2 results from the impaired insulin secretion, impaired insulin action and increased endogenous glucose production. Diabetes evolves through several phases characterized by qualitative and quantitative changes of beta cell secretory function. The aim of our study was to analyze the impact of diabetes duration on beta cell secretory function and insulin resistance. The results indicated significant negative correlation of diabetes duration and fasting insulinemia, as well as beta cell secretory function assessed by HOMA β index. Our study also found significant negative correlation of diabetes duration and insulin resistance assessed by HOMA IR index. Significant positive correlation was established between beta cell secretory capacity (fasting insulinemia and HOMA β and insulin resistance assessed by HOMA IR index, independently of diabetes duration. These results indicate that: beta cell secretory capacity, assessed by HOMA β index, significantly decreases with diabetes duration. In parallel with decrease of fasting insulinemia, reduction of insulin resistance assessed by HOMA IR index was found as well.

  4. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

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    Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

    2009-01-01

    Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...... secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored...

  5. Chronic antidiabetic sulfonylureas in vivo: reversible effects on mouse pancreatic beta-cells.

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    Maria Sara Remedi

    2008-10-01

    Full Text Available Pancreatic beta-cell ATP-sensitive potassium (K ATP channels are critical links between nutrient metabolism and insulin secretion. In humans, reduced or absent beta-cell K ATP channel activity resulting from loss-of-function K ATP mutations induces insulin hypersecretion. Mice with reduced K ATP channel activity also demonstrate hyperinsulinism, but mice with complete loss of K ATP channels (K ATP knockout mice show an unexpected insulin undersecretory phenotype. Therefore we have proposed an "inverse U" hypothesis to explain the response to enhanced excitability, in which excessive hyperexcitability drives beta-cells to insulin secretory failure without cell death. Many patients with type 2 diabetes treated with antidiabetic sulfonylureas (which inhibit K ATP activity and thereby enhance insulin secretion show long-term insulin secretory failure, which we further suggest might reflect a similar progression.To test the above hypotheses, and to mechanistically investigate the consequences of prolonged hyperexcitability in vivo, we used a novel approach of implanting mice with slow-release sulfonylurea (glibenclamide pellets, to chronically inhibit beta-cell K ATP channels. Glibenclamide-implanted wild-type mice became progressively and consistently diabetic, with significantly (p < 0.05 reduced insulin secretion in response to glucose. After 1 wk of treatment, these mice were as glucose intolerant as adult K ATP knockout mice, and reduction of secretory capacity in freshly isolated islets from implanted animals was as significant (p < 0.05 as those from K ATP knockout animals. However, secretory capacity was fully restored in islets from sulfonylurea-treated mice within hours of drug washout and in vivo within 1 mo after glibenclamide treatment was terminated. Pancreatic immunostaining showed normal islet size and alpha-/beta-cell distribution within the islet, and TUNEL staining showed no evidence of apoptosis.These results demonstrate that

  6. The GLP-1 analogue liraglutide improves first-phase insulin secretion and maximal beta-cell secretory capacity over 14 weeks of therapy in subjects with Type 2 diabetes

    DEFF Research Database (Denmark)

    Madsbad, Sten; Vilsbøll, Tina; Brock, Birgitte

    Aims: We investigated the clinical effect of liraglutide, a long- acting GLP-1 analogue, on insulin secretion in Type 2 diabetes. Methods: Thirty-nine subjects (28 completed) from a randomised trial received a hyperglycaemic clamp (20 mM) with intravenous arginine stimulation, and an insulin...... group. Conclusion: In subjects with Type 2 diabetes, 14 weeks’ once-daily liraglutide (1.25 and 1.9 mg/day) markedly improves beta-cell function, significantly increases first-phase insulin secretion and maximal beta-cell secretory capacity....

  7. Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

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    Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

    2016-06-01

    We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  8. Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

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    Holger A Russ

    Full Text Available Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug

  9. Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

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    MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

    2015-01-01

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

  10. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia.

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    Ernesto Bernal-Mizrachi

    2010-08-01

    Full Text Available Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+ influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+ signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes.To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP. To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis.Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes.

  11. Porosome: The Universal Secretory Portal in Cells

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    Jena, Bhanu

    2012-10-01

    In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the ``all or none'' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in 1993 in a News and Views article in Nature, E. Neher wrote ``It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.'' This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the ``Porosome,'' the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nm resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm

  12. Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

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    Ma, Z; Ramanadham, S; Wohltmann, M; Bohrer, A; Hsu, F F; Turk, J

    2001-04-20

    A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

  13. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    Science.gov (United States)

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Differential testosterone secretory capacity of the testes of aggressive and nonaggressive house mice during ontogeny

    OpenAIRE

    de Ruiter, Anne J H; Koolhaas, Jaap M; van Oortmerssen, Geert A; Bohus, Bela

    1992-01-01

    In this study, testosterone secretory capacity of testicular Leydig cells during ontogeny was determined in males of an aggressive and a nonaggressive genetic selection line of wild house mice. Neonates, 23-day-old prepubertals, and adult male mice were studied. A morphometric method was used to quantify 3-beta-hydroxy steroid dehydrogenase (3-beta-HSD)-stained Leydig cells in testicular sections to determine testosterone secretory capacity. We consider this parameter to reflect circulating t...

  15. Influence of beta blockade on gastric acid secretion and changes in gastric mucosal blood flow before and after parietal cell vagotomy in dogs and man

    DEFF Research Database (Denmark)

    Hovendal, C P; Bech, K; Bekker, C

    1983-01-01

    The aim of the present study was, in paired experiments in dogs, to examine the effect of beta-receptor blockade on gastric acid secretion and mucosal blood flow before and after parietal cell vagotomy (PCV). The secretory response to pentagastrin was reduced after vagotomy. beta-Adrenergic block......The aim of the present study was, in paired experiments in dogs, to examine the effect of beta-receptor blockade on gastric acid secretion and mucosal blood flow before and after parietal cell vagotomy (PCV). The secretory response to pentagastrin was reduced after vagotomy. beta...

  16. Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

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    Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

    2009-01-01

    The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

  17. Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice

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    Hongyu Li

    2015-03-01

    Conclusions: Together with our previous studies, the current data reveal a conserved role for BIG3 in regulating alpha- and beta-cell functions. We propose that BIG3 negatively regulates hormone production at the secretory granule biogenesis stage and that such regulatory mechanism may be used in secretory pathways of other endocrine cells.

  18. Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

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    Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

    2016-11-01

    Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

  19. Vildagliptin, a dipeptidyl peptidase-IV inhibitor, improves model-assessed beta-cell function in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Mari, A; Sallas, W M; He, Y L

    2005-01-01

    in diabetic patients, suggesting that more sophisticated measures are necessary to ascertain the influence of vildagliptin on beta-cell function. METHODS: This study examined the effects of 28-d treatment with vildagliptin (100 mg, twice daily; n = 9) vs. placebo (n = 11) on beta-cell function in diabetic...... and other factors. RESULTS: Vildagliptin significantly increased the insulin secretory rate at 7 mmol/liter glucose (secretory tone), calculated from the dose response; the difference in least squares mean (deltaLSM) was 101 +/- 51 pmol.min(-1).m(-2) (P = 0.002). The slope of the beta-cell dose response......, the derivative component, and the potentiation factor were not affected. Vildagliptin also significantly decreased mean prandial glucose (deltaLSM, -1.2 +/- 0.4 mmol/liter; P = 0.01) and glucagon (deltaLSM, -10.7 +/- 4.8 ng/liter; P = 0.03) levels and increased plasma levels of intact GLP-1 (deltaLSM, +10...

  20. Dual role of proapoptotic BAD in insulin secretion and beta cell survival.

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    Danial, Nika N; Walensky, Loren D; Zhang, Chen-Yu; Choi, Cheol Soo; Fisher, Jill K; Molina, Anthony J A; Datta, Sandeep Robert; Pitter, Kenneth L; Bird, Gregory H; Wikstrom, Jakob D; Deeney, Jude T; Robertson, Kirsten; Morash, Joel; Kulkarni, Ameya; Neschen, Susanne; Kim, Sheene; Greenberg, Michael E; Corkey, Barbara E; Shirihai, Orian S; Shulman, Gerald I; Lowell, Bradford B; Korsmeyer, Stanley J

    2008-02-01

    The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

  1. What are the potential benefits of clinical beta-cell imaging in diabetes mellitus?

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    Göke, Burkhard

    2010-05-01

    Previously, studies of the endocrine pancreatic beta-cell were mainly performed ex vivo by morphological means. This data supported the analysis of pathophysiological changes in the pancreatic islet during insults such as diabetes mellitus. Metabolic testing of the pancreatic islet by assaying hormone parameters such als plasma insulin or C-peptide combined with more or less sophisticated calculations allowed conclusions about states of insulin resistance or secretory failure. It also allowed certain correlations of endocrine function with beta-cell mass. Today, with firmer pathophysiological concepts about beta-cell failure, modern protocols of islet transplantation, and drugs on the market coming with promises of preservation or even expansion of beta-cell mass in diabetes mellitus it has become very attractive to search for tools measuring beta-cell mass, if possible even repeatingly in the same organism in vivo. From a clinical point of view, the potential of pancreatic beta-cell mass imaging technologies is looked upon with high expectations. Methodologically, the decisive question is whether it is likely that future beta-cell imaging will provide significant advantages over the metabolic methods already in hand. With new in vivo tools, studies of beta-cell mass and function may offer even new approaches stratifying patients to anti-diabetic therapies.

  2. Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

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    Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

    2011-04-01

    Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

  3. [Expansion of secretory cells in the fallopian tubal epithelium in the early stages of the pathogenesis of ovarian serous carcinomas].

    Science.gov (United States)

    Asaturova, A V; Ezhova, L S; Faizullina, N M; Adamyan, L V; Khabas, G N; Sannikova, M V

    to investigate the frequency of the types of fallopian tubal secretory cell expansion (SCE) in diseases of the reproductive organs and to determine the immunophenotype and biological role of the cells in the early stages of the pathogenesis of high-grade ovarian serous carcinomas (HGOSC). The investigation enrolled 287 patients with extraovarian diseases and ovarian serous tumors varying in grade, whose fallopian tubes were morphologically and immunohistochemically examined using p53, Ki-67, PAX2, Bcl-2, beta-catenin, and ALDH1 markers. The material was statistically processed applying the Mann-Whitney test and χ2 test. The rate of secretory cell proliferation (SCP) (more than 10 consecutive secretory cells) and that of secretory cell overgrowth (SCO) (more than 30 consecutive secretory cells) increase with age in all investigated reproductive system diseases. The rate of SCP in the corpus fimbriatum of the patients with HGOSC was 5.9 times higher than that in those with extraovarian disease (pepithelium (2.8), in SCP (1.3), in SCO (1.2), in serous tubal intraepithelial carcinoma (STIC) (1.0), and in HGOSC (0.9); Bcl-2 was in the intact epithelium (2.2), in SCP (2.1), STIC (0.9), and in HGOSC (0.6), β-catenin was in the intact epithelium (0.5), in SCP (2.85), in SCO (2.95), in STIC (0.6), and in HGOSC (0.5); ALDH1 was in the intact epithelium (0.5), in SCP (2.91), in SCO (2.92), in STIC (1.2), and in HGOSC (0.6). There were statistically significant differences with a 95% confidence interval (pepithelium and pathology (fallopian tube lesions and HGOSC); 2) Bcl-2 between the intact epithelium and SCE (SCP and SCO) and between SCE and HGOSC; 3) beta-catenin between the intact epithelium and SCE (SCP and SCO) and between SCE and HGOSC; 4) ALDH1 between the intact epithelium and SCE, between and SCE and STIC, and between STIC and HGOSC. SCE was shown to be an independent intraepithelial lesion. The incidence of this abnormality increased with age and significantly

  4. Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets

    DEFF Research Database (Denmark)

    Maedler, Kathrin; Størling, Joachim; Sturis, Jeppe

    2004-01-01

    Increasing evidence indicates that a progressive decrease in the functional beta-cell mass is the hallmark of both type 1 and type 2 diabetes. The underlying causes, beta-cell apoptosis and impaired secretory function, seem to be partly mediated by macrophage production of interleukin (IL)-1beta ...

  5. File list: Unc.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 Unclassified Uterus Fallopian tube secret...ory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  6. File list: Unc.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  11. File list: Oth.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  13. File list: DNS.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Pol.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  18. File list: DNS.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 TFs and others Uterus Fallopian tube secret...ory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  20. The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.

    Science.gov (United States)

    Squires, Paul E; Hills, Claire E; Rogers, Gareth J; Garland, Patrick; Farley, Sophia R; Morgan, Noel G

    2004-10-06

    beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect

  1. Use of digitonin permeabilization for characterization of calcium incorporated into. beta. cells in response to glucose

    Energy Technology Data Exchange (ETDEWEB)

    Wesslen, N [Uppsala Univ. (Sweden)

    1985-01-01

    ..beta.. cell-rich pancreatic islets were used to characterize /sup 45/Ca uptake into pools with different sensitivities to permeabilization with digitonin. The plasma and secretory granule membranes were among the membranes most sensitive to digitonin treatment whereas the mitochondria were more resistant. Most of the /sup 45/Ca incorporated in response to glucose was found in a fraction released after exposure to a high concentration of digitonin. The results suggest that glucose promotes active sequestration of calcium in mitochondria and that the uptake of /sup 45/Ca in the secretory granules in secondary to a raised cytosolic Ca/sup 2 +/ activity.

  2. Glucose activates prenyltransferases in pancreatic islet {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Goalstone, Marc [Department of Medicine, University of Colorado, VA Medical Center, Denver, CO 80220 (United States); Kamath, Vasudeva [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States); Kowluru, Anjaneyulu, E-mail: akowluru@med.wayne.edu [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States)

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  3. File list: NoD.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 No description Uterus Fallopian tube secret...ory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  4. File list: NoD.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: NoD.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: NoD.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 No description Uterus Fallopian tube secret...ory epithelial cell http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  7. The Role of Adenoid Mast Cells in the Pathogenesis of Secretory Otitis Media

    Directory of Open Access Journals (Sweden)

    M. Faruk Oktay

    2007-01-01

    Full Text Available To investigate the possible role of adenoid mast cells in the etiology of secretory otitis media. Between 2001-2002, 25 patients with chronic adenoitis and chronic secretory otitis media and 25 patients with isolated adenoid hypertrophy were included to the study. Adenoidectomy performed to the all patients under general anesthesia. Adenoidectomy specimens were evaluated under the light microscopy and the number of mast cells were calculated for each patient. The number of mast cells were compared between two groups. The number of mast cells were between 4-84 in the otitis media with effusion and adenoid hypertrophy group (median:52, however it was between 2-63 (median: 23 in the isolated adenoid hypertrophy group. When comparing the two groups using Mann-Withney U test, the number of mast cells found to be significantly higher in the chronic secretory otitis media group (p<0.001.Based on our findings there is a relationship between increased adenoid mast cells and otitis media with effusion and these cells may have a possible role in the etiology of chronic secretory otitis media.

  8. Aspirin induces morphological transformation to the secretory state in isolated rabbit parietal cells.

    Science.gov (United States)

    Murthy, U K; Levine, R A

    1991-08-01

    The morphological response of rabbit parietal cells to aspirin was evaluated by grading several ultra-structural features including the extent of the tubulovesicular system, intracellular secretory canaliculi, and microvilli. After exposure of isolated parietal cells and gastric glands to aspirin or histamine, there was an approximately twofold increase in the ratio of secretory to nonsecretory parietal cells, and depletion of extracellular Ca2+ abolished the aspirin-induced morphological changes. Morphometry in parietal cells showed that aspirin induced a sixfold increase in secretory canalicular membrane elaboration. Aspirin potentiated histamine-induced parietal cell respiration and aminopyrine uptake ratio but did not increase basal respiration or aminopyrine uptake, suggesting an apparent dissociation from aspirin-induced morphological changes.

  9. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  10. Suppression of cell death by the secretory form of N-terminal ERC/mesothelin.

    Science.gov (United States)

    Wang, Tegexibaiyin; Kajino, Kazunori; Abe, Masaaki; Tan, Ke; Maruo, Masumi; Sun, Guodong; Hagiwara, Yoshiaki; Maeda, Masahiro; Hino, Okio

    2010-08-01

    ERC/mesothelin is highly expressed in malignant mesothelioma, pancreatic cancer, and ovarian cancer. It is cleaved to a 30 kDa N-terminal secretory form (N-ERC) and a 40 kDa C-terminal membranous form (C-ERC). Several functions have been reported for full-length ERC (full-ERC) and C-ERC/mesothelin, such as in cell adhesion and invasion, stimulation of cell proliferation, and the suppression of cell death. However, there have been no studies to date on the function of secretory N-ERC, despite the fact that it is abundantly secreted into the sera of mesothelioma patients. In this study, we investigated whether N-ERC could function as a secretory factor to stimulate tumor progression. Full-, N, or C-ERC was overexpressed in the human hepatocellular carcinoma cell line Huh7 that lacks endogenous expression of ERC/mesothelin. Changes in the rates of cell proliferation and cell death were determined, and the state of signal transducers was examined using various endpoints: total cell counts, trypan blue exclusion rate, BrdU incorporation rate, TUNEL assay, and the phosphorylation of ERK1/2 and Stat3. In cells overexpressing N-ERC, phosphorylation of ERK1/2 was enhanced and the rate of cell death decreased, leading to the increase of cell number. The culture medium containing the secretory N-ERC also had the activity to increase the number of cells. Our data suggested that one of the full-ERC functions reported previously was mediated by the secretory N-ERC.

  11. Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

    Directory of Open Access Journals (Sweden)

    Tiina Rasila

    Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

  12. The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

    Science.gov (United States)

    Gerber, Philipp A; Rutter, Guy A

    2017-04-01

    Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  15. File list: ALL.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. Loss of Sonic hedgehog leads to alterations in intestinal secretory cell maturation and autophagy.

    Directory of Open Access Journals (Sweden)

    Jessica Gagné-Sansfaçon

    Full Text Available BACKGROUND: Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. METHODOLOGY/PRINCIPAL FINDINGS: A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. CONCLUSIONS/SIGNIFICANCE: Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy.

  17. The plant secretory pathway seen through the lens of the cell wall.

    Science.gov (United States)

    van de Meene, A M L; Doblin, M S; Bacic, Antony

    2017-01-01

    Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.

  18. File list: His.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: His.Utr.05.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  3. File list: InP.Utr.10.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: InP.Utr.50.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

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    Lifescience Database Archive (English)

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  6. File list: InP.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell hg19 Input control Uterus Fallopian tube secret...iencedbc.jp/kyushu-u/hg19/assembled/InP.Utr.20.AllAg.Fallopian_tube_secretory_epithelial_cell.bed ...

  7. Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

    DEFF Research Database (Denmark)

    Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik

    1993-01-01

    Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part...... of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double...... labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein...

  8. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

    Science.gov (United States)

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

    2016-10-31

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

  9. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    Science.gov (United States)

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  10. Peroxisome proliferator-activated receptor alpha (PPARalpha) protects against oleate-induced INS-1E beta cell dysfunction by preserving carbohydrate metabolism

    DEFF Research Database (Denmark)

    Frigerio, F; Brun, T; Bartley, C

    2009-01-01

    and investigated key metabolic pathways and genes responsible for metabolism-secretion coupling during a culture period of 3 days in the presence of 0.4 mmol/l oleate. RESULTS: In INS-1E cells, the secretory dysfunction primarily induced by oleate was aggravated by silencing of PPARalpha. Conversely, PPARalpha...... enzyme pyruvate carboxylase. PPARalpha overproduction increased both beta-oxidation and fatty acid storage in the form of neutral triacylglycerol, revealing overall induction of lipid metabolism. These observations were substantiated by expression levels of associated genes. CONCLUSIONS....../INTERPRETATION: PPARalpha protected INS-1E beta cells from oleate-induced dysfunction, promoting both preservation of glucose metabolic pathways and fatty acid turnover....

  11. Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase.

    Science.gov (United States)

    Chen, Jianguo; Jeppesen, Per Bendix; Nordentoft, Iver; Hermansen, Kjeld

    2007-06-01

    Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.

  12. Beta cell adaptation in pregnancy

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    2016-01-01

    Pregnancy is associated with a compensatory increase in beta cell mass. It is well established that somatolactogenic hormones contribute to the expansion both indirectly by their insulin antagonistic effects and directly by their mitogenic effects on the beta cells via receptors for prolactin...... and growth hormone expressed in rodent beta cells. However, the beta cell expansion in human pregnancy seems to occur by neogenesis of beta cells from putative progenitor cells rather than by proliferation of existing beta cells. Claes Hellerström has pioneered the research on beta cell growth for decades...... in the expansion of the beta cell mass in human pregnancy, and the relative roles of endocrine factors and nutrients....

  13. Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function.

    Science.gov (United States)

    Wang, Hongdong; Wang, Xiaping; Zhu, Yunxia; Chen, Fang; Sun, Yujie; Han, Xiao

    2015-11-01

    Although insulin resistance is recognized to contribute to the reproductive and metabolic phenotypes of polycystic ovary syndrome (PCOS), pancreatic beta cell dysfunction plays an essential role in the progression from PCOS to the development of type 2 diabetes. However, the role of insulin secretory abnormalities in PCOS has received little attention. In addition, the precise changes in beta cells and the underlying mechanisms remain unclear. In this study, we therefore attempted to elucidate potential mechanisms involved in beta cell alterations in a rat model of PCOS. Glucose-induced insulin secretion was measured in islets isolated from DHT-treated and control rats. Oxygen consumption rate (OCR), ATP production, and mitochondrial copy number were assayed to evaluate mitochondrial function. Glucose-stimulated insulin secretion is significantly decreased in islets from DHT-treated rats. On the other hand, significant reductions are observed in the expression levels of several key genes involved in mitochondrial biogenesis and in mitochondrial OCR and ATP production in DHT-treated rat islets. Meanwhile, we found that androgens can directly impair beta cell function by inducing mitochondrial dysfunction in vitro in an androgen receptor dependent manner. For the first time, our study demonstrates that increased androgens in female rats can impair glucose-stimulated insulin secretion partly through disruption of pancreatic beta cell mitochondrial function. This work has significance for hyperandrogenic women with PCOS: excess activation of the androgen receptor by androgens may provoke beta cell dysfunction via mitochondrial dysfunction. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...

  15. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  16. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    International Nuclear Information System (INIS)

    Baconnais, S.; Delavoie, F.; Zahm, J.M.; Milliot, M.; Terryn, C.; Castillon, N.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E.; Balossier, G.

    2005-01-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na + absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na + , Mg 2+ , P, S and Cl - ) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR inh -172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF

  17. Identification and characterization of secretory proteins during ionizing radiation-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Han, Na Kyung; Hong, Mi Na; Jung, Seung Hee; Kang, Kyoung Ah; Lee, Jae Seon [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chi, Seong Gil [Korea University, Seoul (Korea, Republic of)

    2011-05-15

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated {beta} alactosidase positivity. Recently a large number of molecular phenotypes such as changes in gene expression, protein processing and chromatin organization have been also described. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence. Here, we show that senescent human breast cancer MCF7 cells promote the proliferation, invasion and migration of neighboring cells

  18. Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

    Directory of Open Access Journals (Sweden)

    Nils Paulmann

    2009-10-01

    Full Text Available While serotonin (5-HT co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/- and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

  19. Disparate effects of p24alpha and p24delta on secretory protein transport and processing.

    Directory of Open Access Journals (Sweden)

    Jeroen R P M Strating

    Full Text Available BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3, -beta(1, -gamma(3 and -delta(2 is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC. METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 or p24delta(2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

  20. The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis

    Directory of Open Access Journals (Sweden)

    Samuel B. Stephens

    2017-09-01

    Full Text Available The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity.

  1. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1993-01-01

    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....

  2. Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease.

    Science.gov (United States)

    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2012-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    International Nuclear Information System (INIS)

    Abu-Hamdah, Rania; Cho, Won Jin; Hoerber, J.K.H.; Jena, Bhanu P.

    2006-01-01

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles

  4. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Hamdah, Rania [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Cho, Won Jin [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Hoerber, J.K.H. [Department of Physics, University of Bristol, Bristol BS8 1TD (United Kingdom); Jena, Bhanu P. [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States)]. E-mail: bjena@med.wayne.edu

    2006-06-15

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 {mu}m in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

  5. Ubiquitin fold modifier 1 (UFM1 and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Katleen Lemaire

    Full Text Available UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116, which we name UFM1-binding protein 1 containing a PCI domain (UFBP1, and CDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3 are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E. siRNA-mediated Ufm1 or Ufbp1 knockdown enhances apoptosis upon ER stress. Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1 participate in preventing ER stress-induced apoptosis in protein secretory cells.

  6. Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects after prolonged culture in a low non-stimulating glucose concentration.

    Science.gov (United States)

    Roma, L P; Pascal, S M; Duprez, J; Jonas, J-C

    2012-08-01

    Pancreatic beta cells chronically exposed to low glucose concentrations show signs of oxidative stress, loss of glucose-stimulated insulin secretion (GSIS) and increased apoptosis. Our aim was to confirm the role of mitochondrial oxidative stress in rat islet cell apoptosis under these culture conditions and to evaluate whether its reduction similarly improves survival and GSIS. Apoptosis, oxidative stress-response gene mRNA expression and glucose-induced stimulation of mitochondrial metabolism, intracellular Ca(2+) concentration and insulin secretion were measured in male Wistar rat islets cultured for 1 week in RPMI medium containing 5-10 mmol/l glucose with or without manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) or N-acetyl-L-: cysteine (NAC). Oxidative stress was measured in islet cell clusters cultured under similar conditions using cytosolic and mitochondrial redox-sensitive green fluorescent protein (roGFP1/mt-roGFP1). Prolonged culture in 5 vs 10 mmol/l glucose increased mt-roGFP1 (but not roGFP1) oxidation followed by beta cell apoptosis and loss of GSIS resulting from reduced insulin content, mitochondrial metabolism, Ca(2+) influx and Ca(2+)-induced secretion. Tolbutamide-induced, but not high K(+)-induced, Ca(2+) influx was also suppressed. Under these conditions, MnTBAP, but not NAC, triggered parallel ~50-70% reductions in mt-roGFP1 oxidation and beta cell apoptosis, but failed to protect against the loss of GSIS despite significant improvement in glucose-induced and tolbutamide-induced Ca(2+) influx. Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects during culture in a low glucose concentration. Thus, targeting beta cell survival may not be sufficient to restore insulin secretion when beta cells suffer from prolonged mitochondrial oxidative stress, e.g. in the context of reduced glucose metabolism.

  7. Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Brandl, Julian

    2017-01-01

    , counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods...... to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional...... regulation of protein secretion than healthy mouse cells.  Conclusions: The RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein...

  8. Microbiota promote secretory cell determination in the intestinal epithelium by modulating host Notch signaling.

    Science.gov (United States)

    Troll, Joshua V; Hamilton, M Kristina; Abel, Melissa L; Ganz, Julia; Bates, Jennifer M; Stephens, W Zac; Melancon, Ellie; van der Vaart, Michiel; Meijer, Annemarie H; Distel, Martin; Eisen, Judith S; Guillemin, Karen

    2018-02-23

    Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer. © 2018. Published by The Company of Biologists Ltd.

  9. Distorted secretory granule composition in mast cells with multiple protease deficiency.

    Science.gov (United States)

    Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

    2013-10-01

    Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

  10. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  11. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    Science.gov (United States)

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

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

    DEFF Research Database (Denmark)

    Frödin, M; Sekine, N; Roche, E

    1995-01-01

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

  13. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Wang

    Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

  14. Secretory structure and histochemistry test of some Zingiberaceae plants

    Science.gov (United States)

    Indriyani, Serafinah

    2017-11-01

    A secretory structure is a structure that produces a plant's metabolite substances. Secretory structures are grouped into an internal and external. Zingiberaceae plants are known as traditional medicine plants and as spice plants due to secretory structures in their tissues. The objective of the research were to describe the secretory structure of Zingiberaceae plants and to discover the qualitatively primary metabolite substances in plant's tissues via histochemistry test. The research was conducted by observation descriptive design, quantitative data including the density of secretory cells per mm². The quantitative data were analyzed by ANOVA and continued by Duncan at α = 5 %. The results showed that the secretory structures in leaves, rhizome, and the root of 14 species of Zingiberaceae plants are found in the mesophyll of leaves and cortex, and also pith in rhizome and roots. The type of secretory structure is internal. Within the root of Zingiber cassumunar Roxb.(bengle), Curcuma domestica Val. (kunyit), Curcuma zedoaria (Berg.) Roscoe (kunyit putih), Zingiber zerumbet (L.) J.E. Smith (lempuyang), Alpiniapurpurata K. Schum (lengkuas merah), and Curcuma aeruginosa Val. (temu ireng) were found amylum grains, while in Kaemferia galanga L. (kencur), Boesen bergiapandurata L. (temu kunci), and Curcuma xanthorrhiza Roxb. (temulawak) there were no amylum grains in the root as well as in the leaves. The roots of bengle had the greatest density of amylum grain, it had 248.1 ± 9.8 secretory cells of amylum grains per mm². Lipids (oil droplets) were found in the root of bengle, Zingiber officinale Roxb. Var. emprit (jahe emprit), Zingiber officinale Roxb. Var. Gajah (jahe gajah), Zingiber officinale Roxb. Var. Rubrum (jahe merah), Keampferia angustifolia L. (kunci pepet), kunyit, kunyit putih, lempuyang, lengkua smerah, Curcuma aeruginosa Val. (temu ireng), and Curcuma mangga Val. and van Zijp (temu mangga); the root of lempuyang had the greatest density of oil

  15. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells

    OpenAIRE

    Kaur, J.; Cutler, D. F.

    2002-01-01

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal...

  16. Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-β.

    Science.gov (United States)

    Mikulic, Josip; Longet, Stéphanie; Favre, Laurent; Benyacoub, Jalil; Corthesy, Blaise

    2017-06-01

    The importance of secretory IgA in controlling the microbiota is well known, yet how the antibody affects the perception of the commensals by the local immune system is still poorly defined. We have previously shown that the transport of secretory IgA in complex with bacteria across intestinal microfold cells results in an association with dendritic cells in Peyer's patches. However, the consequences of such an interaction on dendritic cell conditioning have not been elucidated. In this study, we analyzed the impact of the commensal Lactobacillus rhamnosus, alone or associated with secretory IgA, on the responsiveness of dendritic cells freshly recovered from mouse Peyer's patches, mesenteric lymph nodes, and spleen. Lactobacillus rhamnosus-conditioned mucosal dendritic cells are characterized by increased expression of Toll-like receptor regulatory proteins [including single immunoglobulin interleukin-1 receptor-related molecule, suppressor of cytokine signaling 1, and Toll-interacting molecule] and retinaldehyde dehydrogenase 2, low surface expression of co-stimulatory markers, high anti- versus pro-inflammatory cytokine production ratios, and induction of T regulatory cells with suppressive function. Association with secretory IgA enhanced the anti-inflammatory/regulatory Lactobacillus rhamnosus-induced conditioning of mucosal dendritic cells, particularly in Peyer's patches. At the systemic level, activation of splenic dendritic cells exposed to Lactobacillus rhamnosus was partially dampened upon association with secretory IgA. These data suggest that secretory IgA, through coating of commensal bacteria, contributes to the conditioning of mucosal dendritic cells toward tolerogenic profiles essential for the maintenance of intestinal homeostasis.

  17. Ursodeoxycholic acid attenuates colonic epithelial secretory function

    Science.gov (United States)

    Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

    2013-01-01

    Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

  18. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    Science.gov (United States)

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  19. Generation of Transplantable Beta Cells for Patient-Specific Cell Therapy

    Directory of Open Access Journals (Sweden)

    Xiaojie Wang

    2012-01-01

    Full Text Available Islet cell transplantation offers a potential cure for type 1 diabetes, but it is challenged by insufficient donor tissue and side effects of current immunosuppressive drugs. Therefore, alternative sources of insulin-producing cells and isletfriendly immunosuppression are required to increase the efficiency and safety of this procedure. Beta cells can be transdifferentiated from precursors or another heterologous (non-beta-cell source. Recent advances in beta cell regeneration from somatic cells such as fibroblasts could circumvent the usage of immunosuppressive drugs. Therefore, generation of patient-specific beta cells provides the potential of an evolutionary treatment for patients with diabetes.

  20. The differences in RCAS1 and DFF45 endometrial expression between late proliferative, early secretory, and mid-secretory cycle phases.

    Directory of Open Access Journals (Sweden)

    Jerzy Sikora

    2008-04-01

    Full Text Available RCAS1 expression is related to the regulation of activated immune cells and to connective tissue remodeling within the endometrium. DFF45 seems to play an important role in the apoptotic process, most likely by acting through the regulation of DNA fragmentation. Its expression changes within the endometrium seem to be related to the resistance of endometrial cells to apoptosis. The aim of the present study was to evaluate RCAS1 and DFF45 endometrial expressions during ovulation and the implantation period. RCAS1 and DFF45 expression was assessed by the Western-blot method in endometrial tissue samples obtained from 20 patients. The tissue samples were classified according to the menstrual cycle phases in which they were collected, with a division into three phases: late proliferative, early secretory, and mid-secretory. The lowest level of RCAS1 and the highest level of DFF45 endometrial expression was found during the early secretory cycle phase. Statistically significantly higher RCAS1 and statistically significantly lower DFF45 endometrial expression was identified in the endometrium during the late proliferative as compared to the early secretory cycle phase. Moreover, statistically significantly higher RCAS1 and statistically significantly lower DFF45 expression was found in the endometrium during the mid-secretory as compared to the early secretory cycle phase. The preparation for implantation process in the endometrium is preceded by dynamic changes in endometrial ECM and results from the proper interaction between endometrial and immune cells. The course of this process is conditioned by the immunomodulating activity of endometrial cells and their resistance to immune-mediated apoptosis. These dynamic changes are closely related to RCAS1 and DFF45 expression alterations.

  1. Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E

    2010-09-10

    Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

  2. Secretory cavities and volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): an endemic species collected in the restingas of Rio de Janeiro, Brazil.

    Science.gov (United States)

    Victório, Cristiane Pimentel; Moreira, Claudio B; Souza, Marcelo da Costa; Sato, Alice; Arruda, Rosani do Carmo de Oliveira

    2011-07-01

    In this study, we investigated the leaf anatomy and the composition of volatiles in Myrrhinium atropurpureum var. atropurpureum endemic to Rio de Janeiro restingas. Particularly, leaf secretory structures were described using light microscopy, and histochemical tests were performed from fresh leaves to localize the secondary metabolites. To observe secretory cavities, fixed leaf samples were free-hand sectioned. To evaluate lipophilic compounds and terpenoids the following reagents were employed: Sudans III and IV, Red oil O and Nile blue. Leaf volatiles were characterized by gas chromatography after hydrodistillation (HD) or simultaneous distillation-extraction (SDE). Leaf analysis showed several cavities in mesophyll that are the main sites of lipophilic and terpenoid production. Monoterpenes, which represented more than 80% of the major volatiles, were characterized mainly by alpha- and beta-pinene and 1,8-cineole. In order to provide tools for M. atropurpureum identification, the following distinguishing characteristics were revealed by the following data: 1) adaxial face clear and densely punctuated by the presence of round or ellipsoidal secretory cavities randomly distributed in the mesophyll; 2) the presence of cells overlying the upper neck cells of secretory cavities; 3) the presence of numerous paracytic stomata distributed on the abaxial leaf surface, but absent in vein regions and leaf margin; and 4) non-glandular trichomes on both leaf surfaces. Our study of the compounds produced by the secretory cavities of M. atropurpureum led us to conclude that volatile terpenoid class are the main secretory compounds and that they consist of a high concentration of monoterpenes, which may indicate the phytotherapeutic importance of this plant.

  3. A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway*

    Science.gov (United States)

    Melo, Fabio Rabelo; Waern, Ida; Rönnberg, Elin; Åbrink, Magnus; Lee, David M.; Schlenner, Susan M.; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Turk, Boris; Wernersson, Sara; Pejler, Gunnar

    2011-01-01

    Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin−/− cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis. PMID:21123167

  4. Identification of CTLA2A, DEFB29, WFDC15B, SERPINA1F and MUP19 as Novel Tissue-Specific Secretory Factors in Mouse.

    Directory of Open Access Journals (Sweden)

    Jibin Zhang

    Full Text Available Secretory factors in animals play an important role in communication between different cells, tissues and organs. Especially, the secretory factors with specific expression in one tissue may reflect important functions and unique status of that tissue in an organism. In this study, we identified potential tissue-specific secretory factors in the fat, muscle, heart, lung, kidney and liver in the mouse by analyzing microarray data from NCBI's Gene Expression Omnibus (GEO public repository and searching and predicting their subcellular location in GeneCards and WoLF PSORT, and then confirmed tissue-specific expression of the genes using semi-quantitative PCR reactions. With this approach, we confirmed 11 lung, 7 liver, 2 heart, 1 heart and muscle, 7 kidney and 2 adipose and liver-specific secretory factors. Among these genes, 1 lung-specific gene--CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha, 3 kidney-specific genes--SERPINA1F (serpin peptidase inhibitor, Clade A, member 1F, WFDC15B (WAP four-disulfide core domain 15B and DEFB29 (defensin beta 29 and 1 liver-specific gene--MUP19 (major urinary protein 19 have not been reported as secretory factors. These genes were tagged with hemagglutinin at the 3'end and then transiently transfected to HEK293 cells. Through protein detection in cell lysate and media using Western blotting, we verified secretion of the 5 genes and predicted the potential pathways in which they may participate in the specific tissue through data analysis of GEO profiles. In addition, alternative splicing was detected in transcripts of CTLA2A and SERPINA1F and the corresponding proteins were found not to be secreted in cell culture media. Identification of novel secretory factors through the current study provides a new platform to explore novel secretory factors and a general direction for further study of these genes in the future.

  5. Imaging Polarized Secretory Traffic at the Immune Synapse in Living T Lymphocytes.

    Science.gov (United States)

    Calvo, Víctor; Izquierdo, Manuel

    2018-01-01

    Immune synapse (IS) formation by T lymphocytes constitutes a crucial event involved in antigen-specific, cellular and humoral immune responses. After IS formation by T lymphocytes and antigen-presenting cells, the convergence of secretory vesicles toward the microtubule-organizing center (MTOC) and MTOC polarization to the IS are involved in polarized secretion at the synaptic cleft. This specialized mechanism appears to specifically provide the immune system with a fine strategy to increase the efficiency of crucial secretory effector functions of T lymphocytes, while minimizing non-specific, cytokine-mediated stimulation of bystander cells, target cell killing and activation-induced cell death. The molecular bases involved in the polarized secretory traffic toward the IS in T lymphocytes have been the focus of interest, thus different models and several imaging strategies have been developed to gain insights into the mechanisms governing directional secretory traffic. In this review, we deal with the most widely used, state-of-the-art approaches to address the molecular mechanisms underlying this crucial, immune secretory response.

  6. Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

    Science.gov (United States)

    Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

    1990-09-01

    Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

  7. Co-culture of neural crest stem cells (NCSC and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death.

    Directory of Open Access Journals (Sweden)

    Anongnad Ngamjariyawat

    Full Text Available PURPOSE: Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. PROCEDURES: Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry, nitrite production (Griess reagent, protein localization (immunofluorescence and protein phosphorylation (flow cytometry. RESULTS: We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii NCSC-derived laminin production; (iii decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv decreased beta-TC6 cell phosphorylation of ERK(T202/Y204, FAK(Y397 and FAK(Y576. Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. CONCLUSION: In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell

  8. Beta cell proliferation and growth factors

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis; Svensson, C; Møldrup, Annette

    1999-01-01

    Formation of new beta cells can take place by two pathways: replication of already differentiated beta cells or neogenesis from putative islet stem cells. Under physiological conditions both processes are most pronounced during the fetal and neonatal development of the pancreas. In adulthood little...... increase in the beta cell number seems to occur. In pregnancy, however, a marked hyperplasia of the beta cells is observed both in rodents and man. Increased mitotic activity has been seen both in vivo and in vitro in islets exposed to placental lactogen (PL), prolactin (PRL) and growth hormone (GH...... and activation of the tyrosine kinase JAK2 and the transcription factors STAT1 and 3. The activation of the insulin gene however also requires the distal part of the receptor and activation of calcium uptake and STAT5. In order to identify putative autocrine growth factors or targets for growth factors we have...

  9. A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

    International Nuclear Information System (INIS)

    Adrian, T.E.; Goldenring, J.R.; Oddsdottir, M.; Zdon, M.J.; Zucker, K.A.; Lewis, J.J.; Modlin, I.M.

    1989-01-01

    A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [ 14 C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [ 14 C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [ 14 C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes

  10. The bioactive effects of casein proteins on enteroendocrine cell health, proliferation and incretin hormone secretion.

    Science.gov (United States)

    Gillespie, Anna L; Green, Brian D

    2016-11-15

    Previous studies suggest that casein exerts various anti-diabetic effects. However, it is not known which casein proteins are bioactive, nor their effects on enteroendocrine cells. This study evaluated the effects of intact whole casein, intact individual proteins (alpha, beta and kappa casein) and hydrolysates on an enteroendocrine cell line. High content analysis accurately monitored changes in cell health and intracellular glucagon-like peptide-1 (GLP-1) content. Cheese ripening duration and GLP-1 secretory responses were also considered. Beta casein significantly stimulated enteroendocrine cell proliferation and all caseins were potent GLP-1 secretagogues (except kappa casein). Interestingly the GLP-1 secretory activity was almost always lost or significantly reduced upon hydrolysis with proteolytic enzymes. Only pepsin-derived beta casein hydrolysates had significantly increased potency compared with the intact protein, but this was diminished with prolonged hydrolysis. In conclusion casein proteins are not detrimental to enteroendocrine cells, and alpha and beta casein are particularly beneficial stimulating proliferation and GLP-1 secretion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study

    DEFF Research Database (Denmark)

    Zander, Mette; Madsbad, Sten; Madsen, Jan Lysgaard

    2002-01-01

    subcutaneous infusion of GLP-1 (n=10) or saline (n=10) for 6 weeks. Before (week 0) and at weeks 1 and 6, they underwent beta-cell function tests (hyperglycaemic clamps), 8 h profiles of plasma glucose, insulin, C-peptide, glucagon, and free fatty acids, and appetite and side-effect ratings on 100 mm visual...... analogue scales; at weeks 0 and 6 they also underwent dexascanning, measurement of insulin sensitivity (hyperinsulinaemic euglycaemic clamps), haemoglobin A(1c), and fructosamine. The primary endpoints were haemoglobin A(1c) concentration, 8-h profile of glucose concentration in plasma, and beta......-cell function (defined as the first-phase response to glucose and the maximum insulin secretory capacity of the cell). Analyses were per protocol. FINDINGS: One patient assigned saline was excluded because no veins were accessible. In the remaining nine patients in that group, no significant changes were...

  12. Diabetic beta-cells can achieve self-protection against oxidative stress through an adaptive up-regulation of their antioxidant defenses.

    Directory of Open Access Journals (Sweden)

    Grégory Lacraz

    Full Text Available BACKGROUND: Oxidative stress (OS, through excessive and/or chronic reactive oxygen species (ROS, is a mediator of diabetes-related damages in various tissues including pancreatic beta-cells. Here, we have evaluated islet OS status and beta-cell response to ROS using the GK/Par rat as a model of type 2 diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Localization of OS markers was performed on whole pancreases. Using islets isolated from 7-day-old or 2.5-month-old male GK/Par and Wistar control rats, 1 gene expression was analyzed by qRT-PCR; 2 insulin secretion rate was measured; 3 ROS accumulation and mitochondrial polarization were assessed by fluorescence methods; 4 antioxidant contents were quantified by HPLC. After diabetes onset, OS markers targeted mostly peri-islet vascular and inflammatory areas, and not islet cells. GK/Par islets revealed in fact protected against OS, because they maintained basal ROS accumulation similar or even lower than Wistar islets. Remarkably, GK/Par insulin secretion also exhibited strong resistance to the toxic effect of exogenous H(2O(2 or endogenous ROS exposure. Such adaptation was associated to both high glutathione content and overexpression (mRNA and/or protein levels of a large set of genes encoding antioxidant proteins as well as UCP2. Finally, we showed that such a phenotype was not innate but spontaneously acquired after diabetes onset, as the result of an adaptive response to the diabetic environment. CONCLUSIONS: The GK/Par model illustrates the effectiveness of adaptive response to OS by beta-cells to achieve self-tolerance. It remains to be determined to what extend such islet antioxidant defenses upregulation might contribute to GK/Par beta-cell secretory dysfunction.

  13. Chronology of endocrine differentiation and beta-cell neogenesis.

    Science.gov (United States)

    Miyatsuka, Takeshi

    2016-01-01

    Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

  14. The regulated secretory pathway in CD4(+ T cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse.

    Directory of Open Access Journals (Sweden)

    Clare Jolly

    2011-09-01

    Full Text Available Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1 at the virological synapse (VS is an efficient mode of dissemination between CD4(+ T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+ T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS. Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL and we show that the HIV-1 envelope glycoprotein (Env localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+ T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+ T cells to enhance its dissemination.

  15. Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

    Directory of Open Access Journals (Sweden)

    F D’Amico

    2009-12-01

    Full Text Available The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA, Ulex europaeus agglutinin I (UEA-I, Glycine max agglutinin (SBA, Arachys hypogaea agglutinin (PNA]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory gran- ules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.

  16. Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

    International Nuclear Information System (INIS)

    Brooks, J.C.; Brooks, M.

    1985-01-01

    Permeabilized cells treated with the adenosine triphosphate analog, ( 35 S)adenosine-5'-0-3(3-thiotriphosphate) ((γ- 35 S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35 S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35 S from the cells over a 20 min period. Treatment of permeabilized cells with ATPγS inhibited secretion and 35 S incorporation into the cells. Pretreatment with ATPγS resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35 S from (γ- 35 S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables

  17. Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Abou Hachem, Maher; Næsted, Henrik

    2010-01-01

    biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha...

  18. Proton microprobe analysis of pancreatic. beta. cells

    Energy Technology Data Exchange (ETDEWEB)

    Lindh, U [Uppsala Univ. (Sweden). Gustaf Werner Inst.; Juntti-Berggren, L; Berggren, P O; Hellman, B [Uppsala Univ. (Sweden)

    1985-01-01

    Freeze-dried pancreas sections from obese hyperglycemic mice were subjected to proton bombardment and the elemental contents in the ..beta.. cells and the exocrine part were obtained from the characteristic X-rays emitted. Quantitative data were provided for 18 different elements. The mole ratio between K and Na exceeded 10, implying that neither the sample preparation nor the irradiation had induced significant diffuse changes. With the demonstration of this high K/Na ratio it seems likely that also the ..beta.. cells are equipped with an efficient Na/sup +//K/sup +/ pump. The ..beta.. cells contained about 70 mmoles Cl per litre cell water. Observed amounts of Ca and Mg were equivalent to those previously recorded by electrothermal atomic absorption spectroscopy. The significant role of Zn for the storage of insulin was emphasized by the demonstration of 3 times as much of this element in the ..beta.. cells as compared with the exocrine pancreas. In addition, the sensitivity of the proton microprobe enabled measurements of various trace elements such as Rb, Cr, Cu, Al and Pb not previously demonstrated in the pancreatic ..beta.. cells.

  19. Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers

    Directory of Open Access Journals (Sweden)

    Kabeerdoss Jayakanthan

    2011-12-01

    Full Text Available Abstract Background Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet. Findings 26 women aged 18-21 (median 19 years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (109 in 200 ml for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184 and returned to normal after cessation of probiotic yoghurt intake. Conclusions Bifidobacterium lactis Bb12® increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.

  20. The islet beta-cell: fuel responsive and vulnerable.

    Science.gov (United States)

    Nolan, Christopher J; Prentki, Marc

    2008-10-01

    The pancreatic beta-cell senses blood nutrient levels and is modulated by neurohormonal signals so that it secretes insulin according to the need of the organism. Nutrient sensing involves marked metabolic activation, resulting in the production of coupling signals that promote insulin biosynthesis and secretion. The beta-cell's high capacity for nutrient sensing, however, necessitates reduced protection to nutrient toxicity. This potentially explains why in susceptible individuals, chronic fuel surfeit results in beta-cell failure and type 2 diabetes. Here we discuss recent insights into first, the biochemical basis of beta-cell signaling in response to glucose, amino acids and fatty acids, and second, beta-cell nutrient detoxification. We emphasize the emerging role of glycerolipid/fatty acid cycling in these processes.

  1. MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana.

    Science.gov (United States)

    Arsovski, Andrej A; Villota, Maria M; Rowland, Owen; Subramaniam, Rajagopal; Western, Tamara L

    2009-01-01

    Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.

  2. A Plant-Based Dietary Intervention Improves Beta-Cell Function and Insulin Resistance in Overweight Adults: A 16-Week Randomized Clinical Trial.

    Science.gov (United States)

    Kahleova, Hana; Tura, Andrea; Hill, Martin; Holubkov, Richard; Barnard, Neal D

    2018-02-09

    The aim of this study was to test the effect of a plant-based dietary intervention on beta-cell function in overweight adults with no history of diabetes. Participants ( n = 75) were randomized to follow a low-fat plant-based diet ( n = 38) or to make no diet changes ( n = 37) for 16 weeks. At baseline and 16 weeks, beta-cell function was quantified with a mathematical model. Using a standard meal test, insulin secretory rate was calculated by C-peptide deconvolution. The Homeostasis Model Assessment (HOMA-IR) index was used to assess insulin resistance while fasting. A marked increase in meal-stimulated insulin secretion was observed in the intervention group compared with controls (interaction between group and time, Gxt, p effect -1.0 (95% CI, -1.2 to -0.8); Gxt, p = 0.004). Changes in HOMA-IR correlated positively with changes in body mass index (BMI) and visceral fat volume ( r = 0.34; p = 0.009 and r = 0.42; p = 0.001, respectively). The latter remained significant after adjustment for changes in BMI ( r = 0.41; p = 0.002). Changes in glucose-induced insulin secretion correlated negatively with BMI changes ( r = -0.25; p = 0.04), but not with changes in visceral fat. Beta-cell function and insulin sensitivity were significantly improved through a low-fat plant-based diet in overweight adults.

  3. Effect of Semen on Vaginal Fluid Cytokines and Secretory Leukocyte Protease Inhibitor

    Directory of Open Access Journals (Sweden)

    Kathy J. Agnew

    2008-01-01

    Methods: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test. Results: of 138 subjects, 28 (20% had acid phosphatase detected; of these, only 19 (68% reported recent intercourse and 3 (11% had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase. Conclusions: proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal SLPI concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations.

  4. Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation

    International Nuclear Information System (INIS)

    Willner, Jochen; Vordermark, Dirk; Schmidt, Michael; Gassel, Andreamaria; Flentje, Michael; Wirtz, Hubert

    2003-01-01

    Purpose: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo. Methods and Materials: Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using 3 H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index. Results: During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G 2 /M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls. Conclusion: In contrast to literature data, we observed no direct

  5. Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line

    DEFF Research Database (Denmark)

    Hou, X; Dietrich, J; Kuhlmann, J

    1994-01-01

    not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association......The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still...... with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti...

  6. Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

    International Nuclear Information System (INIS)

    Antoine, M.H.; Hermann, M.; Herchuelz, A.; Lebrun, P.

    1991-01-01

    Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the K+ channel openers. The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry

  7. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    International Nuclear Information System (INIS)

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-01-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates

  8. Foodborne cereulide causes beta-cell dysfunction and apoptosis.

    Directory of Open Access Journals (Sweden)

    Roman Vangoitsenhoven

    Full Text Available To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function.Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6 and rat (INS-1E beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1. Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS. Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests.24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05. Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05 and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein. Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05 and reactive oxygen species increased by more than twofold (P<0.05 following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively.Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell.

  9. Sperm-storage defects and live birth in Drosophila females lacking spermathecal secretory cells.

    Directory of Open Access Journals (Sweden)

    Sandra L Schnakenberg

    2011-11-01

    Full Text Available Male Drosophila flies secrete seminal-fluid proteins that mediate proper sperm storage and fertilization, and that induce changes in female behavior. Females also produce reproductive-tract secretions, yet their contributions to postmating physiology are poorly understood. Large secretory cells line the female's spermathecae, a pair of sperm-storage organs. We identified the regulatory regions controlling transcription of two genes exclusively expressed in these spermathecal secretory cells (SSC: Spermathecal endopeptidase 1 (Send1, which is expressed in both unmated and mated females, and Spermathecal endopeptidase 2 (Send2, which is induced by mating. We used these regulatory sequences to perform precise genetic ablations of the SSC at distinct time points relative to mating. We show that the SSC are required for recruiting sperm to the spermathecae, but not for retaining sperm there. The SSC also act at a distance in the reproductive tract, in that their ablation: (1 reduces sperm motility in the female's other sperm-storage organ, the seminal receptacle; and (2 causes ovoviviparity--the retention and internal development of fertilized eggs. These results establish the reproductive functions of the SSC, shed light on the evolution of live birth, and open new avenues for studying and manipulating female fertility in insects.

  10. The transcriptional corepressor MTGR1 regulates intestinal secretory lineage allocation.

    Science.gov (United States)

    Parang, Bobak; Rosenblatt, Daniel; Williams, Amanda D; Washington, Mary K; Revetta, Frank; Short, Sarah P; Reddy, Vishruth K; Hunt, Aubrey; Shroyer, Noah F; Engel, Michael E; Hiebert, Scott W; Williams, Christopher S

    2015-03-01

    Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation. © FASEB.

  11. Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

    Science.gov (United States)

    Palozza, Paola; Bellovino, Diana; Simone, Rossella; Boninsegna, Alma; Cellini, Francesco; Monastra, Giovanni; Gaetani, Sancia

    2009-07-01

    Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

  12. A Plant-Based Dietary Intervention Improves Beta-Cell Function and Insulin Resistance in Overweight Adults: A 16-Week Randomized Clinical Trial

    Directory of Open Access Journals (Sweden)

    Hana Kahleova

    2018-02-01

    Full Text Available The aim of this study was to test the effect of a plant-based dietary intervention on beta-cell function in overweight adults with no history of diabetes. Participants (n = 75 were randomized to follow a low-fat plant-based diet (n = 38 or to make no diet changes (n = 37 for 16 weeks. At baseline and 16 weeks, beta-cell function was quantified with a mathematical model. Using a standard meal test, insulin secretory rate was calculated by C-peptide deconvolution. The Homeostasis Model Assessment (HOMA-IR index was used to assess insulin resistance while fasting. A marked increase in meal-stimulated insulin secretion was observed in the intervention group compared with controls (interaction between group and time, Gxt, p < 0.001. HOMA-IR index fell significantly (p < 0.001 in the intervention group (treatment effect −1.0 (95% CI, −1.2 to −0.8; Gxt, p = 0.004. Changes in HOMA-IR correlated positively with changes in body mass index (BMI and visceral fat volume (r = 0.34; p = 0.009 and r = 0.42; p = 0.001, respectively. The latter remained significant after adjustment for changes in BMI (r = 0.41; p = 0.002. Changes in glucose-induced insulin secretion correlated negatively with BMI changes (r = −0.25; p = 0.04, but not with changes in visceral fat. Beta-cell function and insulin sensitivity were significantly improved through a low-fat plant-based diet in overweight adults.

  13. Pancreatic Beta-Cell Purification by Altering FAD and NAD(PH Metabolism

    Directory of Open Access Journals (Sweden)

    P. de Vos

    2008-07-01

    Full Text Available Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD and nicotinamide-adenine dinucleotide phosphate (NAD(PH autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(PH fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(PH contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(PH fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

  14. TGF-beta and 'adaptive' Foxp3(+) regulatory T cells.

    Science.gov (United States)

    Chen, Wanjun; Konkel, Joanne E

    2010-02-01

    In naïve T cells transforming growth factor-beta (TGF-beta) induces Foxp3, a transcription factor essential for programming and developing T regulatory cells (Treg cells). This finding reveals a physiological factor which can turn on the Foxp3 gene and establishes an experimental approach to induce antigen-specific Treg cells as a potential therapy for human diseases. While this role for TGF-beta is well confirmed, several critical questions remain largely unanswered and await further investigation. In this regard, it is imperative to understand the molecular pathways by which TGF-beta signaling initiates and regulates Foxp3 expression. It is also important to elucidate which factors and/or cytokines influence the TGF-beta-mediated conversion of naïve T cells and how to create an immunologically regulatory milieu to facilitate Treg cell generation in vivo. In this short article, we will highlight the key findings and recent progress in the field, discuss the molecular mechanisms underlying the TGF-beta-mediated induction of Foxp3, and attempt to outline the challenges ahead.

  15. Progress in molecular nuclear medicine imaging of pancreatic beta cells

    International Nuclear Information System (INIS)

    Wu Haifei; Yin Hongyan; Liu Shuai; Zhang Yifan

    2010-01-01

    Diabetes mellitus is a common and frequently occurring disease which seriously threaten the health of human beings. Type 1 and type 2 diabetes respectively results from being destroyed and insufficient beta-cell mass. The associated symptoms appear until 50%-60% decrease of beta-cell mass. Because pancreas is deeply located in the body, with few beta-cell mass, the current methods of clinical diagnosis are invasive and late. So diagnosis of metabolism disease of beta-cell early non-invasively becomes more and more popular, imaging diagnosis of diabetes mellitus becomes the focus of researches, but how to estimate the mass of beta-cell still an important subject in imaging technology. (authors)

  16. Secretory diarrhea.

    Science.gov (United States)

    Schiller, L R

    1999-10-01

    Diarrhea, defined as loose stools, occurs when the intestine does not complete absorption of electrolytes and water from luminal contents. This can happen when a nonabsorbable, osmotically active substance is ingested ("osmotic diarrhea") or when electrolyte absorption is impaired ("secretory diarrhea"). Most cases of acute and chronic diarrhea are due to the latter mechanism. Secretory diarrhea can result from bacterial toxins, reduced absorptive surface area caused by disease or resection, luminal secretagogues (such as bile acids or laxatives), circulating secretagogues (such as various hormones, drugs, and poisons), and medical problems that compromise regulation of intestinal function. Evaluation of patients with secretory diarrhea must be tailored to find the likely causes of this problem. Specific and nonspecific treatment can be valuable.

  17. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Jee [Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyeonggi-do (Korea, Republic of); Chun, Ji Na; Jung, Sun-Ah [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of); Cho, Jin Won [Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lee, Joon H., E-mail: joonhlee@konyang.ac.kr [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  18. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    Science.gov (United States)

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  19. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures

    DEFF Research Database (Denmark)

    Tanner, V A; Ploug, Thorkil; Tao-Cheng, J H

    1996-01-01

    substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types...... of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV...

  20. Enhancing pancreatic Beta-cell regeneration in vivo with pioglitazone and alogliptin.

    Directory of Open Access Journals (Sweden)

    Hao Yin

    Full Text Available Pancreatic beta-cells retain limited ability to regenerate and proliferate after various physiologic triggers. Identifying therapies that are able to enhance beta-cell regeneration may therefore be useful for the treatment of both type 1 and type 2 diabetes.In this study we investigated endogenous and transplanted beta-cell regeneration by serially quantifying changes in bioluminescence from beta-cells from transgenic mice expressing firefly luciferase under the control of the mouse insulin I promoter. We tested the ability of pioglitazone and alogliptin, two drugs developed for the treatment of type 2 diabetes, to enhance beta-cell regeneration, and also defined the effect of the immunosuppression with rapamycin and tacrolimus on transplanted islet beta mass.Pioglitazone is a stimulator of nuclear receptor peroxisome proliferator-activated receptor gamma while alogliptin is a selective dipeptidyl peptidase IV inhibitor. Pioglitazone alone, or in combination with alogliptin, enhanced endogenous beta-cell regeneration in streptozotocin-treated mice, while alogliptin alone had modest effects. In a model of syngeneic islet transplantation, immunosuppression with rapamycin and tacrolimus induced an early loss of beta-cell mass, while treatment with insulin implants to maintain normoglycemia and pioglitazone plus alogliptin was able to partially promote beta-cell mass recovery.These data highlight the utility of bioluminescence for serially quantifying functional beta-cell mass in living mice. They also demonstrate the ability of pioglitazone, used either alone or in combination with alogliptin, to enhance regeneration of endogenous islet beta-cells as well as transplanted islets into recipients treated with rapamycin and tacrolimus.

  1. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  2. Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

    Science.gov (United States)

    Parmar, Mehtab S; Mishra, Smruti Ranjan; Somal, Anjali; Pandey, Sriti; Kumar, G Sai; Sarkar, Mihir; Chandra, Vikash; Sharma, G Taru

    2017-05-01

    The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Assessment of affinities of beta-CIT, beta-CIT-FE, and beta-CIT-FP for monoamine transporters permanently expressed in cell lines

    International Nuclear Information System (INIS)

    Okada, Tomoya; Fujita, Masahiro; Shimada, Shoichi; Sato, Kohji; Schloss, Patrick; Watanabe, Yoshiyuki; Itoh, Yasushi; Tohyama, Masaya; Nishimura, Tsunehiko

    1998-01-01

    We investigated the effects of three cocaine analogs, beta-CIT (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane), beta-CIT-FE (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(2-fluoroethyl)-nortropane), and beta-CIT-FP (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane), on the uptake of [ 3 H]dopamine(DA), serotonin(5-HT), and 1-norepinephrine (NE) using cell lines permanently expressing DA, 5-HT, and NE transporters, respectively, to determine their affinities for these three transporters. We generated cell lines stably expressing DA, 5-HT, and NE transporters, respectively, by the Chen-Okayama method, and then tested the abilities of (-)cocaine, beta-CIT, beta-CIT-FE, beta-CIT-FP, and clomipramine to inhibit the uptake of [ 3 H]DA, 5-HT, and 1-NE. Ki values of beta-CIT, beta-CIT-FE, and beta-CIT-FP for [ 3 H]DA, 5-HT, 1-NE uptake were 6, 29, and 33 nM, 91, 133, and 130 nM, and 28, 113 and 70 nM, respectively, whereas those of cocaine and clomipramine were 316, 581, and 176 nM and > 10,000, 437, and 851 nM, respectively. Beta-CIT, beta-CIT-FE, and beta-CIT-FP were shown to be potent DA, 5-HT, and NE uptake inhibitors. Beta-CIT and beta-CIT-FP were highly potent and selective dopamine uptake inhibitors, and therefore might be useful for imaging of DA transporter with single photon emission computed tomography (SPECT) or positron emission tomography (PET)

  4. [Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A].

    Science.gov (United States)

    German, G P; Chernokhvostova, E V; Gol'derman, S Ia

    1975-10-01

    A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.

  5. Mechanisms of pancreatic beta-cell growth and regeneration

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1989-01-01

    Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found...... of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells....... that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements...

  6. Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

    Science.gov (United States)

    Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

    2017-01-01

    Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  7. cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells

    DEFF Research Database (Denmark)

    Juhl, Kirstine; Høy, Marianne; Olsen, Hervør L

    2003-01-01

    Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha...... from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination...... of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited...

  8. Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

    1988-10-25

    DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

  9. Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

    Science.gov (United States)

    Wang, A C; Fu, L

    2001-03-01

    To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

  10. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    Science.gov (United States)

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  11. Characterization of a Commercial Silicon Beta Cell

    International Nuclear Information System (INIS)

    Foxe, Michael P.; Hayes, James C.; Mayer, Michael F.; McIntyre, Justin I.; Sivels, Ciara B.; Suarez, Rey

    2016-01-01

    Silicon detectors are of interest for the verification of the Comprehensive Nuclear-Test-Ban Treaty (CTBT) due to their enhanced energy resolution compared to plastic scintillators beta cells. Previous work developing a figure-of-merit (FOM) for comparison of beta cells suggests that the minimum detectable activity (MDA) could be reduced by a factor of two to three with the use of silicon detectors. Silicon beta cells have been developed by CEA (France) and Lares Ltd. (Russia), with the PIPSBox developed by CEA being commercially available from Canberra for approximately $35k, but there is still uncertainty about the reproducibility of the capabilities in the field. PNNL is developing a high-resolution beta-gamma detector system in the shallow underground laboratory, which will utilize and characterize the operation of the PIPSBox detector. Throughout this report, we examine the capabilities of the PIPSBox as developed by CEA. The lessons learned through the testing and use of the PIPSBox will allow PNNL to strategically develop a silicon detector optimized to better suit the communities needs in the future.

  12. Non-Cell Autonomous Effects of the Senescence-Associated Secretory Phenotype in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Tareq Saleh

    2018-05-01

    Full Text Available In addition to promoting various forms of cell death, most conventional anti-tumor therapies also promote senescence. There is now extensive evidence that therapy-induced senescence (TIS might be transient, raising the concern that TIS could represent an undesirable outcome of therapy by providing a mechanism for tumor dormancy and eventual disease recurrence. The senescence-associated secretory phenotype (SASP is a hallmark of TIS and may contribute to aberrant effects of cancer therapy. Here, we propose that the SASP may also serve as a major driver of escape from senescence and the re-emergence of proliferating tumor cells, wherein factors secreted from the senescent cells contribute to the restoration of tumor growth in a non-cell autonomous fashion. Accordingly, anti-SASP therapies might serve to mitigate the deleterious outcomes of TIS. In addition to providing an overview of the putative actions of the SASP, we discuss recent efforts to identify and eliminate senescent tumor cells.

  13. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

    2016-05-15

    Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression

  14. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

    Science.gov (United States)

    Morosky, Stefanie; Lennemann, Nicholas J.

    2016-01-01

    ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of

  15. Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

    Science.gov (United States)

    Dang, Donna; Prasad, Hari; Rao, Rajini

    2017-11-01

    Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

  16. IFN-beta inhibits T cell activation capacity of central nervous system APCs

    DEFF Research Database (Denmark)

    Teige, Ingrid; Liu, Yawei; Issazadeh-Navikas, Shohreh

    2006-01-01

    We have previously investigated the physiological effects of IFN-beta on chronic CNS inflammation and shown that IFN-beta(-/-) mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-beta(+/-) littermates. This result was shown to be associated with a higher activation...... state of the glial cells and a higher T cell cytokine production in the CNS. Because this state suggested a down-regulatory effect of IFN-beta on CNS-specific APCs, these results were investigated further. We report that IFN-beta pretreatment of astrocytes and microglia (glial cells) indeed down......-modulate their capacity to activate autoreactive Th1 cells. First, we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion while maintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-beta before...

  17. Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

    2009-01-01

    Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study...... in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep...... into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP...

  18. Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain

    DEFF Research Database (Denmark)

    Johansen, Torben; Knudsen, T; Bertelsen, Niels Haldor

    1990-01-01

    The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity. The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition...... of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following...

  19. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    DEFF Research Database (Denmark)

    Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel

    2012-01-01

    glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal.......5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...

  20. The physiology of rodent beta-cells in pancreas slices.

    Science.gov (United States)

    Rupnik, M

    2009-01-01

    Beta-cells in pancreatic islets form complex syncytia. Sufficient cell-to-cell electrical coupling seems to ensure coordinated depolarization pattern and insulin release that can be further modulated by rich innervation. The complex structure and coordinated action develop after birth during fast proliferation of the endocrine tissue. These emergent properties can be lost due to various reasons later in life and can lead to glucose intolerance and diabetes mellitus. Pancreas slice is a novel method of choice to study the physiology of beta-cells still embedded in their normal cellulo-social context. I present major advantages, list drawbacks and provide an overview on recent advances in our understanding of the physiology of beta-cells using the pancreas slice approach.

  1. On the coherent behavior of pancreatic beta cell clusters

    Energy Technology Data Exchange (ETDEWEB)

    Loppini, Alessandro, E-mail: a.loppini@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Capolupo, Antonio, E-mail: capolupo@sa.infn.it [Physics Department, University of Salerno, Fisciano, 84084 (Italy); Cherubini, Christian, E-mail: c.cherubini@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); International Center for Relativistic Astrophysics, University Campus Bio-Medico, Via A. del Portillo 21, I-00128, Rome (Italy); Gizzi, Alessio, E-mail: a.gizzi@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Bertolaso, Marta, E-mail: m.bertolaso@unicampus.it [Faculty of Engineering and Institute of Philosophy of Scientific and Technological Practice, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Filippi, Simonetta, E-mail: s.filippi@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); International Center for Relativistic Astrophysics, University Campus Bio-Medico, Via A. del Portillo 21, I-00128, Rome (Italy); Vitiello, Giuseppe, E-mail: vitiello@sa.infn.it [Physics Department, University of Salerno, Fisciano, 84084 (Italy)

    2014-09-12

    Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory. - Highlights: • Beta cells in pancreas are coupled oscillators able to synchronize their activity. • We analyze scale free self-similarity features for beta cells. • We adopt the algebraic approach of coherent state formalism. • We show that coherent molecular domains arise from functional conditions.

  2. Role of MicroRNAs in Islet Beta-Cell Compensation and Failure during Diabetes

    Directory of Open Access Journals (Sweden)

    Valérie Plaisance

    2014-01-01

    Full Text Available Pancreatic beta-cell function and mass are markedly adaptive to compensate for the changes in insulin requirement observed during several situations such as pregnancy, obesity, glucocorticoids excess, or administration. This requires a beta-cell compensation which is achieved through a gain of beta-cell mass and function. Elucidating the physiological mechanisms that promote functional beta-cell mass expansion and that protect cells against death, is a key therapeutic target for diabetes. In this respect, several recent studies have emphasized the instrumental role of microRNAs in the control of beta-cell function. MicroRNAs are negative regulators of gene expression, and are pivotal for the control of beta-cell proliferation, function, and survival. On the one hand, changes in specific microRNA levels have been associated with beta-cell compensation and are triggered by hormones or bioactive peptides that promote beta-cell survival and function. Conversely, modifications in the expression of other specific microRNAs contribute to beta-cell dysfunction and death elicited by diabetogenic factors including, cytokines, chronic hyperlipidemia, hyperglycemia, and oxidized LDL. This review underlines the importance of targeting the microRNA network for future innovative therapies aiming at preventing the beta-cell decline in diabetes.

  3. Distribution and structure of internal secretory reservoirs on the vegetative organs of Inula helenium L. (Asteraceae

    Directory of Open Access Journals (Sweden)

    Aneta Sulborska

    2012-12-01

    Full Text Available The aim of the study was to investigate the structure and topography of endogenous secretory tissues of Inula helenium L. By using light and electron microscopy, morphological and anatomical observations of stems, leaves and rhizomes were made. It was shown that in the stems secretory cavities were situated in the vicinity of phloem and xylem bundles. The number of the reservoirs reached its maximum value (34 at shoot flowerig termination, whereas the cavities with the largest diameter were observed at full flowering stage (44.6 µm. In the leaf petioles and midribs, the reservoirs also accompanied the vascular bundles, and their number and size increased along with the growth of the assimilation organs. Observations of the cross sections of the rhizomes revealed the presence of several rings of secretory reservoirs. The measurements of the cavities showed that as a rule the reservoirs with a larger dimension were located in the phelloderm, whereas the smallest ones in the xylem area. The secretory cavities located in the stems and leaves developed by schizogenesis, whereas the rhizome reservoirs were probably formed schizolisygenously. The cells lining the reservoirs formed a one - four-layered epithelium. Observed in TEM, the secretory cells of the mature cavities located in the rhizomes were characterised by the presence of a large central vacuole, whereas the protoplast was largely degraded. Fibrous elements of osmophilic secretion and numerous different coloured vesicles could be distinguished in it. The cell walls formed, from the side of the reservoir lumen, ingrowths into the interior of the epithelial cells. Between the cell wall and the plasmalemma of the glandular cells, a brighter periplasmatic zone with secretory vesicles was observed.

  4. Proliferation of sorted human and rat beta cells

    DEFF Research Database (Denmark)

    Parnaud, G; Bosco, D; Berney, T

    2008-01-01

    The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors.......The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors....

  5. RFP tags for labeling secretory pathway proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Liyang; Zhao, Yanhua [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Xi; Peng, Jianxin [College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xu, Pingyong, E-mail: pyxu@ibp.ac.cn [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Huan, Shuangyan, E-mail: shuangyanhuan@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Mingshu, E-mail: mingshu1984@gmail.com [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  6. Secretory structures of Ipomoea asarifolia: anatomy and histochemistry

    Directory of Open Access Journals (Sweden)

    Fabiano M. Martins

    2012-02-01

    Full Text Available Ipomoea asarifolia (Desr. Roem. & Schult., Convolvulaceae, is a weed that infests agricultural areas and is toxic to cattle. In spite of its toxicity, the leaves of this plant are used in traditional remedies in the state of Bahia, Brazil. The present work describes the leaf anatomy of I. asarifolia and characterizes the exudates of its secretory structures. The leaves have a unistratified epidermis composed of ordinary cells with straight to slightly sinuous anticlinal walls and thin cuticles. Paracytic stomata are found on both surfaces of the leaves at the same level as the ordinary epidermal cells. Trichomes producing polysaccharide secretions occur on the petiole and leaf blade and are considered colleters. The mesophyll is dorsiventral and the vascular bundle of the central vein is bicollateral. Two opposed nectaries occur on the petiole near the leaf blade. Each nectary is composed of a small canal with internal ramifications and numerous secretory trichomes. The laticiferous glands are articulated, not anastomosed, and are composed of large diameter cells with thin cell walls. The secretions of the laticiferous glands are lipidic.

  7. Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin.

    Directory of Open Access Journals (Sweden)

    Imane Song

    Full Text Available One week of treatment with EGF and gastrin (EGF/G was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of

  8. Acquired TGF beta 1 sensitivity and TGF beta 1 expression in cell lines established from a single small cell lung cancer patient during clinical progression

    DEFF Research Database (Denmark)

    Nørgaard, P; Damstrup, L; Rygaard, K

    1996-01-01

    Three small cell lung cancer cell lines established from a single patient during longitudinal follow-up were examined for in vitro expression of TGF beta and TGF beta receptors, i.e. the components of an autocrine loop. GLC 14 was established prior to treatment, GLC 16 on relapse after chemotherapy...... was found in GLC 16 and GLC 19. These cell lines were also growth inhibited by exogenously administrated TGF beta 1. TGF beta 1 mRNA and protein in its latent form was only expressed in the radiotherapy-resistant cell line, GLC 19. The results indicate that disease progression in this patient was paralleled...... II receptor gene, as examined by Southern blotting. Also, the type I receptor could not be detected by ligand binding assay in this cell line, despite expression of mRNA for this receptor. This agrees with previous findings that type I receptor cannot bind TGF beta 1 without co-expression of the type...

  9. Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

    DEFF Research Database (Denmark)

    Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

    2003-01-01

    decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

  10. The potential role of SOCS-3 in the interleukin-1beta-induced desensitization of insulin signaling in pancreatic beta-cells

    DEFF Research Database (Denmark)

    Emanuelli, Brice; Glondu, Murielle; Filloux, Chantal

    2004-01-01

    insulin signaling is required for the optimal beta-cell function, we assessed the effect of IL-1beta on the insulin pathway in a rat pancreatic beta-cell line. We show that IL-1beta decreases insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS...

  11. TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

    Science.gov (United States)

    Farmer, John T; Weigent, Douglas A

    2006-03-01

    Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

  12. Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells

    DEFF Research Database (Denmark)

    Ortis, Fernanda; Naamane, Najib; Flamez, Daisy

    2010-01-01

    by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h......-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression...... of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine...

  13. Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

    Science.gov (United States)

    Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

    2017-07-17

    Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

  14. Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

    Science.gov (United States)

    Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

    2012-01-01

    Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

  15. The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

    International Nuclear Information System (INIS)

    Woynillowicz, Amanda K.; Raha, Sandeep; Nicholson, Catherine J.; Holloway, Alison C.

    2012-01-01

    The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.

  16. The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

    Energy Technology Data Exchange (ETDEWEB)

    Woynillowicz, Amanda K. [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Raha, Sandeep [Department of Pediatrics, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Nicholson, Catherine J. [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Holloway, Alison C., E-mail: hollow@mcmaster.ca [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada)

    2012-11-15

    The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.

  17. Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

    Science.gov (United States)

    Hur, Yong Suk; Yoo, Seung Hyun

    2015-01-01

    The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

  18. Beta-Cell Replacement: Pancreas and Islet Cell Transplantation.

    Science.gov (United States)

    Niclauss, Nadja; Meier, Raphael; Bédat, Benoît; Berishvili, Ekaterine; Berney, Thierry

    2016-01-01

    Pancreas and islet transplantation are 2 types of beta-cell replacement therapies for type 1 diabetes mellitus. Since 1966, when pancreas transplantation was first performed, it has evolved to become a highly efficient procedure with high success rates, thanks to advances in surgical technique and immunosuppression. Pancreas transplantation is mostly performed as simultaneous pancreas-kidney transplantation in patients with end-stage nephropathy secondary to diabetes. In spite of its efficiency, pancreas transplantation is still a major surgical procedure burdened by high morbidity, which called for the development of less invasive and hazardous ways of replacing beta-cell function in the past. Islet transplantation was developed in the 1970s as a minimally invasive procedure with initially poor outcomes. However, since the report of the 'Edmonton protocol' in 2000, the functional results of islet transplantation have substantially and constantly improved and are about to match those of whole pancreas transplantation. Islet transplantation is primarily performed alone in nonuremic patients with severe hypoglycemia. Both pancreas transplantation and islet transplantation are able to abolish hypoglycemia and to prevent or slow down the development of secondary complications of diabetes. Pancreas transplantation and islet transplantation should be seen as two complementary, rather than competing, therapeutic approaches for beta-cell replacement that are able to optimize organ donor use and patient care. © 2016 S. Karger AG, Basel.

  19. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan Trige

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quies...

  20. Giant renin secretory granules in beige mouse renal afferent arterioles

    DEFF Research Database (Denmark)

    Jensen, B L; Rasch, Ruth; Nyengaard, Jens Randel

    1997-01-01

    The mutant beige mouse (C57BL/6 bg) has a disease characterised by abnormally enlarged cytoplasmic granules in a variety of cells. With the purpose of establishing a suitable cellular model for studying renin secretion, the present study was undertaken to compare renin granule morphology in beige...... (average granular volume 0.681 microm3), whereas 1-2 large granules were present per cell in beige mice. The volume of afferent arteriole that contained secretory granules was lower in the beige mice. We conclude that the beige mouse synthesizes, stores and releases active renin. Renin secretory granules...... in beige mice are grossly enlarged with 1-2 granules per juxtaglomerular cell. Compared with control mice, a similar amount of total renin granule volume per afferent arteriole is contained in a smaller part of beige mouse afferent arteriole. Granular cells from beige mice could therefore be a valuable...

  1. Do post-translational beta cell protein modifications trigger type 1 diabetes?

    DEFF Research Database (Denmark)

    Størling, Joachim; Overgaard, Anne Julie; Brorsson, Caroline Anna

    2013-01-01

    beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading...... diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from...... the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1...

  2. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  3. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

  4. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Birgitte Holst

    Full Text Available Secretory vesicles in endocrine cells store hormones such as growth hormone (GH and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs domain protein PICK1 (protein interacting with C kinase 1 as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of

  5. Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.; Ari, Krakowski; Luo, Kunxin; Chen, David J.; Li, Song

    2004-08-08

    Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

  6. Airway Secretory microRNAome Changes during Rhinovirus Infection in Early Childhood.

    Directory of Open Access Journals (Sweden)

    Maria J Gutierrez

    Full Text Available Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome and examine the changes during rhinovirus (RV infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood.Nasal airway secretions were obtained from children (≤3 yrs. old during PCR-confirmed RV infections (n = 10 and age-matched controls (n = 10. Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC differentiated at air-liquid interface (ALI. Bioinformatics tools were used to determine the unified (nasal and bronchial signature airway secretory miRNAome and changes during RV infection in children.Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV.Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway

  7. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

  8. Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages.

    Science.gov (United States)

    Weiss, Carola; Rubach, Malte; Lang, Roman; Seebach, Elisabeth; Blumberg, Simone; Frank, Oliver; Hofmann, Thomas; Somoza, Veronika

    2010-02-10

    As the consumption of coffee beverages sometimes is reported to cause gastric irritation, for which an increased stomach acid secretion is one of the promoting factors, different processing technologies such as steam-treatment have been developed to reduce putative stomach irritating compounds. There is evidence-based data neither on the effect of detailed processing variations nor on individual coffee components affecting the proton secretory activity (PSA). This work aimed at developing a screening model suitable for investigating the effects of commercial coffee beverages and components thereof on human parietal cells. Human gastric cancer cells (HGT-1) were treated with reconstituted freeze-dried coffee beverages prepared from customary coffee products such as regular coffee (RC, n = 4), mild bean coffee (MBC, n = 5), stomach friendly coffee (SFC, n = 4), and SFC decaffeinated (SFCD, n = 3). PSA was analyzed by flow cytometry using the pH-sensitive dye SNARF-AM. Treatment of the cells with MBC did not result in a PSA different from RC treatment (p stomach irritating compounds revealed significantly reduced contents of (beta)N-alkanoyl-5-hydroxytryptamides, caffeine, N-methylpyridinium, and catechol in SFCD compared to RC. However, none of these compounds seem to act as the sole key bioactive reducing the PSA of SFCD, since their contents in MBC and SFC samples were not different from those in RC samples, although the PSA of these beverages was significantly lower than that of reconstituted freeze-dried RC beverage.

  9. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  10. Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

    Science.gov (United States)

    Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

    2009-04-15

    Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

  11. Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

    Science.gov (United States)

    Dolz, Manuel; Bailbé, Danielle; Giroix, Marie-Hélène; Calderari, Sophie; Gangnerau, Marie-Noelle; Serradas, Patricia; Rickenbach, Katharina; Irminger, Jean-Claude; Portha, Bernard

    2005-11-01

    Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness.

  12. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

    Energy Technology Data Exchange (ETDEWEB)

    Kover, Karen, E-mail: kkover@cmh.edu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Tasch, James; Hager, Melissa [Kansas City University Medical Biosciences, Kansas City, MO (United States); Clements, Mark; Moore, Wayne V. [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States)

    2015-06-19

    Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

  13. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

    International Nuclear Information System (INIS)

    Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

    2015-01-01

    Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H 2 O 2 assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H 2 O 2 levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP

  14. Ins1 Cre knock-in mice for beta cell-specific gene recombination

    OpenAIRE

    Thorens Bernard; Tarussio David; Maestro Miguel Angel; Maestro Miguel Angel; Rovira Meritxell; Rovira Meritxell; Heikkilä Eija; Ferrer Jorge; Ferrer Jorge; Ferrer Jorge

    2013-01-01

    Aims/hypothesis Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cel...

  15. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-01-01

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

  16. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-12-11

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    International Nuclear Information System (INIS)

    Plopper, C.G.; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-01-01

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O 3 ). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined

  18. Influence of continuous light and darkness on the secretory ...

    Indian Academy of Sciences (India)

    Unknown

    rent phases of the secretory process were demonstrated. (Srivastava 1999). ..... in the pineal supportive cells of deep sea fish, Nezumia liolepis (McNulty 1976) and .... factors as light and temperature. Melatonin and ... Brain Res. 52 271–296.

  19. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Cedric Cortijo

    2012-12-01

    Full Text Available Planar cell polarity (PCP refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

  20. T Cell-Mediated Beta Cell Destruction: Autoimmunity and Alloimmunity in the Context of Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Adam L. Burrack

    2017-12-01

    Full Text Available Type 1 diabetes (T1D results from destruction of pancreatic beta cells by T cells of the immune system. Despite improvements in insulin analogs and continuous blood glucose level monitoring, there is no cure for T1D, and some individuals develop life-threatening complications. Pancreas and islet transplantation have been attractive therapeutic approaches; however, transplants containing insulin-producing cells are vulnerable to both recurrent autoimmunity and conventional allograft rejection. Current immune suppression treatments subdue the immune system, but not without complications. Ideally a successful approach would target only the destructive immune cells and leave the remaining immune system intact to fight foreign pathogens. This review discusses the autoimmune diabetes disease process, diabetic complications that warrant a transplant, and alloimmunity. First, we describe the current understanding of autoimmune destruction of beta cells including the roles of CD4 and CD8 T cells and several possibilities for antigen-specific tolerance induction. Second, we outline diabetic complications necessitating beta cell replacement. Third, we discuss transplant recognition, potential sources for beta cell replacement, and tolerance-promoting therapies under development. We hypothesize that a better understanding of autoreactive T cell targets during disease pathogenesis and alloimmunity following transplant destruction could enhance attempts to re-establish tolerance to beta cells.

  1. Reduced incretin effect in type 2 diabetes: cause or consequence of the diabetic state?

    DEFF Research Database (Denmark)

    Knop, Filip K; Vilsbøll, Tina; Højberg, Patricia V

    2007-01-01

    We aimed to investigate whether the reduced incretin effect observed in patients with type 2 diabetes is a primary event in the pathogenesis of type 2 diabetes or a consequence of the diabetic state. Eight patients with chronic pancreatitis and secondary diabetes (A1C mean [range] of 6.9% [6...... intravenous glucose infusion, respectively. The incretin effect (100% x [beta-cell secretory response to oral glucose tolerance test - intravenous beta-cell secretory response]/beta-cell secretory response to oral glucose tolerance test) was significantly (P ... with chronic pancreatitis and secondary diabetes (31 +/- 4%) compared with patients with chronic pancreatitis and NGT (68 +/- 3) and healthy subjects (60 +/- 4), respectively. In the type 2 diabetes group, the incretin effect amounted to 36 +/- 6%, significantly (P

  2. Imaging the Beta-cell mass: why and how

    DEFF Research Database (Denmark)

    Saudek, Frantisek; Brogren, Carl-Henrik; Manohar, Srirang

    2008-01-01

    is called dihydrotetrabenazine (DTBZ), antibodies to zinc transporter (ZnT-8) and the monoclonal antibody IC2. While DTBZ and antibodies to ZnT-8 showed binding activities to more than beta-cells, the anti-IC2 monoclonal antibody showed binding properties exclusively to insulin-producing beta...

  3. Re-exposure to beta cell autoantigens in pancreatic allograft recipients with preexisting beta cell autoantibodies.

    Science.gov (United States)

    Mujtaba, Muhammad Ahmad; Fridell, Jonathan; Book, Benita; Faiz, Sara; Sharfuddin, Asif; Wiebke, Eric; Rigby, Mark; Taber, Tim

    2015-11-01

    Re-exposure to beta cell autoantigens and its relevance in the presence of donor-specific antibodies (DSA) in pancreatic allograft recipients is not well known. Thirty-three patients requiring a pancreas transplant were enrolled in an IRB approved study. They underwent prospective monitoring for DSA and beta cell autoantibody (BCAA) levels to GAD65, insulinoma-associated antigen 2 (IA-2), insulin (micro-IAA [mIAA]), and islet-specific zinc transporter isoform-8 (ZnT8). Twenty-five (75.7%) had pre-transplant BCAA. Twenty had a single antibody (mIAA n = 15, GAD65 n = 5); five had two or more BCAA (GAD65 + mIAA n = 2, GAD65 + mIAA+IA-2 n = 2, GA65 + mIAA+IA-2 + ZnT8 = 1). No changes in GAD65 (p > 0.29), IA-2 (>0.16), and ZnT8 (p > 0.07) were observed between pre-transplant and post-transplant at 6 or 12 months. A decrease in mIAA from pre- to post-6 months (p BCAA was observed at one yr. Seven (21.0%) developed de novo DSA. The incidence of DSA was 24% in patients with BCAA vs. 25% in patients without BCAA (p = 0.69). Pancreatic allograft function of patients with vs. without BCAA, and with and without BCAA + DSA was comparable until last follow-up (three yr). Re-exposure to beta cell autoantigens by pancreas transplant may not lead to increased levels or development of new BCAA or pancreatic allograft dysfunction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Secretory IgA (SIgA: designed for antimicrobial defence

    Directory of Open Access Journals (Sweden)

    Per eBrandtzaeg

    2013-08-01

    Full Text Available Prevention of infections by vaccination remains a compelling goal to improve public health. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion -- a term coined for non-inflammatory antibody shielding of internal body surfaces, mediated principally by secretory immunoglobulin A (SIgA. The exported antibodies are polymeric, mainly IgA dimers (pIgA, produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein -- the secretory component (SC. A common SC-dependent transport mechanism for pIgA and pentameric IgM was then proposed, implying that membrane SC acts as a receptor, now usually called the polymeric Ig receptor (pIgR. From the basolateral surface, pIg-pIgR complexes are taken up by endocytosis and then extruded into the lumen after apical cleavage of the receptor -- bound SC having stabilizing and innate functions in the secretory antibodies. Mice deficient for pIgR show that this is the only receptor responsible for epithelial export of IgA and IgM. These knockout mice show a variety of defects in their mucosal defensce and changes in their intestinal microbiota. In the gut, induction of B cells occurs in gut-associated lymphoid tissue (GALT, particularly the Peyer’s patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the activated memory/effector B cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue (NALT but by different homing receptors. This compartmentalization is a challenge for mucosal vaccination, as are the mechanisms used by the mucosal immune system to discriminate between commensal symbionts (mutualism, pathobionts and overt pathogens (elimination.

  5. Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs

    Directory of Open Access Journals (Sweden)

    Jonathan L. S. Esguerra

    2014-11-01

    Full Text Available Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D. The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.

  6. Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units.

    Science.gov (United States)

    Cutler, L S; Christian, C P; Rendell, J K

    1987-01-01

    The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.

  7. Ectopic production of beta-HCG by a maxillary squamous cell carcinoma.

    Science.gov (United States)

    Scholl, P D; Jurco, S; Austin, J R

    1997-12-01

    Paraneoplastic syndromes of the head and neck are rare. Hypercalcemia and leukocytosis have been described. The literature was reviewed, and a case of a squamous cell carcinoma of the maxilla producing beta human chorionic gonadotropin (beta-HCG) is presented. A 47-year-old white man with a T4N1M0 squamous cell carcinoma of the left maxilla was treated with a maxillectomy and neck dissection for an N1 positive neck. After completing his planned radiotherapy, he developed distant metastases, which included an axillary node that stained positive for human beta-HCG. Retrospective review of the primary specimen showed beta-HCG positivity in an anaplastic component of the tumor along with vascular invasion. The first case in the literature of a paraneoplastic syndrome with beta-HCG production in association with squamous cell carcinoma of the maxilla is presented. This case history fits the aggressive nature of beta HCG producing tumors elsewhere in the body.

  8. Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Ringholm, Lene; Søstrup, Birgitte

    2014-01-01

    is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.......OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones...... there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women...

  9. Pancreatic beta-cell lipotoxicity induced by overexpression of hormone-sensitive lipase

    DEFF Research Database (Denmark)

    Winzell, Maria Sörhede; Svensson, Håkan; Enerbäck, Sven

    2003-01-01

    Lipid perturbations associated with triglyceride overstorage in beta-cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone-sensitive lipase, which is expressed and enzymatically active in beta-cells, in the development of lipotoxicity, we generated transgenic...... mice overexpressing hormone-sensitive lipase specifically in beta-cells. Transgenic mice developed glucose intolerance and severely blunted glucose-stimulated insulin secretion when challenged with a high-fat diet. As expected, both lipase activity and forskolin-stimulated lipolysis was increased...

  10. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

  11. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance

    DEFF Research Database (Denmark)

    Holst, Birgitte; Madsen, Kenneth L; Jansen, Anna M

    2013-01-01

    by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate...

  12. Beta Cell Workshop 2013 Kyoto

    DEFF Research Database (Denmark)

    Heller, R Scott; Madsen, Ole D; Nielsen, Jens Høiriis

    2013-01-01

    The very modern Kyoto International Conference Center provided the site for the 8th workshop on Beta cells on April 23-26, 2013. The preceding workshops were held in Boston, USA (1991); Kyoto, Japan (1994); Helsingør, Denmark (1997); Helsinki, Finland (2003); El Perello, Spain (2006); Peebles...

  13. The Relationship between the Ionic Composition of the Environment and the Secretory Activity of the Endocrine Cell Types of Stannius Corpuscles in the Teleost Gasterosteus aculeatus

    NARCIS (Netherlands)

    Wendelaar Bonga, S.E.; Greven, J.A.A.; Veenhuis, M.

    1976-01-01

    The corpuscles of Stannius of threespined sticklebacks contain two glandular cell types of presumed endocrine nature. To elucidate the function of both cell types the secretory activity of the cells was studied in fully adapted seawater and freshwater fishes and in specimens transferred from sea

  14. Rat beta-LPH, gamma-LPH and beta-endorphin biosynthesized by isolated cells of pars intermedia and pars distalis

    International Nuclear Information System (INIS)

    Gianoulakis, C.; Seidah, N.G.; Routhier, R.; Chretien, M.

    1980-01-01

    Rats pars intermedia cells were incubated for 3 h with the following amino-acids: a) 35 S-methionine and 3 H-phenylalamine; b) 3 H-valine; and c) 3 H-valine and 3 H-lysine. Radioactive gamma-lipotropin, beta-lipotropin and beta-endorphin were purified on carboxy- methyl-cellulose and characterized by polyacrylamide disc gel electrophoresis af pH 4.5, molecular weight estimation and microsequencing. Rat gamma-lipotropin was shown to differ slightly from ovine gamma-lipotropin in its NH 2 -terminal amino acid sequence, in containing no methionine and having phenylalanine at position 6, valine at positions 13 and 27, and lysine at position 20. The same variations were observed in the sequence of rat beta-lipotropin, while rat beta-endorphin was shown to be identical to the ovine beta-endorphin. Following a 3-h pulse of rat pars distalis, the cells were extracted with care to avoid beta-lipotropin degradation by proteolytic enzymes. A peptide was purified and identified to be rat beta-endorphin, thus demonstrating that beta-endorphin is biosynthesized in pars distalis and is not an extraction artifact. (author)

  15. Progressive quality control of secretory proteins in the early secretory compartment by ERp44.

    Science.gov (United States)

    Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

    2014-10-01

    ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. © 2014. Published by The Company of Biologists Ltd.

  16. Dysregulation of Dicer1 in Beta Cells Impairs Islet Architecture and Glucose Metabolism

    Directory of Open Access Journals (Sweden)

    Amitai D. Mandelbaum

    2012-01-01

    Full Text Available microRNAs (miRNAs play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas.

  17. Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    Energy Technology Data Exchange (ETDEWEB)

    Zitta, Karina; Brandt, Berenice [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Wuensch, Annegret [Institute of Molecular Animal Breeding and Biotechnology, Ludwig Maximilians University, Munich (Germany); Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Albrecht, Martin, E-mail: Albrecht@anaesthesie.uni-kiel.de [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany)

    2010-09-03

    Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined

  18. Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

    Science.gov (United States)

    Merwin, J R; Anderson, J M; Kocher, O; Van Itallie, C M; Madri, J A

    1990-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with

  19. Strain-dependent differences in sensitivity of rat beta-cells to interleukin 1 beta in vitro and in vivo

    DEFF Research Database (Denmark)

    Reimers, J I; Andersen, H U; Mauricio, D

    1996-01-01

    /kg) or vehicle for 5 days. All the strains investigated were susceptible to IL-1 beta-induced changes in body weight, food intake, temperature, and plasma glucagon and corticosterone. However, IL-1 beta induced hyperglycemia and impairment of beta-cell glucose responsiveness in WK/Mol and LS/Mol rats...

  20. Characterization of estrogen receptors alpha and beta in uterine leiomyoma cells.

    Science.gov (United States)

    Valladares, Francisco; Frías, Ignacio; Báez, Delia; García, Candelaria; López, Francisco J; Fraser, James D; Rodríguez, Yurena; Reyes, Ricardo; Díaz-Flores, Lucio; Bello, Aixa R

    2006-12-01

    Cellular and subcellular localization of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in uterine leiomyomas. Retrospective study. University of La Laguna (ULL) and Canary University Hospital (HUC). Premenopausal and postmenopausal women with uterine leiomyomas. Hysterectomy and myomectomy. Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellular location in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial, and connective tissue cells with nuclear location in all cases studied; variations were only found in the muscle cells for this receptor. Estrogens operate in leiomyoma smooth muscle cells through different receptors, alpha and beta. However they only act through the ERbeta in endothelial and connective cells.

  1. A human beta cell line with drug inducible excision of immortalizing transgenes

    Science.gov (United States)

    Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

    2015-01-01

    Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

  2. Demonstration of interleukin-1 beta transcripts in acute myeloblastic leukemic cells by in situ hybridization.

    Science.gov (United States)

    Nakamura, M; Kanakura, Y; Furukawa, Y; Ernst, T J; Griffin, J D

    1990-07-01

    The cells from some patients with acute myeloblastic leukemia will secrete autostimulatory cytokines in tissue culture without the addition of stimulators such as phorbol 12-myristate 13-acetate. Production of interleukin-1 beta (IL-1 beta), for example, has been observed in up to 50% of cases. In order to investigate the nature of the cell secreting IL-1 beta in AML, we used an antisense RNA probe to detect specific IL-1 beta transcripts in individual leukemic cells by in situ hybridization. In fresh, uncultured cells, IL-1 beta transcripts were observed in 1-40% of undifferentiated leukemic blast cells in 17 of 19 cases. In situ hybridization was at least as sensitive as Northern blot analysis in detecting IL-1 beta transcripts. No correlation of IL-1 beta transcript expression with FAB classification was observed. Normal blood and bone marrow mononuclear cells did not contain cells expressing IL-1 beta transcripts. These results support the concept that the regulation of cytokine genes in AML cells is aberrant.

  3. A Simple Matter of Life and Death—The Trials of Postnatal Beta-Cell Mass Regulation

    Directory of Open Access Journals (Sweden)

    Elena Tarabra

    2012-01-01

    Full Text Available Pancreatic beta-cells, which secrete the hormone insulin, are the key arbiters of glucose homeostasis. Defective beta-cell numbers and/or function underlie essentially all major forms of diabetes and must be restored if diabetes is to be cured. Thus, the identification of the molecular regulators of beta-cell mass and a better understanding of the processes of beta-cell differentiation and proliferation may provide further insight for the development of new therapeutic targets for diabetes. This review will focus on the principal hormones and nutrients, as well as downstream signalling pathways regulating beta-cell mass in the adult. Furthermore, we will also address more recently appreciated regulators of beta-cell mass, such as microRNAs.

  4. Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

    Science.gov (United States)

    Guo, Jinya; Miao, Yansong; Cai, Yi

    2017-01-01

    Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

  5. Quantitation of secretory protein levels by radioimmunoassay

    International Nuclear Information System (INIS)

    Klein, J.L.; Dawson, J.R.

    1978-01-01

    A radioimmunoassay was designed for the detection of secretory protein, a component of secretory immunoglobulin A, in human serum. The assay uses free secretory protein isolated from human colostrum, and antisera raised in rabbits to be purified antigen. The mean level of secretory protein in the control group was 2.34+-0.41 μg/ml (mean+-S.E.M.). The level in cord blood was slightly lower (0.74+-0.26 μg/ml), while the level in patients with ovarian carcinoma was significantly increased (12.67+-1.43 μg/ml). Pregnant women have increasingly secretory protein levels with increasing length of gestation (5.86+-2.02, 11.55+-1.30 and 17.00+-1.16 μg/ml for the first, second and third trimesters, respectively. (Auth.)

  6. Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

    Science.gov (United States)

    Spens, Erika; Häggström, Lena

    2009-05-20

    NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

  7. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  8. Isolation of beta-glucan from the cell wall of Saccharomyces cerevisiae.

    Science.gov (United States)

    Shokri, Hojjatollah; Asadi, Farzad; Khosravi, Ali Reza

    2008-03-20

    Beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae (S. cerevisiae), has been found to enhance immune functions. At present study, we developed an optimal procedure to extract and purify beta-glucan. At first, yeast cells were grown in sabouraud dextrose agar and then cultured in yeast extract-peptone-glucose (YPG) broth. After incubation, cells were harvested, washed and disrupted by means of sonication method. The obtained cell walls were used to prepare alkali-soluble beta-glucan (glucan-S1). In this regard, 2% sodium hydroxide (NaOH) and 3% acetic acid were used in alkaline-acid extraction, respectively. This preparation contained 2.4% protein. In the next step, DEAE sephacel chromatography was used to remove remaining proteins (glucan-S2). Subsequently this preparation was applied into concanavalin-A sepharose column to remove manann. Finally, beta-glucan free of mannoprotein complexes was prepared (glucan-S3).

  9. Comparison of ion transport by cultured secretory and absorptive canine airway epithelia

    DEFF Research Database (Denmark)

    Boucher, R C; Larsen, Erik Hviid

    1988-01-01

    The use of primary cell culture techniques to predict the function of native respiratory epithelia was tested in studies of dog airway epithelia. Epithelial cells from Cl- secretory (tracheal) and Na+ absorptive (bronchial) airway regions were isolated by enzymatic digestion, plated on collagen...

  10. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    Science.gov (United States)

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  11. Regulation of pancreatic beta-cell mass and proliferation by SOCS-3

    DEFF Research Database (Denmark)

    Lindberg, K; Rønn, S G; Tornehave, D

    2005-01-01

    Growth hormone and prolactin are important growth factors for pancreatic beta-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in beta-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (ST...

  12. Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function

    DEFF Research Database (Denmark)

    Gimond, C; van Der Flier, A; van Delft, S

    1999-01-01

    different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated...... for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts...

  13. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    Directory of Open Access Journals (Sweden)

    Sonia Gullón

    Full Text Available Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase and a Tat-dependent model protein (agarase in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  14. Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca2+ and GTPγS

    International Nuclear Information System (INIS)

    Balletta, Andrea; Lorenz, Dorothea; Rummel, Andreas; Gerhard, Ralf; Bigalke, Hans; Wegner, Florian

    2015-01-01

    Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca 2+ and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (C m ) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell “stimulated” C m increase, as well as on “constitutive” secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3–24 h exhibited a significant reduction of the mean absolute and relative C m increase in response to free-Ca 2+ and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity

  15. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

    International Nuclear Information System (INIS)

    Pi Jingbo; Zhang Qiang; Fu Jingqi; Woods, Courtney G.; Hou Yongyong; Corkey, Barbara E.; Collins, Sheila; Andersen, Melvin E.

    2010-01-01

    This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H 2 O 2 , act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function.

  16. A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines.

    Directory of Open Access Journals (Sweden)

    Nan Sook Lee

    Full Text Available Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+ beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

  17. Effect of long-term transfusion therapy on the glycometabolic status and pancreatic beta cell function in patients with beta Thalassemia major

    Directory of Open Access Journals (Sweden)

    Kamalakshi G Bhat

    2014-01-01

    Full Text Available Background: Diabetes mellitus is a major complication of iron overload in patients with beta thalassemia major. Design: This is a descriptive study conducted in a Tertiary Care Teaching Hospital to analyze beta cell function and insulin resistance, and their relation to iron overload status in beta thalassemia major. Fasting glucose, two-hour post load glucose, fasting insulin, alanine amino transaminase (ALT, and ferritin were used as outcome measures. The homeostatic model assessment (HOMA model was used to calculate the beta cell function and insulin resistance index. Results: Of the 30 cases, 20% had impaired fasting glucose, 3.3% had impaired glucose tolerance, and none had diabetes. Fasting glucose was not significant between the cases and controls (P = 0.113. Fasting insulin (P = 0.001, ferritin (P = 0.001, and ALT (P = 0.001 levels were significantly high in the cases. Insulin resistance index was significantly higher in the cases (P = 0.001 as also the beta cell function (P = 0.001. With increase in age and the number of units transfused there is a decline in beta cell function, fasting insulin, and insulin resistance after attaining the maximum level. This suggests that initial insulin resistance is followed by insulin depletion due to loss of beta cell function, leading to diabetes mellitus. Conclusion: Impaired glucose tolerance (IGT and insulin resistance precede the onset of insulin-dependent diabetes and adequate chelation therapy is essential for delaying the onset or for prevention of diabetes.

  18. Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion

    DEFF Research Database (Denmark)

    Piwko-Czuchra, Aleksandra; Koegel, Heidi; Meyer, Hannelore

    2009-01-01

    BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in thei...... of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis....... that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon...... was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations...

  19. Semi-automated digital measurement as the method of choice for beta cell mass analysis.

    Directory of Open Access Journals (Sweden)

    Violette Coppens

    Full Text Available Pancreas injury by partial duct ligation (PDL activates beta cell differentiation and proliferation in adult mouse pancreas but remains controversial regarding the anticipated increase in beta cell volume. Several reports unable to show beta cell volume augmentation in PDL pancreas used automated digital image analysis software. We hypothesized that fully automatic beta cell morphometry without manual micrograph artifact remediation introduces bias and therefore might be responsible for reported discrepancies and controversy. However, our present results prove that standard digital image processing with automatic thresholding is sufficiently robust albeit less sensitive and less adequate to demonstrate a significant increase in beta cell volume in PDL versus Sham-operated pancreas. We therefore conclude that other confounding factors such as quality of surgery, selection of samples based on relative abundance of the transcription factor Neurogenin 3 (Ngn3 and tissue processing give rise to inter-laboratory inconsistencies in beta cell volume quantification in PDL pancreas.

  20. Signaling from the secretory granule to the nucleus: Uhmk1 and PAM.

    Science.gov (United States)

    Francone, Victor P; Ifrim, Marius F; Rajagopal, Chitra; Leddy, Christopher J; Wang, Yanping; Carson, John H; Mains, Richard E; Eipper, Betty A

    2010-08-01

    Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

  1. Anti-Proliferative and Apoptotic Effects of Beta-Ionone in Human Leukemia Cell Line K562

    Directory of Open Access Journals (Sweden)

    Zohreh Faezizadeh

    2016-06-01

    Full Text Available Background Beta-ionone is an aroma compound found in the Rosaceae family. Some evidence supported that beta-ionone has a great potential for cancer prevention. To date, the anti-proliferative and apoptotic effects of beta-ionone in human leukemia cell line K562 were not studied. Objectives Hence, we investigated whether beta-ionone could inhibit cell growth and induce apoptosis in the K562 cells. Materials and Methods In this experimental study, human leukemia cell line K562 was cultured and anti-proliferation effect of beta-ionone with different doses (25 - 400 µm at different times (24 - 96 hours on treated cells was evaluated by the MTT assay. To determine apoptosis rate, the Hoechst 33342 staining and flow cytometry was performed. Results The MTT assay showed that beta-ionone inhibited proliferation of K562 cells in a dose-dependent manner significantly (P = 0.0008. Moreover, the increased apoptotic rate was found after incubation of K562 cells with 200 µm beta-ionone. The Hoechst staining and flow cytometry analysis indicated that beta-ionone could increase apoptosis of K562 cells in a dose-dependent manner. Conclusions The results demonstrated that beta-ionone has anti-proliferative and apoptotic effects on K562 cells, and in the future may be used in the treatment of some leukemia sub-types.

  2. Failure of anti-T-cell receptor V beta antibodies to consistently identify a malignant T-cell clone in Sézary syndrome.

    Science.gov (United States)

    Bigler, R D; Boselli, C M; Foley, B; Vonderheid, E C

    1996-11-01

    Monoclonal antibodies (MAbs) reacting with the human T cell receptor (TCR) V beta or V alpha region have been shown to be almost as specific as a private idiotypic MAb in identifying T cell clones. When available, V beta-specific MAbs offer the ease of immunofluorescence analysis to identify and quantitate expanded malignant or nonmalignant T cell populations without requiring polymerase chain reaction (PCR) technology to evaluate expression of V beta gene families. The V beta expression of peripheral blood lymphocytes from twenty-three consecutive patients with Sézary syndrome has been analyzed by reverse transcriptase (RT)-PCR. Ten patients had malignant T cell clones that expressed a TCR V beta corresponding to a commercially available anti-V beta antibody. Immunofluorescence staining with anti-V beta MAbs showed a direct correlation with RT-PCR results in seven of ten patients. No false positive reactivity was noted on immunofluorescence staining with any MAb. Cells from three patients, however, did not react with the corresponding anti-V beta MAb. These three cases expressed a TCR V beta from gene families containing a single member, ie, V beta 14, V beta 18, and V beta 20, yet MAbs reported to be specific for these regions failed to react with the T cell clone from these patients. Sequencing of the PCR product in these cases confirmed the RT-PCR results. Cells from two patients expressed a TCR using V beta 5.1-D beta 1.1 genes with different J-C segments. One patient's cells reacted with an anti-V beta 5.1 MAb (LC4) whereas the other patient's cells bound one-tenth the amount of this same MAb. These results indicate that currently available anti-TCR V region MAbs may not react consistently with T cell clones expressing the corresponding V region or may react with a low affinity making detection difficult. Differences in the J-C junction or in CDR3 may influence the binding of these MAbs. Until the false negative rate is reduced and the fine specificity and

  3. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F

    2001-01-01

    of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m......RNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates....

  4. The Importance of REST for Development and Function of Beta Cells

    DEFF Research Database (Denmark)

    Martin, David; Grapin-Botton, Anne

    2017-01-01

    that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST......Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among...... these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes...

  5. Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion.

    Directory of Open Access Journals (Sweden)

    Aleksandra Piwko-Czuchra

    Full Text Available BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.

  6. A Figure-of-Merit for Beta Cell Detector Characterization

    Energy Technology Data Exchange (ETDEWEB)

    Foxe, Michael P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Miller, Brian W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Suarez, Rey [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hayes, James C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-09-02

    In order to decrease the minimum detectable activities (MDAs) of beta-gamma radioxenon detectors, it is important to increase the ability to resolve the individual isotopes. One proposed method for doing this is to increase the energy resolution of the beta cell through the use of silicon detectors. While silicon detectors can improve the energy resolution, it is accompanied with a decrease in detection efficiency compared to plastic scintillator beta cells. Due to the uncertainty on the impact of the competing variables, we have developed a figure-of-merit (FOM) capable of determining the impact of detector parameters on the MDAs. By utilizing the FOM to analyze different detectors, we are able to directly compare current and future detectors and estimate their impact on the radioxenon MDAs.

  7. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kandaswamy, Krishna Kumar [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Graduate School for Computing in Medicine and Life Sciences, University of Luebeck, 23538 Luebeck (Germany); Pugalenthi, Ganesan [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Hartmann, Enno; Kalies, Kai-Uwe [Centre for Structural and Cell Biology in Medicine, Institute of Biology, University of Luebeck, 23538 Luebeck (Germany); Moeller, Steffen [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Suganthan, P.N. [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Martinetz, Thomas, E-mail: martinetz@inb.uni-luebeck.de [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany)

    2010-01-15

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  8. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    International Nuclear Information System (INIS)

    Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Hartmann, Enno; Kalies, Kai-Uwe; Moeller, Steffen; Suganthan, P.N.; Martinetz, Thomas

    2010-01-01

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  9. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...

  10. TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

    Science.gov (United States)

    Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

    2010-01-01

    Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

  11. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  12. Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

    Science.gov (United States)

    Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

    2008-01-01

    The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

  13. High fat programming of beta cell compensation, exhaustion, death and dysfunction.

    Science.gov (United States)

    Cerf, Marlon E

    2015-03-01

    Programming refers to events during critical developmental windows that shape progeny health outcomes. Fetal programming refers to the effects of intrauterine (in utero) events. Lactational programming refers to the effects of events during suckling (weaning). Developmental programming refers to the effects of events during both fetal and lactational life. Postnatal programming refers to the effects of events either from birth (lactational life) to adolescence or from weaning (end of lactation) to adolescence. Islets are most plastic during the early life course; hence programming during fetal and lactational life is most potent. High fat (HF) programming is the maintenance on a HF diet (HFD) during critical developmental life stages that alters progeny metabolism and physiology. HF programming induces variable diabetogenic phenotypes dependent on the timing and duration of the dietary insult. Maternal obesity reinforces HF programming effects in progeny. HF programming, through acute hyperglycemia, initiates beta cell compensation. However, HF programming eventually leads to chronic hyperglycemia that triggers beta cell exhaustion, death and dysfunction. In HF programming, beta cell dysfunction often co-presents with insulin resistance. Balanced, healthy nutrition during developmental windows is critical for preserving beta cell structure and function. Thus early positive nutritional interventions that coincide with the development of beta cells may reduce the overwhelming burden of diabetes and metabolic disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Tumor associated macrophages protect colon cancer cells from TRAIL-induced apoptosis through IL-1beta-dependent stabilization of Snail in tumor cells.

    Directory of Open Access Journals (Sweden)

    Pawan Kaler

    2010-07-01

    Full Text Available We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3 halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3 sensitizes tumor cells to TRAIL

  15. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

    Science.gov (United States)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

    2009-08-07

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

  16. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

    2013-11-15

    The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

  17. Initial slope of human tumor cell survival curves: its modification by the oxic cell sensitizer beta-arabinofuranosyladenine

    International Nuclear Information System (INIS)

    Chavaudra, N.; Halimi, M.; Parmentier, C.; Gaillard, N.; Grinfeld, S.; Malaise, E.P.

    1989-01-01

    The initial slope of the survival curve, which is a characteristic of each tumor cell line, varies with the histological group of the tumor. It is one of the factors on which clinical radioresponsiveness depends. The DNA dependant DNA polymerase inhibitor beta-ara A acts as an oxic cell sensitizer. This study was carried out on human tumor cell lines to look for a correlation between the degree of radiosensitization induced by beta-ara A and the radiosensitivity of a given cell line. Six human tumor cell lines with different radiosensitivities were used (the survival rate at 2 Gy and D ranged from 20 to 73% and from 1.2 to 3.2 Gy, respectively). beta-ara A had a major toxic effect on all cell lines but this varied greatly from one cell line to another and was concentration dependant; this toxic effect was taken into account when calculating the surviving fractions. For all cell lines, beta-ara A acted as an oxic radiosensitizer and the radiosensitization was concentration dependant. Analysis of the survival curves of the 6 cell lines using the linear quadratic model showed that concentrations of beta-ara A between 200 and 1000 microM induced an increase in the linear component while the quadratic component underwent no systematic change. The sensitizing enhancement ratio (SER) measured from the Ds ratios, varied greatly from one line to another. For example, at a concentration of 500 microM, the extreme values of Ds ratios were 1.5 and 2.6. The radiosensitization is greater, the higher the radiosensitivity of the cell line studied during exponential growth. The results do not favor the use of beta-ara A in the treatment of intrinsically radioresistant human tumors

  18. Initial slope of human tumor cell survival curves: its modification by the oxic cell sensitizer beta-arabinofuranosyladenine

    Energy Technology Data Exchange (ETDEWEB)

    Chavaudra, N.; Halimi, M.; Parmentier, C.; Gaillard, N.; Grinfeld, S.; Malaise, E.P.

    1989-05-01

    The initial slope of the survival curve, which is a characteristic of each tumor cell line, varies with the histological group of the tumor. It is one of the factors on which clinical radioresponsiveness depends. The DNA dependant DNA polymerase inhibitor beta-ara A acts as an oxic cell sensitizer. This study was carried out on human tumor cell lines to look for a correlation between the degree of radiosensitization induced by beta-ara A and the radiosensitivity of a given cell line. Six human tumor cell lines with different radiosensitivities were used (the survival rate at 2 Gy and D ranged from 20 to 73% and from 1.2 to 3.2 Gy, respectively). beta-ara A had a major toxic effect on all cell lines but this varied greatly from one cell line to another and was concentration dependant; this toxic effect was taken into account when calculating the surviving fractions. For all cell lines, beta-ara A acted as an oxic radiosensitizer and the radiosensitization was concentration dependant. Analysis of the survival curves of the 6 cell lines using the linear quadratic model showed that concentrations of beta-ara A between 200 and 1000 microM induced an increase in the linear component while the quadratic component underwent no systematic change. The sensitizing enhancement ratio (SER) measured from the Ds ratios, varied greatly from one line to another. For example, at a concentration of 500 microM, the extreme values of Ds ratios were 1.5 and 2.6. The radiosensitization is greater, the higher the radiosensitivity of the cell line studied during exponential growth. The results do not favor the use of beta-ara A in the treatment of intrinsically radioresistant human tumors.

  19. Microculture system for studying monolayers of functional beta-cells.

    Science.gov (United States)

    Dobersen, M J; Scharff, J E; Notkins, A L

    1980-04-01

    A method is described for growing monolayers of newborn rat beta-cells in microculture trays. After disruption of the pancreas with collagenase, islets were isolated by Ficoll density gradient centrifugation, trypsinized to obtain individual cells, and plated in 96-well tissue culture trays. The cells were incubated for the first 3 days in growth medium containing 0.1 mM 3-isobutyl-1-methylxanthine to promote monolayer formation. The cultures could be maintained in a functional state, as defined by their responsiveness to known modulators of insulin secretion, for at least 2 weeks. As few as 1 X 10(3) islet cells/well gave results that were reproducible within +/- 10%. It is suggested that the microculture system for islet cells might prove to be a rapid and reproducible screening technique for studying drugs, viruses, or other agents that affect beta-cell function.

  20. Profile of blood glucose and ultrastucture of beta cells pancreatic islet in alloxan compound induced rats

    Directory of Open Access Journals (Sweden)

    I Nyoman Suarsana

    2010-06-01

    Full Text Available Diabetes is marked by elevated levels of blood glucose, and progressive changes of the structure of pancreatic islet histopathology. The objective of this research was to analyse the glucose level and histophatological feature in pancreatic islet in alloxan compound induced rats. A total of ten male Spraque Dawley rats of 2 months old were used in this study. The rats were divided into two groups: (1 negative control group (K-, and (2 positif induced alloxan group (diabetic group =DM. The rats were induced by a single dose intraperitonial injection of alloxan compound 120 mg/kg of body weight. The treatment was conducted for 28 days. Blood glucose levels of rats were analysed at 0, 4, 7, 14, 21, and 28 days following treatment. At the end of the experiment, rats were sacrificed by cervical dislocation. Pancreas was collected for analysis of histopathological study by Immunohistochemical technique, and ultrastructural study using transmission electron microscope (TEM. The result showed that Langerhans islet of diabetic rat (rat of DM group showed a marked reduction of size, number of Langerhans islet of diabetic rat decrease, and characterized by hyperglycemic condition. By using TEM, beta cells of DM group showed the rupture of mitochondrial membrane, the lost of cisternal structure of inner membrane of mitocondria, reduction of insulin secretory granules, linkage between cells acinar with free Langerhans islet, and the caryopicnotic of nucleus.

  1. The effect of interferon-{beta} on mouse neural progenitor cell survival and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Hirsch, Marek [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Knight, Julia [Neuroscience Department, University of Vermont College of Medicine, Burlington, VT (United States); Tobita, Mari; Soltys, John; Panitch, Hillel [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Mao-Draayer, Yang, E-mail: yang.mao-draayer@vtmednet.org [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States)

    2009-10-16

    Interferon-{beta} (IFN-{beta}) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-{beta} on the central nervous system (CNS) are not well understood. To determine whether IFN-{beta} has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-{beta} and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFN{alpha}/{beta} receptor (IFNAR). In response to IFN-{beta} treatment, no effect was observed on differentiation or proliferation. However, IFN-{beta} treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-{beta} treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-{beta} can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

  2. Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

    Science.gov (United States)

    Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

    2017-01-01

    Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

  3. Visualizing pancreatic {beta}-cell mass with [{sup 11}C]DTBZ

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Norman Ray [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Souza, Fabiola [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Witkowski, Piotr [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Maffei, Antonella [Institute of Genetics and Biophysics ' Adriano Buzzati-Traverso' , CNR, Naples 80131 (Italy); Raffo, Anthony [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Herron, Alan [Center for Comparative Medicine and The Department of Pathology, Baylor College of Medicine, Houston, TX 77030 (United States); Kilbourn, Michael [Department of Radiology, University of Michigan, Ann Arbor, MI 48109-0638 (United States); Jurewicz, Agata [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Herold, Kevan [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Liu, Eric [Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, MD 20854 (United States); Hardy, Mark Adam [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Van Heertum, Ronald [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Harris, Paul Emerson [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States)]. E-mail: peh1@columbia.edu

    2006-10-15

    {beta}-Cell mass (BCM) influences the total amount of insulin secreted, varies by individual and by the degree of insulin resistance, and is affected by physiologic and pathologic conditions. The islets of Langerhans, however, appear to have a reserve capacity of insulin secretion and, overall, assessments of insulin and blood glucose levels remain poor measures of BCM, {beta}-cell function and progression of diabetes. Thus, novel noninvasive determinations of BCM are needed to provide a quantitative endpoint for novel therapies of diabetes, islet regeneration and transplantation. Built on previous gene expression studies, we tested the hypothesis that the targeting of vesicular monoamine transporter 2 (VMAT2), which is expressed by {beta} cells, with [{sup 11}C]dihydrotetrabenazine ([{sup 11}C]DTBZ), a radioligand specific for VMAT2, and the use of positron emission tomography (PET) can provide a measure of BCM. In this report, we demonstrate decreased radioligand uptake within the pancreas of Lewis rats with streptozotocin-induced diabetes relative to their euglycemic historical controls. These studies suggest that quantitation of VMAT2 expression in {beta} cells with the use of [{sup 11}C]DTBZ and PET represents a method for noninvasive longitudinal estimates of changes in BCM that may be useful in the study and treatment of diabetes.

  4. Secretory immunity with special reference to the oral cavity

    Directory of Open Access Journals (Sweden)

    Per Brandtzaeg

    2013-03-01

    Full Text Available The two principal antibody classes present in saliva are secretory IgA (SIgA and IgG; the former is produced as dimeric IgA by local plasma cells (PCs in the stroma of salivary glands and is transported through secretory epithelia by the polymeric Ig receptor (pIgR, also named membrane secretory component (SC. Most IgG in saliva is derived from the blood circulation by passive leakage mainly via gingival crevicular epithelium, although some may be locally produced in the gingiva or salivary glands. Gut-associated lymphoid tissue (GALT and nasopharynx-associated lymphoid tissue (NALT do not contribute equally to the pool of memory/effector B cells differentiating to mucosal PCs throughout the body. Thus, enteric immunostimulation may not be the best way to activate the production of salivary IgA antibodies although the level of specific SIgA in saliva may still reflect an intestinal immune response after enteric immunization. It remains unknown whether the IgA response in submandibular/sublingual glands is better related to B-cell induction in GALT than the parotid response. Such disparity is suggested by the levels of IgA in submandibular secretions of AIDS patients, paralleling their highly upregulated intestinal IgA system, while the parotid IgA level is decreased. Parotid SIgA could more consistently be linked to immune induction in palatine tonsils/adenoids (human NALT and cervical lymph nodes, as supported by the homing molecule profile observed after immune induction at these sites. Several other variables influence the levels of antibodies in salivary secretions. These include difficulties with reproducibility and standardization of immunoassays, the impact of flow rate, acute or chronic stress, protein loss during sample handling, and uncontrolled admixture of serum-derived IgG and monomeric IgA. Despite these problems, saliva is an easily accessible biological fluid with interesting scientific and clinical potentials.

  5. Infusion of Autologous Retrodifferentiated Stem Cells into Patients with Beta-Thalassemia

    Directory of Open Access Journals (Sweden)

    Ilham Saleh Abuljadayel

    2006-01-01

    Full Text Available Beta-thalassemia is a genetic, red blood cell disorder affecting the beta-globin chain of the adult hemoglobin gene. This results in excess accumulation of unpaired alpha-chain gene products leading to reduced red blood cell life span and the development of severe anemia. Current treatment of this disease involves regular blood transfusion and adjunct chelation therapy to lower blood transfusion–induced iron overload. Fetal hemoglobin switching agents have been proposed to treat genetic blood disorders, such as sickle cell anemia and beta-thalassemia, in an effort to compensate for the dysfunctional form of the beta-globin chain in adult hemoglobin. The rationale behind this approach is to pair the excess normal alpha-globin chain with the alternative fetal gamma-chain to promote red blood cell survival and ameliorate the anemia. Reprogramming of differentiation in intact, mature, adult white blood cells in response to inclusion of monoclonal antibody CR3/43 has been described. This form of retrograde development has been termed “retrodifferentiation”, with the ability to re-express a variety of stem cell markers in a heterogeneous population of white blood cells. This form of reprogramming, or reontogeny, to a more pluripotent stem cell state ought to recapitulate early hematopoiesis and facilitate expression of a fetal and/or adult program of hemoglobin synthesis or regeneration on infusion and subsequent redifferentiation. Herein, the outcome of infusion of autologous retrodifferentiated stem cells (RSC into 21 patients with beta-thalassemia is described. Over 6 months, Infusion of 3-h autologous RSC subjected to hematopoietic-conducive conditions into patients with beta-thalassemia reduced mean blood transfusion requirement, increased mean fetal hemoglobin synthesis, and significantly lowered mean serum ferritin. This was always accompanied by an increase in mean corpuscular volume (MCV, mean corpuscular hemoglobin (MCH, and mean

  6. Dynamics and Synchrony of Pancreatic beta-cells and Islets

    DEFF Research Database (Denmark)

    Pedersen, Morten Gram

    2006-01-01

    description of these processes and their interactions would provide important input in the search for a better treatment of the disease. The thesis describes several aspects of mathematical modeling of beta-cells relevant for the understanding of glucose stimulated insulin secretion. It consists...... and the synchronized behavior of many coupled beta-cells as well as to the synchrony of islets. Rather than developing new biophysical models, the thesis investigates existing models, their integration and simplifications, and analyzed the corresponding dynamics, in order to use these models for investigating...

  7. Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

    Energy Technology Data Exchange (ETDEWEB)

    Selmi, Anna [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Malinowski, Mariusz [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Brutkowski, Wojciech [Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw (Poland); Bednarek, Radoslaw [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland)

    2012-08-15

    Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

  8. Hydroxyurea responses in clinically varied beta, HbE-beta thalassaemia and sickle cell anaemia patients of Eastern India.

    Science.gov (United States)

    Chatterjee, Tridip; Chakravarty, Amit; Chakravarty, Sudipa

    2018-05-01

    The haematological and clinical response to hydroxyurea was estimated in HbE-beta, beta thalassaemia and sickle cell anaemia patients of Eastern India, with variable clinical severity and transfusion requirement to determine whether hydroxyurea can help these patients to maintain their steady haemoglobin level without blood transfusions. Three hundred patients (189 HbE-beta thalassaemia, 95 beta thalassaemia and 16 other haemoglobinopathies including sickle cell anaemia) were selected for hydroxyurea therapy and were followed up for 48-60 months. Results suggest significant response to hydroxyurea therapy in 19 beta and 99 HbE-beta patients in the transfusion-dependent group (GR-I). All of them became transfusion-independent while on hydroxyurea therapy. The majority of responding patients were IVS1-5(G-C) in one of their alleles in HbE-beta cases (83 out of 119). Though IVS1-5(G-C) was found to be the commonest mutation in our selected patients, the mutational background of the patients does not found to have any significant correlation with the response category towards hydroxyurea as per the results observed in our study. But, the drug works pretty well in most of the transfusion-dependent patients, as these patients were withdrawn from regular blood transfusion. At the same time, partial or no response to the drug hydroxyurea was also recorded in our study.

  9. Occurrence of thymosin beta4 in human breast cancer cells and in other cell types of the tumor microenvironment

    DEFF Research Database (Denmark)

    Larsson, L.-I.; Holck, Susanne

    2007-01-01

    that there is a considerable heterogeneity in the cellular distribution of thymosin beta4 in breast cancer. In most tumors examined, cancer cells showed low or intermediate reactivity for thymosin beta4, whereas leukocytes and macrophages showed intense reactivity. In addition, endothelial cells showed variable reactivity...... to thymosin beta4, whereas myofibroblasts were negative. There was no correlation between the intensity of tumor cell staining and histological grade, whereas there was a tendency toward a correlation between endothelial cell staining and grade. These results demonstrate that multiple cell types within...

  10. Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass

    DEFF Research Database (Denmark)

    Gelling, Richard W; Vuguin, Patricia M; Du, Xiu Quan

    2009-01-01

    in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release...

  11. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ebi, Masahide [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan)

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  12. Cell specificity of the cytoplasmic Ca2+ response to tolbutamide is impaired in beta-cells from hyperglycemic mice

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Larsson-Nyrén, Gerd; Lindström, Per

    2006-01-01

    We recently reported that the timing and magnitude of the nutrient-induced Ca(2+) response are specific and reproducible for each isolated beta-cell. We have now used tolbutamide and arginine to test if the cell specificity exists also for the response to non-nutrient stimulation of beta-cells an...

  13. Use of antibodies against the variable regions of the T-cell receptor alpha/beta heterodimer for the study of cutaneous T-cell lymphomas.

    Science.gov (United States)

    Ralfkiaer, E; Wollf-Sneedorff, A; Vejlsgaard, G L

    1991-11-01

    Recent studies have suggested that antibodies against the variable (V) regions of the T-cell antigen receptor (TCR) may be used as markers for clonality and malignancy in T-cell infiltrates. We have investigated this by examining biopsy samples from 45 patients with cutaneous T-cell lymphomas (CTCL) for reactivity with seven antibodies against different V-gene families on the TCR alpha/beta heterodimer, i.e. ICI (V beta 5a), W112 (V beta 5b), OT145 (V beta 6a), 16G8 (V beta 8a), S511 (V beta 12a), F1 (V alpha 2a) and LC4 (alpha beta Va). Serial biopsies were available in 13 patients and a total of 62 samples were studied. The neoplastic cells in five cases were positive for either V beta 5 (one case), V beta 6 (one case), V beta 8 (two cases) or V beta 12 (one case). In the remaining 40 cases, no staining was seen of the neoplastic cells. These findings indicate that while antibodies against the TCR V-regions may be used as clonotypic markers for certain T-cell neoplasms, there is as yet not a sufficient number of anti-TCR V-region antibodies available for the routine diagnosis of these conditions.

  14. Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

    Science.gov (United States)

    Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

  15. Sustained beta-cell dysfunction but normalized islet mass in aged thrombospondin-1 deficient mice.

    Directory of Open Access Journals (Sweden)

    Carl Johan Drott

    Full Text Available Pancreatic islet endothelial cells have in recent years been shown to support beta-cell mass and function by paracrine interactions. Recently, we identified an islets endothelial-specific glycoprotein, thrombospondin-1 (TSP-1, that showed to be of importance for islet angiogenesis and beta-cell function in young mice. The present study aimed to investigate long-term consequences for islet morphology and beta-cell function of TSP-1 deficiency. Islet and beta-cell mass were observed increased at 10-12 weeks of age in TSP-1 deficient mice, but were normalized before 16 weeks of age when compared to wild-type controls. Islet vascularity was normal in 10-12 and 16-week-old TSP-1 deficient animals, whereas islets of one-year-old animals lacking TSP-1 were hypervascular. Beta-cell dysfunction in TSP-1 deficient animals was present at similar magnitudes between 10-12 and 52 weeks of age, as evaluated by glucose tolerance tests. The insulin secretion capacity in vivo of islets in one-year-old TSP-1 deficient animals was only ∼15% of that in wild-type animals. Using a transplantation model, we reconstituted TSP-1 in adult TSP-deficient islets. In contrast to neonatal TSP-1 deficient islets that we previously reported to regain function after TSP-1 reconstitution, adult islets failed to recover. We conclude that TSP-1 deficiency in islets causes changing vascular and endocrine morphological alterations postnatally, but is coupled to a chronic beta-cell dysfunction. The beta-cell dysfunction induced by TSP-1 deficiency is irreversible if not substituted early in life.

  16. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

    International Nuclear Information System (INIS)

    Rhodes, C.J.; Lucas, C.A.; Mutkoski, R.L.; Orci, L.; Halban, P.A.

    1987-01-01

    Isolated rat pancreatic islets were pulse-labeled for 5 min with [ 3 H]leucine then chased for 25 min, during which time endogenously labeled [ 3 H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [ 3 H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect

  17. Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids.

    Science.gov (United States)

    Dembele, Korami; Nguyen, K Hoa; Hernandez, Tiffany A; Nyomba, B L Grégoire

    2009-04-01

    Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic beta-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on beta-cell survival and metabolism. We treated the rat beta-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F beta-cells. However, ethanol causes beta-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human beta-cells.

  18. Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

    Science.gov (United States)

    Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

    2014-01-01

    Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC

  19. Identification and characterization of secretory proteins during ionizing radiation-induced premature senescence

    International Nuclear Information System (INIS)

    Han, Na Kyung; Hong, Mi Na; Jung, Seung Hee; Kang, Kyoung Ah; Lee, Jae Seon; Chi, Seong Gil

    2011-01-01

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated β alactosidase positivity. Recently a large number of molecular phenotypes such as changes in gene expression, protein processing and chromatin organization have been also described. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence. Here, we show that senescent human breast cancer MCF7 cells promote the proliferation, invasion and migration of neighboring cells

  20. Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.

    Science.gov (United States)

    Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B

    2009-11-01

    We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation.

  1. Autocrine production of beta-chemokines protects CMV-Specific CD4 T cells from HIV infection.

    Directory of Open Access Journals (Sweden)

    Joseph P Casazza

    2009-10-01

    Full Text Available Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

  2. Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

    Science.gov (United States)

    Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

    2011-01-15

    Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that

  3. Adrenergic regulation of cytoplasmic structures related to secretory processes in pig pinealocytes-an ultrastructural, quantitative study.

    Science.gov (United States)

    Przybylska-Gornowicz, Barbara; Lewczuk, Bogdan; Ziółkowska, Natalia; Prusik, Magdalena

    2017-10-01

    Two structures, considered as secretory in nature, are present in the pinealocytes in of the domestic pig show the presence of two structures, which are considered as secretory in nature - the dense core vesicles (DCV) and the membrane bounded (dense) bodies (MBB). The latter are extremely numerous in pig pinealocytes (they occupy 6-20% of the cytoplasm), and the number of MBB changes under different physiological and experimental conditions. Norepinephrine is the main neurotransmitter that regulates the secretion of pineal melatonin. The present study was carried out to 1) clarify whether the DCV and their source - the Golgi apparatus (GA) - as well as the MBB are controlled by norepinephrine, 2) determine the effect of adrenergic stimulation on these structures, and 3) identify the receptors involved in the regulation of these structures. The studies were performed using a static organ culture of pig pineal explants. The explants were incubated in a control medium between 08:00 and 20:00 and in a medium with 10μM norepinephrine or alpha- or beta-adrenoceptor agonists between 20:00 and 08:00 on five consecutive days. The tissues were subsequently prepared for ultrastructural analysis. The results distinctly showed that the DCV, GA and MBB in pig pinealocytes are under adrenergic control. The stimulation of the beta-adrenoceptors resulted in an increase in the numerical density of the DCV and a decrease in the relative volume of the GA in the perikarya, while the incubation with agonists of the alpha1-adrenoceptors was ineffective. The relative volume of the MBB in the perikarya significantly decreased after treatment with both beta-agonists and alpha1-agonists, which suggested the involvement of two types of adrenoceptors in the regulation of these structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Secretory proteins of the pulmonary extracellular lining

    International Nuclear Information System (INIS)

    Gupta, R.P.; Patton, S.E.; Eddy, M.; Smits, H.L.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.R.

    1986-01-01

    The objective of this investigation was to identify proteins in the pulmonary extracellular lining (EL) that are secreted by cells of the pulmonary epithelium. Pulmonary lavage effluents from the lungs of rabbits were centrifuged to remove all cells and particulate materials. Serum proteins were removed by repeatedly passing concentrated lavage effluent fluid through an affinity column containing IgG fraction of goat anti-rabbit (whole serum) antiserum bound to Sepharose-4B. Nonserum proteins accounted for 21.3 +/- 10.3% of the total soluble proteins in pulmonary lavage effluents. Serum free lavage effluents (SFL) contained 25 identifiable proteins as determined by using SDS-PAGE under reducing conditions. Of these proteins approximately 73% was accounted for by a single protein with MW of 66 kd. The secretory nature of the proteins present in SFL was investigated by studying the incorporation of 35 S-methionine into proteins released by lung slices and trachea followed by SDS-PAGE and autoradiography. Many, but not all proteins present in SFL were identified as proteins secreted by pulmonary tissues. The major secretory proteins appeared to have MWs of 59, 53, 48, 43, 24, 14, and 6 kd under reducing conditions. These data demonstrate the presence of several proteins in the pulmonary extracellular lining that appear to be secreted by the pulmonary epithelium

  5. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    Science.gov (United States)

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. © 2015 The Authors.

  6. Targeting of beta 1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells

    NARCIS (Netherlands)

    Dickreuter, E.; Eke, I.; Krause, M.; Borgmann, K.; van Vugt, M. A.; Cordes, N.

    2016-01-01

    beta 1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of beta 1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of beta 1 integrin targeting on the repair of radiation-induced

  7. Redox Homeostasis in Pancreatic beta Cells

    Czech Academy of Sciences Publication Activity Database

    Ježek, Petr; Dlasková, Andrea; Plecitá-Hlavatá, Lydie

    2012-01-01

    Roč. 2012, č. 2012 (2012), s. 932838 ISSN 1942-0900 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GPP304/10/P204 Institutional support: RVO:67985823 Keywords : beta cells * reactive oxygen species homeostasis * mitochondria Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 3.393, year: 2012

  8. Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and beta-cell protection

    NARCIS (Netherlands)

    Christensen, D.P.; Gysemans, C.; Lundh, M.; Dahllof, M.S.; Noesgaard, D.; Schmidt, S.F.; Mandrup, S; Birkbak, N.; Workman, C.T.; Piemonti, L.; Blaabjerg, L.; Monzani, V.; Fossati, G.; Mascagni, P.; Paraskevas, S.; Aikin, R.A.; Billestrup, N.; Grunnet, L.G.; Dinarello, C.A.; Mathieu, C.; Mandrup-Poulsen, T.

    2014-01-01

    Type 1 diabetes is due to destruction of pancreatic beta-cells. Lysine deacetylase inhibitors (KDACi) protect beta-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes

  9. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Coppé

    2010-02-01

    Full Text Available Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP, which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen, do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3% oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

  10. Eustachian tube three-dimensional reconstruction of secretory otitis media

    International Nuclear Information System (INIS)

    Yu Yafeng; Zhou Weirong; Bao Xueping; Li Min; Hu Zhenmin

    2006-01-01

    Objective: To study relationship between Eustachian tube and secretory otitis media and to explore the pathogeny of secretory otitis by three-dimensional reconstruction of Eustachian tube. Methods: Thirty cases of secretory otitis media (male 19, female 11) were selected randomly. Everyone was checked by otoscope and audiometry. Their bilateral Eustachian tubes were scanning by helix CT while making Valsalva's action. All images were passed on to work station to make three-dimensional reconstruction. Results: Four patients were found have Eustachian tube diseases, while most of patients' Eustachian tubes ventilated normally. Conclusions: Three-dimensional reconstruction of Eustachian tube can open out some pathogens of some secretory otitis medias. It will be helpful to diagnosis and therapy of secretory otitis media. (authors)

  11. GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

    Science.gov (United States)

    Panka, David J; Cho, Daniel C; Atkins, Michael B; Mier, James W

    2008-01-11

    Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

  12. Insulin replacement restores the vesicular secretory apparatus in the diabetic rat lacrimal gland

    Directory of Open Access Journals (Sweden)

    Ana Carolina Dias

    2015-06-01

    Full Text Available ABSTRACT Purpose: In the lacrimal gland (LG acinar cells, signaling regulates the release of secretory vesicles through specific Rab and SNARE exocytotic proteins. In diabetes mellitus (DM, the LGs are dysfunctional. The aim of this work was to determine if secretory apparatus changes were associated with any effects on the secretory vesicles (SV in diabetic rats as well as the expression levels of constituent Rab and members of the SNARE family, and if insulin supplementation reversed those changes. Methods: DM was induced in male Wistar rats with an intravenous dose of streptozotocin (60 mg/kg. One of the two diabetic groups was then treated every other day with insulin (1 IU. A third control group was injected with vehicle. After 10 weeks, Western blotting and RT-PCR were used to compared the Rab and SNARE secretory factor levels in the LGs. Transmission electron microscopy evaluated acinar cell SV density and integrity. Results: In the diabetes mellitus group, there were fewer and enlarged SV. The Rab 27b, Rab 3d, and syntaxin-1 protein expression declined in the rats with diabetes mellitus. Insulin treatment restored the SV density and the Rab 27b and syntaxin expression to their control protein levels, whereas the Vamp 2 mRNA expression increased above the control levels. Conclusions: Diabetes mellitus LG changes were associated with the declines in protein expression levels that were involved in supporting exocytosis and vesicular formation. They were partially reversed by insulin replacement therapy. These findings may help to improve therapeutic management of dry eye in diabetes mellitus.

  13. Post-secretory fate of host defence components in mucus.

    Science.gov (United States)

    Salathe, Matthias; Forteza, Rosanna; Conner, Gregory E

    2002-01-01

    Airway mucus is a complex mixture of secretory products that provide a multifaceted defence against infection. Among many antimicrobial substances, mucus contains a peroxidase identical to milk lactoperoxidase (LPO) that is produced by goblet cells and submucosal glands. Airway secretions contain the substrates for LPO, namely thiocyanate and hydrogen peroxide, at concentrations sufficient for production of the biocidal compound hypothiocyanite, a fact confirmed by us in vitro. In vivo, inhibition of airway LPO in sheep significantly inhibits bacterial clearance, suggesting that the LPO system is a major contributor to host defences. Since secretory products including LPO are believed to be steadily removed by mucociliary clearance, their amount and availability on the surface is thought to be controlled solely by secretion. In contrast to this paradigm, new data suggest that LPO and other substances are retained at the ciliary border of the airway epithelium by binding to surface-associated hyaluronan, thereby providing an apical, fully active enzyme pool. Thus, hyaluronan, secreted from submucosal gland cells, plays a previously unrecognized pivotal role in mucosal host defence by retaining LPO and possibly other substances important for first line host defence at the apical surface 'ready for use' and protected from ciliary clearance.

  14. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  15. New Therapeutic Approaches to Prevent or Delay Beta-Cell Failure in Diabetes

    Directory of Open Access Journals (Sweden)

    Ionica Floriana Elvira

    2015-09-01

    Full Text Available Background and aims: The most recent estimates of International Diabetes Federation indicate that 382 million people have diabetes, and the incidence of this disease is increasing. While in type 1 diabetes mellitus (T1DM beta-cell death is autoimmunemediated, type 2 diabetes mellitus (T2DM results from an interaction between genetic and environmental factors that impair beta-cell function and insulin action. Many people with T2DM remain unaware of their illness for a long time because symptoms may take years to appear or be recognized, while the body is affected by excess blood glucose. These patients are often diagnosed only when diabetes complications have already developed. The aim of this article was to perform a review based on literature data on therapeutic modalities to prevent/delay beta cell function decline. Material and Methods: We searched MEDLINE from 2000 to the present to identify the therapeutic approaches to prevent or delay beta-cell failure in patients with T2DM. Results and conclusions: Several common polymorphisms in genes linked to monogenic forms of diabetes appear to influence the response to T2DM pharmacotherapy. Recent studies report the role of the G protein coupled receptor 40 (GPR40, also known as Free Fatty Acids Receptor 1 (FFAR1 in the regulation of beta-cell function- CNX-011-67 (a GPR40 agonist has the potential to provide good and durable glycemic control in T2DM patients.

  16. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

    DEFF Research Database (Denmark)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

    2009-01-01

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

  17. Diagnostic significance of red cell indices in beta-thalassaemia trait

    International Nuclear Information System (INIS)

    Hussain, Z.; Malik, N.; Chughtai, A.S.

    2005-01-01

    The purpose of this study was to evaluate the formulae for the diagnosis of beta-thalassemia trait cases in settings where electrophoreses is not available. The study included 50 cases of beta-thalassaemia trait already diagnosed by Hb electrophoresis. CBC samples were analyzed on Sysmex K4500 and red cell indices were used to evaluate formulae for differentiating beta thalassaemia trait from iron deficiency anemia. The formula MCV/RBC and MCH/RBC identified 56% of the cases. Formula MCV - (5 x Hb)- RBC - 8.4 identified 54% of beta thalassemia trait cases. The formula MCV x MCH identified 92% of cases. RBC indices given by 100 electronic counters can be used to differentiate iron deficiency anemia from beta-thalassaemia trait at least provisionally in areas where Hb electrophoresis is not available. (author)

  18. Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1beta-mediated toxicity through inhibition of multiple nuclear factor-kappaB-regulated proapoptotic pathways

    DEFF Research Database (Denmark)

    Karlsen, Allan Ertman; Heding, P E; Frobøse, H

    2004-01-01

    The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback reg...... regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown....

  19. Secretory IgM Exacerbates Tumor Progression by Inducing Accumulations of MDSCs in Mice.

    Science.gov (United States)

    Tang, Chih-Hang Anthony; Chang, Shiun; Hashimoto, Ayumi; Chen, Yi-Ju; Kang, Chang Won; Mato, Anthony R; Del Valle, Juan R; Gabrilovich, Dmitry I; Hu, Chih-Chi Andrew

    2018-06-01

    Chronic lymphocytic leukemia (CLL) cells can secrete immunoglobulin M. However, it is not clear whether secretory IgM (sIgM) plays a role in disease progression. We crossed the Eμ-TCL1 mouse model of CLL, in which the expression of human TCL1 oncogene was driven by the V(H) promoter-Ig(H)-Eμ enhancer, with MD4 mice whose B cells produced B-cell receptor (membrane-bound IgM) and sIgM with specificity for hen egg lysozyme (HEL). CLL cells that developed in these MD4/Eμ-TCL1 mice reactivated a parental Ig gene allele and secreted IgM, and did not recognize HEL. The MD4/Eμ-TCL1 mice had reduced survival, increased myeloid-derived suppressor cells (MDSC), and decreased numbers of T cells. We tested whether sIgM could contribute to the accumulation of MDSCs by crossing μS -/- mice, which could not produce sIgM, with Eμ-TCL1 mice. The μS -/- /Eμ-TCL1 mice survived longer than Eμ-TCL1 mice and developed decreased numbers of MDSCs which were less able to suppress proliferation of T cells. We targeted the synthesis of sIgM by deleting the function of XBP-1s and showed that targeting XBP-1s genetically or pharmacologically could lead to decreased sIgM, accompanied by decreased numbers and reduced functions of MDSCs in MD4/Eμ-TCL1 mice. Additionally, MDSCs from μS -/- mice grafted with Lewis lung carcinoma were inefficient suppressors of T cells, resulting in slower tumor growth. These results demonstrate that sIgM produced by B cells can upregulate the functions of MDSCs in tumor-bearing mice to aggravate cancer progression. In a mouse model of CLL, production of secretory IgM led to more MDSCs, fewer T cells, and shorter survival times for the mice. Thus, secretory IgM may aggravate the progression of this cancer. Cancer Immunol Res; 6(6); 696-710. ©2018 AACR . ©2018 American Association for Cancer Research.

  20. Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis.

    Science.gov (United States)

    Mir-Coll, Joan; Duran, Jordi; Slebe, Felipe; García-Rocha, Mar; Gomis, Ramon; Gasa, Rosa; Guinovart, Joan J

    2016-05-01

    Glycogen accumulation occurs in beta cells of diabetic patients and has been proposed to partly mediate glucotoxicity-induced beta cell dysfunction. However, the role of glycogen metabolism in beta cell function and its contribution to diabetes pathophysiology remain poorly understood. We investigated the function of beta cell glycogen by studying glucose homeostasis in mice with (1) defective glycogen synthesis in the pancreas; and (2) excessive glycogen accumulation in beta cells. Conditional deletion of the Gys1 gene and overexpression of protein targeting to glycogen (PTG) was accomplished by Cre-lox recombination using pancreas-specific Cre lines. Glucose homeostasis was assessed by determining fasting glycaemia, insulinaemia and glucose tolerance. Beta cell mass was determined by morphometry. Glycogen was detected histologically by periodic acid-Schiff's reagent staining. Isolated islets were used for the determination of glycogen and insulin content, insulin secretion, immunoblots and gene expression assays. Gys1 knockout (Gys1 (KO)) mice did not exhibit differences in glucose tolerance or basal glycaemia and insulinaemia relative to controls. Insulin secretion and gene expression in isolated islets was also indistinguishable between Gys1 (KO) and controls. Conversely, despite effective glycogen overaccumulation in islets, mice with PTG overexpression (PTG(OE)) presented similar glucose tolerance to controls. However, under fasting conditions they exhibited lower glycaemia and higher insulinaemia. Importantly, neither young nor aged PTG(OE) mice showed differences in beta cell mass relative to age-matched controls. Finally, a high-fat diet did not reveal a beta cell-autonomous phenotype in either model. Glycogen metabolism is not required for the maintenance of beta cell function. Glycogen accumulation in beta cells alone is not sufficient to trigger the dysfunction or loss of these cells, or progression to diabetes.

  1. Secretory activity and endocrine regulation of male accessory glands in the blood-sucking bug Panstrongylus megistus (Hemiptera: Reduviidae

    Directory of Open Access Journals (Sweden)

    Lêda Regis

    1987-01-01

    Full Text Available The epithelial cells of Panstrongylus megistus male accessory glands (MAG present ultrastructural characteristics of a secretory cell. Their secretory products are accumulated in the lumen of the four MAG lobes. During the first 8 days of adult life a strong secretion activity occurs, accumulating enough material to produce the first spermatophore. Cerebral neurosecretions as well as juvenile hormone are both involved in MAG secretory activity regulation. Juvenile hormone seems to be the responsible for the stimulation of most protein synthesis in male accessory glands. Cerebral neurosecretion seems to be necessary to stimulate juvenile hormone production and release by the corpus allatum. Furthermore, neurosecretion is required for some polypeptides synthesis by MAG. Although topic application of precocene II to adult males does not reproduce the same effects on MAG as does allatectomy, this compound causes strong reduction on male reproductive capacity.

  2. Synaptic Control of Secretory Trafficking in Dendrites

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    Cyril Hanus

    2014-06-01

    Full Text Available Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK. Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport.

  3. T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

    DEFF Research Database (Denmark)

    Dunon, D; Kaufman, J; Salomonsen, J

    1990-01-01

    beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

  4. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    DEFF Research Database (Denmark)

    Klemm, Robin W; Ejsing, Christer S.; Surma, Michal A

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane...... trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN...... than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery....

  5. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  6. Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin

    DEFF Research Database (Denmark)

    Wu, Xunwei; Quondamatteo, Fabio; Lefever, Tine

    2006-01-01

    for differentiation of skin progenitor cells into HF lineage and that it regulates the turnover of beta-catenin. In the absence of Cdc42, degradation of beta-catenin was increased corresponding to a decreased phosphorylation of GSK3beta at Ser 9 and an increased phosphorylation of axin, which is known to be required...... for binding of beta-catenin to the degradation machinery. Cdc42-mediated regulation of beta-catenin turnover was completely dependent on PKCzeta, which associated with Cdc42, Par6, and Par3. These data suggest that Cdc42 regulation of beta-catenin turnover is important for terminal differentiation of HF...

  7. Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

    Science.gov (United States)

    Balic, Anamaria; Mina, Mina

    2011-01-01

    Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

  8. Role of growth factors in control of pancreatic beta cell mass: focus on betatrophin.

    Science.gov (United States)

    Levitsky, Lynne L; Ardestani, Goli; Rhoads, David B

    2014-08-01

    Betatrophin is a newly described hormone, which potently stimulates beta cell replication in mice. This discovery has engendered great hope that it could prove clinically important in the treatment of type 1 and type 2 diabetes. Betatrophin, a 198-amino acid protein secreted by liver and adipose tissue, stimulates growth of pancreatic beta cell mass in insulin-resistant mice. Betatrophin has previously been named RIFL, lipasin, and ANGPLT8, and its salutory effects on lipid metabolism have been described in mouse and human studies. Serum betatrophin levels in humans correlate with improved adipose tissue lipid storage and lower serum triglyceride levels in the fed state, but do not correlate with insulin resistance or carbohydrate tolerance in humans. Betatrophin has not yet been shown to have an effect on beta cell replication in human pancreatic islets. Many endocrine and paracrine factors, of which betatrophin is the newest described, increase beta cell mass in murine models. None of these factors, including betatrophin, have displayed the same activity in clinical studies. This may reflect a profound species difference in beta cell regeneration pathways in mice and humans.

  9. Hypoglycemic and beta cell protective effects of andrographolide analogue for diabetes treatment

    Directory of Open Access Journals (Sweden)

    Larrick James W

    2009-07-01

    Full Text Available Abstract Background While all anti-diabetic agents can decrease blood glucose level directly or indirectly, few are able to protect and preserve both pancreatic beta cell mass and their insulin-secreting functions. Thus, there is an urgent need to find an agent or combination of agents that can lower blood glucose and preserve pancreatic beta cells at the same time. Herein, we report a dual-functional andrographolide-lipoic acid conjugate (AL-1. The anti-diabetic and beta cell protective activities of this novel andrographolide-lipoic acid conjugate were investigated. Methods In alloxan-treated mice (a model of type 1 diabetes, drugs were administered orally once daily for 6 days post-alloxan treatment. Fasting blood glucose and serum insulin were determined. Pathologic and immunohistochemical analysis of pancreatic islets were performed. Translocation of glucose transporter subtype 4 in soleus muscle was detected by western blot. In RIN-m cells in vitro, the effect of AL-1 on H2O2-induced damage and reactive oxidative species production stimulated by high glucose and glibenclamide were measured. Inhibition of nuclear factor kappa B (NF-κB activation induced by IL-1β and IFN-γ was investigated. Results In alloxan-induced diabetic mouse model, AL-1 lowered blood glucose, increased insulin and prevented loss of beta cells and their dysfunction, stimulated glucose transport protein subtype 4 (GLUT4 membrane translocation in soleus muscles. Pretreatment of RIN-m cells with AL-1 prevented H2O2-induced cellular damage, quenched glucose and glibenclamide-stimulated reactive oxidative species production, and inhibited cytokine-stimulated NF-κB activation. Conclusion We have demonstrated that AL-1 had both hypoglycemic and beta cell protective effects which translated into antioxidant and NF-κB inhibitory activity. AL-1 is a potential new anti-diabetic agent.

  10. Differential Recognition of CD1d-[alpha]-Galactosyl Ceramide by the V[beta]8.2 and V[beta]7 Semi-invariant NKT T Cell Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Pellicci, Daniel G.; Patel, Onisha; Kjer-Nielsen, Lars; Pang, Siew Siew; Sullivan, Lucy C.; Kyparissoudis, Konstantinos; Brooks, Andrew G.; Reid, Hugh H.; Gras, Stephanie; Lucet, Isabelle S.; Koh, Ruide; Smyth, Mark J.; Mallevaey, Thierry; Matsuda, Jennifer L.; Gapin, Laurent; McCluskey, James; Godfrey, Dale I.; Rossjohn, Jamie; PMCI-A; Monash; UCHSC; Melbourne

    2009-09-02

    The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR{alpha} chain is typically invariant, the {beta} chain expression is more diverse, where three V{beta} chains are commonly expressed in mice. We report the structures of V{alpha}14-V{beta}8.2 and V{alpha}14-V{beta}7 NKT TCRs in complex with CD1d-{alpha}-galactosylceramide ({alpha}-GalCer) and the 2.5 {angstrom} structure of the human NKT TCR-CD1d-{alpha}-GalCer complex. Both V{beta}8.2 and V{beta}7 NKT TCRs and the human NKT TCR ligated CD1d-{alpha}-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V{beta} domains of the V{beta}8.2 and V{beta}7 NKT TCR-CD1d complexes resulted in altered TCR{beta}-CD1d-mediated contacts and modulated recognition mediated by the invariant {alpha} chain. Mutagenesis studies revealed the differing contributions of V{beta}8.2 and V{beta}7 residues within the CDR2{beta} loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V{beta} usage in NKT cells.

  11. A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

    Science.gov (United States)

    Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

    2015-11-24

    DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

  12. Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

    Directory of Open Access Journals (Sweden)

    Lee JY

    2015-08-01

    Full Text Available Jue Yeon Lee,1,* Jin Sook Suh,2,* Jung Min Kim,1 Jeong Hwa Kim,1 Hyun Jung Park,1 Yoon Jeong Park,1,2 Chong Pyoung Chung1 1Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Chungcheongbuk-do, Republic of Korea; 2Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Human beta-defensins (hBDs are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to

  13. Hypothyroidism in utero stimulates pancreatic beta cell proliferation and hyperinsulinaemia in the ovine fetus during late gestation.

    Science.gov (United States)

    Harris, Shelley E; De Blasio, Miles J; Davis, Melissa A; Kelly, Amy C; Davenport, Hailey M; Wooding, F B Peter; Blache, Dominique; Meredith, David; Anderson, Miranda; Fowden, Abigail L; Limesand, Sean W; Forhead, Alison J

    2017-06-01

    Thyroid hormones are important regulators of growth and maturation before birth, although the extent to which their actions are mediated by insulin and the development of pancreatic beta cell mass is unknown. Hypothyroidism in fetal sheep induced by removal of the thyroid gland caused asymmetric organ growth, increased pancreatic beta cell mass and proliferation, and was associated with increased circulating concentrations of insulin and leptin. In isolated fetal sheep islets studied in vitro, thyroid hormones inhibited beta cell proliferation in a dose-dependent manner, while high concentrations of insulin and leptin stimulated proliferation. The developing pancreatic beta cell is therefore sensitive to thyroid hormone, insulin and leptin before birth, with possible consequences for pancreatic function in fetal and later life. The findings of this study highlight the importance of thyroid hormones during pregnancy for normal development of the fetal pancreas. Development of pancreatic beta cell mass before birth is essential for normal growth of the fetus and for long-term control of carbohydrate metabolism in postnatal life. Thyroid hormones are also important regulators of fetal growth, and the present study tested the hypotheses that thyroid hormones promote beta cell proliferation in the fetal ovine pancreatic islets, and that growth retardation in hypothyroid fetal sheep is associated with reductions in pancreatic beta cell mass and circulating insulin concentration in utero. Organ growth and pancreatic islet cell proliferation and mass were examined in sheep fetuses following removal of the thyroid gland in utero. The effects of triiodothyronine (T 3 ), insulin and leptin on beta cell proliferation rates were determined in isolated fetal ovine pancreatic islets in vitro. Hypothyroidism in the sheep fetus resulted in an asymmetric pattern of organ growth, pancreatic beta cell hyperplasia, and elevated plasma insulin and leptin concentrations. In pancreatic

  14. Development of biomarker specific of pancreatic beta cells (incretin radiolabelled) for image of beta functional mass in diabetic and obese: study in animal model

    International Nuclear Information System (INIS)

    Seo, Daniele

    2017-01-01

    Increased prevalence of obesity worldwide, has become a vast concern, stimulating investigations focusing prevention and therapy of this condition. The association of type 2 diabetes or insulin resistance aggravates the prognosis of obesity. Even patients successfully submitted to bariatric or metabolic surgery, may not be cured of diabetes, as improvement of circulating values of glucose and insulin not necessarily reflects recovery of pancreatic beta cell mass. There is no consensus about how to estimate beta cell mass in vivo. Available tools suffer from low sensitivity and specificity, often being as well cumbersome and expensive. Radiolabeled incretins, such as glucagon-like-peptide 1 (GLP-1) analogs, seem to be promising options for the measurement of beta cell mass in diabetes and insulinoma. The objective of this study was the development of two conjugates of GLP-1 analog, radiolabeled with 99m Technetium, as a noninvasive imaging method for the estimation of pancreatic beta cell mass, in the presence of obesity. Animal models were selected, including hyperlipidic diet-induced obesity, diet restricted obesity, and as controls, alloxan diabetes. Results indicated that both radiotracers achieved over 97% radiochemical yield. The most successful product was 99m Tc-HYNIC-βAla-Exendin-4. Low beta cell mass uptake occurred in diet-induced obesity. Diet-restricted obesity, with substantial shedding of excess body weight, was followed by remarkable decrease of fasting blood glucose, however beta cell mass uptake was only mildly improved. Future studies are recommended in obesity, type 2 diabetes, and dieting, including bariatric and metabolic operations. (author)

  15. Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

    International Nuclear Information System (INIS)

    Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

    2005-01-01

    Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype

  16. Sickle Cell Beta-Plus Thalassemia with Subcapsular Hematoma of the Spleen

    Directory of Open Access Journals (Sweden)

    Suyash Dahal

    2017-01-01

    Full Text Available While splenic complications like hypersplenism, sequestration crisis, and infarction are commonly reported in sickle cell variants like sickle cell beta-plus thalassemia, splenic rupture with hematoma is rare. We present a case of a 32-year-old young male who presented with dull left upper quadrant pain who was found to have multiple subcapsular splenic lacerations and hematoma on abdominal imaging. Hemoglobin electrophoresis confirmed sickle cell beta-plus thalassemia in the patient. There was no history of trauma, and rest of the workup for possible cause of spontaneous rupture of spleen was negative. With the patient refusing splenectomy, he was managed conservatively. Clinicians need to be aware of this rare complication of sickle cell variants.

  17. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Somanath, Sangeeta [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Partridge, Christopher J. [Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Churchill Hospital, University of Oxford, Oxford, OX3 7LJ (United Kingdom); Marshall, Catriona [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Rowe, Tony [CSL Limited, 45 Poplar Road, Parkville, Victoria 3052 (Australia); Turner, Mark D., E-mail: mark.turner@ntu.ac.uk [Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS (United Kingdom)

    2016-04-29

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  18. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    International Nuclear Information System (INIS)

    Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona; Rowe, Tony; Turner, Mark D.

    2016-01-01

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  19. Effect on pancreatic beta cells and nerve cells by low let x-ray

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kwang Hun [Dept. of Nuclear Medicine, Kyungbuk National University Hospital, Daegu (Korea, Republic of); Kim, Kgu Hwan [Dept. of Radiological Technology, Daegu health College, Daegu (Korea, Republic of)

    2014-03-15

    Cultured pancreatic beta cells and nerve cells, it is given normal condition of 10% FBS (fetal bovine serum), 11.1 mM glucose and hyperglycemia condition of 1% FBS, 30 mM glucose. For low LET X-ray irradiated with 0.5 Gy/hr dose-rate(total dose: 0.5 to 5 Gy). Survival rates were measured by MTT assay. When non irradiated, differentiated in the pancreatic beta cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a small reduction. However increasing the total dose of X-ray, the survival rate of normal conditions decreased slightly compared to the survival rate of hyperglycemia conditions, the synergistic effect was drastically reduced. When non irradiated, undifferentiated in the nerve cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a large reduction. As the cumulative dose of X-ray normal conditions and hyperglycemia were all relatively rapid cell death. But the rate of decreased survivals by almost parallel to the reduction proceed and it didn't show synergistic effect.

  20. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

    Science.gov (United States)

    D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

    2006-11-01

    Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

  1. Ionizing radiation predisposes non-malignant human mammaryepithelial cells to undergo TGF beta-induced epithelial to mesenchymaltransition

    Energy Technology Data Exchange (ETDEWEB)

    Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Costes, Sylvain; Gascard, Philippe; Mott, Joni D.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2007-04-06

    Transforming growth factor {beta}1 (TGF{beta}) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF{beta}, activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF{beta}-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGF{beta} (0.4 ng/ml), or double-treated. All double-treated (IR+TGF{beta}) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, {beta}-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though IR increased chronic TGF{beta} signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  2. Serum adipokines as biomarkers of beta-cell function in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Pham, Minh Nguyet; Kolb, Hubert; Mandrup-Poulsen, Thomas

    2013-01-01

    We investigated the adipokines adiponectin, leptin and resistin as serum biomarkers of beta-cell function in patients with type 1 diabetes.......We investigated the adipokines adiponectin, leptin and resistin as serum biomarkers of beta-cell function in patients with type 1 diabetes....

  3. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    DEFF Research Database (Denmark)

    Holmes, Michael V; Simon, Tabassome; Exeter, Holly J

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.......This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease....

  4. Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

    Science.gov (United States)

    Zheng, Xiaofen; De Paiva, Cintia S; Rao, Kavita; Li, De-Quan; Farley, William J; Stern, Michael; Pflugfelder, Stephen C

    2010-09-01

    To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where

  5. Identification of Secreted Proteins from Ionizing Radiation-Induced Senescent MCF7 Cells Using Comparative Proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Han, Na Kyung; Kim, Han Na; Hong, Mi Na; Park, Su Min; Lee, Jae Seon [Korea Institue of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chi, Seong Gil [Korea University, Seoul (Korea, Republic of)

    2010-05-15

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated beta-galactosidase positivity and over the years a large number of molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence-associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence

  6. The inhibition of myometrial contractions by chlorinated herbicides (atrazine and linuron), and their disruptive effect on the secretory functions of uterine and ovarian cells in cow, in vitro.

    Science.gov (United States)

    Wrobel, Michał H; Mlynarczuk, Jaroslaw

    2017-10-01

    The effect of atrazine and linuron, the popular and widely used chlorinated herbicides, on both myometrial contractions and secretory functions of bovine uterus and ovaries in vitro, was investigated. The pesticides inhibited (Pherbicides affected PGs secretion from myometrium and PGF2α from endometrium. Only the lowest dose of both tested compounds decreased PGE2 secretion from endometrium. The pesticides increased (P<0.05) the OT secretion from granulosa. However, only linuron stimulated (P<0.05) the OT secretion from the luteal cells, and it increased (P<0.05) the expression of mRNA for the OT precursor. Both compounds stimulated (P<0.05) the secretion of testosterone and atrazine increased (P<0.05) also the secretion of estradiol from the granulosa cells. While atrazine and linuron reduced (P<0.05) the progesterone secretion from the luteal cells. The data show that atrazine and linuron altered the secretory functions of ovarian cells and inhibited the myometrial contractions in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

    International Nuclear Information System (INIS)

    Friesen, Claudia; Lubatschofski, Annelie; Debatin, Klaus-Michael; Kotzerke, Joerg; Buchmann, Inga; Reske, Sven N.

    2003-01-01

    Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x L , a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies. (orig.)

  8. Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome

    DEFF Research Database (Denmark)

    Feizi, Amir; Gatto, Francesco; Uhlén, Mathias

    2017-01-01

    Protein secretory pathway in eukaryal cells is responsible for delivering functional secretory proteins. The dysfunction of this pathway causes a range of important human diseases from congenital disorders to cancer. Despite the piled-up knowledge on the molecular biology and biochemistry level...... in specific gene families of the secretory pathway. We also inspected the potential functional link between detected extreme genes and the corresponding tissues enriched secretome. As a result, the detected extreme genes showed correlation with the enrichment of the nature and number of specific post......-translational modifications in each tissue's secretome. Our findings conciliate both the housekeeping and tissue-specific nature of the protein secretory pathway, which we attribute to a fine-tuned regulation of defined gene families to support the diversity of secreted proteins and their modifications....

  9. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

    Science.gov (United States)

    Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

    2017-03-01

    Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Massumi

    Full Text Available The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs. We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

  12. Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

    Directory of Open Access Journals (Sweden)

    Beecken Wolf-Dietrich

    2005-01-01

    Full Text Available Abstract Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a, alpha2beta1 (CD49b, alpha3beta1 (CD49c, alpha4beta1 (CD49d, alpha5beta1 (CD49e, and alpha6beta1 (CD49f receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.

  13. Polymorphisms within novel risk loci for type 2 diabetes determine beta-cell function.

    Directory of Open Access Journals (Sweden)

    Harald Staiger

    Full Text Available BACKGROUND: Type 2 diabetes arises when insulin resistance-induced compensatory insulin secretion exhausts. Insulin resistance and/or beta-cell dysfunction result from the interaction of environmental factors (high-caloric diet and reduced physical activity with a predisposing polygenic background. Very recently, genetic variations within four novel genetic loci (SLC30A8, HHEX, EXT2, and LOC387761 were reported to be more frequent in subjects with type 2 diabetes than in healthy controls. However, associations of these variations with insulin resistance and/or beta-cell dysfunction were not assessed. METHODOLOGY/PRINCIPAL FINDINGS: By genotyping of 921 metabolically characterized German subjects for the reported candidate single nucleotide polymorphisms (SNPs, we show that the major alleles of the SLC30A8 SNP rs13266634 and the HHEX SNP rs7923837 associate with reduced insulin secretion stimulated by orally or intravenously administered glucose, but not with insulin resistance. In contrast, the other reported type 2 diabetes candidate SNPs within the EXT2 and LOC387761 loci did not associate with insulin resistance or beta-cell dysfunction, respectively. CONCLUSIONS/SIGNIFICANCE: The HHEX and SLC30A8 genes encode for proteins that were shown to be required for organogenesis of the ventral pancreas and for insulin maturation/storage, respectively. Therefore, the major alleles of type 2 diabetes candidate SNPs within these genetic loci represent crucial alleles for beta-cell dysfunction and, thus, might confer increased susceptibility of beta-cells towards adverse environmental factors.

  14. Genome-scale modeling of the protein secretory machinery in yeast

    DEFF Research Database (Denmark)

    Feizi, Amir; Österlund, Tobias; Petranovic, Dina

    2013-01-01

    The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking....... Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm...

  15. Zinc as a paracrine effector in pancreatic islet cell death.

    Science.gov (United States)

    Kim, B J; Kim, Y H; Kim, S; Kim, J W; Koh, J Y; Oh, S H; Lee, M K; Kim, K W; Lee, M S

    2000-03-01

    Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.

  16. The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together.

    Science.gov (United States)

    Bäck, Nils; Kanerva, Kristiina; Kurutihalli, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E; Eipper, Betty A

    2017-08-01

    Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V.

    2014-01-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

  18. Central infusion of leptin improves insulin resistance and suppresses beta-cell function, but not beta-cell mass, primarily through the sympathetic nervous system in a type 2 diabetic rat model.

    Science.gov (United States)

    Park, Sunmin; Ahn, Il Sung; Kim, Da Sol

    2010-06-05

    We investigated whether hypothalamic leptin alters beta-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states. The 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300ng/kgbw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3h as a short-term study. After finishing the infusion study, ICV leptin (3mug/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed. Acute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic beta-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual beta-cell size and concomitantly increased beta-cell apoptosis in Sham rats. Leptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic beta-cell mass.

  19. Beta cell 5'-shifted isomiRs are candidate regulatory hubs in type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Jeanette Baran-Gale

    Full Text Available Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA transcriptome, which is in large part due to the diversity of miRNA sequence variants ("isomiRs". Changes to a miRNA's seed sequence (nucleotides 2-8, including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6 cells (widely used as a surrogate for the study of pancreatic beta cells and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1 the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2 miRNA loci can generate multiple highly expressed isomiRs with different 5'-start positions (5'-isomiRs; (3 isomiRs with shifted start positions (5'-shifted isomiRs are highly expressed, and can be as abundant as their unshifted counterparts (5'-reference miRNAs. Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5'-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by in silico target prediction and in vitro transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5'-shifted isomiRs to T2D.

  20. Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

    Directory of Open Access Journals (Sweden)

    Valentina Rapisarda

    2017-03-01

    Full Text Available Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3 is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS, independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.

  1. Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

    Science.gov (United States)

    Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

    2015-09-02

    In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In

  2. CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner

    DEFF Research Database (Denmark)

    Huising, Mark O; van der Meulen, Talitha; Vaughan, Joan M

    2009-01-01

    Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that acti...

  3. Isoreserpine promotes {beta}-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gwak, Jungsug; Song, Taeyun [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Jie-Young; Yun, Yeon-Sook [Laboratory of Radiation Cancer Science, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Choi, Il-Whan [Department of Microbiology, Center for Viral Disease Research, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Jeong, Yongsu [Department of Genetic Engineering, and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701 (Korea, Republic of); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan 614-735 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

    2009-09-25

    Aberrant accumulation of intracellular {beta}-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular {beta}-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular {beta}-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of {beta}-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

  4. SIRT6-mediated transcriptional suppression of Txnip is critical for pancreatic beta cell function and survival in mice.

    Science.gov (United States)

    Qin, Kunhua; Zhang, Ning; Zhang, Zhao; Nipper, Michael; Zhu, Zhenxin; Leighton, Jake; Xu, Kexin; Musi, Nicolas; Wang, Pei

    2018-04-01

    Better understanding of how genetic and epigenetic components control beta cell differentiation and function is key to the discovery of novel therapeutic approaches to prevent beta cell dysfunction and failure in the progression of type 2 diabetes. Our goal was to elucidate the role of histone deacetylase sirtuin 6 (SIRT6) in beta cell development and homeostasis. Sirt6 endocrine progenitor cell conditional knockout and beta cell-specific knockout mice were generated using the Cre-loxP system. Mice were assayed for islet morphology, glucose tolerance, glucose-stimulated insulin secretion and susceptibility to streptozotocin. Transcriptional regulatory functions of SIRT6 in primary islets were evaluated by RNA-Seq analysis. Reverse transcription-quantitative (RT-q)PCR and immunoblot were used to verify and investigate the gene expression changes. Chromatin occupancies of SIRT6, H3K9Ac, H3K56Ac and active RNA polymerase II were evaluated by chromatin immunoprecipitation. Deletion of Sirt6 in pancreatic endocrine progenitor cells did not affect endocrine morphology, beta cell mass or insulin production but did result in glucose intolerance and defective glucose-stimulated insulin secretion in mice. Conditional deletion of Sirt6 in adult beta cells reproduced the insulin secretion defect. Loss of Sirt6 resulted in aberrant upregulation of thioredoxin-interacting protein (TXNIP) in beta cells. SIRT6 deficiency led to increased acetylation of histone H3 lysine residue at 9 (H3K9Ac), acetylation of histone H3 lysine residue at 56 (H3K56Ac) and active RNA polymerase II at the promoter region of Txnip. SIRT6-deficient beta cells exhibited a time-dependent increase in H3K9Ac, H3K56Ac and TXNIP levels. Finally, beta cell-specific SIRT6-deficient mice showed increased sensitivity to streptozotocin. Our results reveal that SIRT6 suppresses Txnip expression in beta cells via deacetylation of histone H3 and plays a critical role in maintaining beta cell function and viability

  5. Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

    Science.gov (United States)

    Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

    2017-04-01

    We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. The effect of training on responses of beta-endorphin and other pituitary hormones to insulin-induced hypoglycemia

    DEFF Research Database (Denmark)

    Mikines, K J; Kjær, Michael; Hagen, C

    1985-01-01

    in untrained (25 +/- 6 mU X l-1) subjects (P less than 0.05). Levels of thyrotropin (TSH) changed in neither of the groups. It is concluded that, in contrast to what has been formerly proposed, training does not result in a general increase in secretory capacity of the anterior pituitary gland. TSH responds......We studied whether the previously reported intensified beta-endorphin response to exercise after training might result from a training-induced general increase in anterior pituitary secretory capacity. Identical hypoglycemia was induced by insulin infusion in 7 untrained (VO2max 49 +/- 4 ml X (kg X...

  7. Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

    Science.gov (United States)

    Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

    2010-01-01

    The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

  8. COBRA-LIKE2, a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE family, plays a role in cellulose deposition in arabidopsis seed coat mucilage secretory cells.

    Science.gov (United States)

    Ben-Tov, Daniela; Abraham, Yael; Stav, Shira; Thompson, Kevin; Loraine, Ann; Elbaum, Rivka; de Souza, Amancio; Pauly, Markus; Kieber, Joseph J; Harpaz-Saad, Smadar

    2015-03-01

    Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. © 2015 American Society of Plant Biologists. All Rights Reserved.

  9. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2012-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  10. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2011-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  11. Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells

    DEFF Research Database (Denmark)

    Gromada, J; Dissing, S; Kofod, Hans

    1995-01-01

    The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open pro...

  12. PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration

    Directory of Open Access Journals (Sweden)

    Yaxi Zhu

    2017-11-01

    Full Text Available Abstract Transcription factors regulate gene expression through binding to specific enhancer sequences. Pancreas/duodenum homeobox protein 1 (PDX1, Neurogenin-3 (NEUROG3, and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA are transcription factors critical for beta cell development and maturation. NEUROG3 is expressed in endocrine progenitor cells and controls islet differentiation and regeneration. PDX1 is essential for the development of pancreatic exocrine and endocrine cells including beta cells. PDX1 also binds to the regulatory elements and increases insulin gene transcription. Likewise, MAFA binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. In addition to those natural roles in beta cell development and maturation, ectopic expression of PDX1, NEUROG3, and/or MAFA has been successfully used to reprogram various cell types into insulin-producing cells in vitro and in vivo, such as pancreatic exocrine cells, hepatocytes, and pluripotent stem cells. Here, we review biological properties of PDX1, NEUROG3, and MAFA, and their applications and limitations for beta cell regenerative approaches. The primary source literature for this review was acquired using a PubMed search for articles published between 1990 and 2017. Search terms include diabetes, insulin, trans-differentiation, stem cells, and regenerative medicine.

  13. Radiation-induced secretory protein, clusterin. Its inductive mechanism and biological significance

    International Nuclear Information System (INIS)

    Suzuki, Masatoshi; Boothman, D.A.

    2007-01-01

    This paper describes biochemistry of secretory clusterin (C), its radiation-inductive mechanism and biological significance. C is a glycoprotein found to be secreted from cells given various stresses like radiation and ultraviolet (UV)-ray, and participates to red cell clustering. Human C gene locates on the chromosome 8p21-p12, C has MW of 60 kDa, its precursor undergoes the degrading processing to α- and β-chains to form their heterodimer before glycosylation, and the C is finally secreted. So many other names have been given to C due to its numerous functions which have been discovered in other fields, such as apolipoprotein J. C is abundant in plasma, milk, urine, cerebrospinal fluid, semen, etc. Within 24 hr after X-ray irradiation, extracellular insulin-like growth factor-1 (IGF-1) level is elevated, and through its binding to the receptor, Src/MAPK signaling participates to C expression. Nuclear C, also induced by radiation, is a splicing variant of C and not secreted from cells. C is induced by radiation with as low dose as 2 cGy, which is different from induction of nuclear C. Secreted C is incorporated in cells by endocytosis and promotes the intracellular survival reaction through IGF-1 receptor/MAPK/Egr-1 pathway, whereas nuclear C induces cell apoptosis via unknown mechanism. Further studies are required for elucidation of the roles of secretory and nuclear C in cellular radiation responses. (R.T.)

  14. A post-translational balancing act: the good and the bad of SUMOylation in pancreatic islets.

    Science.gov (United States)

    MacDonald, Patrick E

    2018-04-01

    Post-translational modification of proteins contributes to the control of cell function and survival. The balance of these in insulin-producing pancreatic beta cells is important for the maintenance of glucose homeostasis. Protection from the damaging effects of reactive oxygen species is required for beta cell survival, but if this happens at the expense of insulin secretory function then the ability of islets to respond to changing metabolic conditions may be compromised. In this issue of Diabetologia, He et al ( https://doi.org/10.1007/s00125-017-4523-9 ) show that post-translational attachment of small ubiquitin-like modifier (SUMO) to target lysine residues (SUMOylation) strikes an important balance between the protection of beta cells from oxidative stress and the maintenance of insulin secretory function. They show that SUMOylation is required to stabilise nuclear factor erythroid 2-related factor 2 (NRF2) and increase antioxidant gene expression. Decreasing SUMOylation in beta cells impairs their antioxidant capacity, causes cell death, hyperglycaemia, and increased sensitivity to streptozotocin-induced diabetes, while increasing SUMOylation is protective. However, this protection from overt diabetes occurs in concert with glucose intolerance due to impaired beta cell function. A possible role for SUMOylation as a key factor balancing beta cell protection vs beta cell responsiveness to metabolic cues is discussed in this Commentary.

  15. Insulin-like growth factors and pancreas beta cells.

    NARCIS (Netherlands)

    Haeften, T.W. van; Twickler, M.

    2004-01-01

    Abstract Insulin-like growth factors (IGFs) have been implicated in normal growth, and especially foetal pancreas beta-cell development. As low birth weight has been implicated in the development of obesity and type 2 diabetes, much research has evolved into the importance of IGF and their

  16. Insulin-like growth factors and pancreas beta cells

    NARCIS (Netherlands)

    van Haeften, T. W.; Twickler, TB

    Insulin-like growth factors (IGFs) have been implicated in normal growth, and especially foetal pancreas beta-cell development. As low birth weight has been implicated in the development of obesity and type 2 diabetes, much research has evolved into the importance of IGF and their signalling

  17. Insulin-like growth factors and pancreas beta cells

    NARCIS (Netherlands)

    van Haeften, T. W.; Twickler, Th B.

    2004-01-01

    Abstract Insulin-like growth factors (IGFs) have been implicated in normal growth, and especially foetal pancreas beta-cell development. As low birth weight has been implicated in the development of obesity and type 2 diabetes, much research has evolved into the importance of IGF and their

  18. Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines

    International Nuclear Information System (INIS)

    Plummer, Howard K III; Yu, Qiang; Cakir, Yavuz; Schuller, Hildegard M

    2004-01-01

    Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta 2 -adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K + flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer

  19. LGR5 and Nanog identify stem cell signature of pancreas beta cells which initiate pancreatic cancer.

    Science.gov (United States)

    Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Polin, Nava; Givol, David

    2013-04-05

    Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Expression of the alpha 6 beta 4 integrin by squamous cell carcinomas and basal cell carcinomas: possible relation to invasive potential?

    DEFF Research Database (Denmark)

    Rossen, K; Dahlstrøm, K K; Mercurio, A M

    1994-01-01

    We have studied the expression of alpha 6 beta 4 integrin, a carcinoma laminin receptor in ten squamous cell carcinomas (SCCs) and ten basal cell carcinomas (BCCs) of the skin in order to examine whether changes in alpha 6 beta 4 integrin expression may be related to invasive and metastatic...... potential. Monoclonal antibodies specific for each subunit were applied on cryosections, using a three step indirect peroxidase technique. In normal epidermis the basal cells expressed both the alpha 6 and the beta 4 subunits, and the expression was polarized against the basement membrane. In SCCs...

  1. Effects of transforming growth factor-beta on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5

    International Nuclear Information System (INIS)

    Morris, J.C. III; Ranganathan, G.; Hay, I.D.; Nelson, R.E.; Jiang, N.S.

    1988-01-01

    Transforming growth factor-beta (TGF beta) has been shown to influence the growth and differentiation of many widely varied cell types in vitro, including some that are endocrinologically active. We have investigated the previously unknown effects of this unique growth factor in the differentiated rat thyroid follicular cell line FRTL-5. The cells demonstrated specific, high affinity binding of TGF beta, and as with other epithelial cells, the growth of these thyroid follicular cells was potently inhibited by addition of TGF beta to the culture medium. TGF beta caused a significant reduction in TSH-sensitive adenylate cyclase activity in the cells. The addition of (Bu)2cAMP along with the growth factor to cultures partially reversed the characteristic morphological changes seen with TGF beta, but did not reverse the growth inhibition. To further investigate the possible mechanisms of the effects of TGF beta on the cells, we measured the influence of the growth factor on [125I]TSH binding. TGF beta did not compete for specific TSH-binding sites; however, exposure of the cells to TGF beta for 12 or more h resulted in a dose-dependent down-regulation of TSH receptors that was fully reversible. While cellular proliferation was potently inhibited by TGF beta, differentiated function, as manifest by iodine-trapping ability, was stimulated by the growth factor. This stimulation of iodine uptake was independent of, and additive to, the stimulatory effects of TSH. Finally, FRTL-5 cells in serum-free medium and in response to TSH were shown to secrete TGF beta-like activity that competed for [125I]TGF beta in a RRA. These studies suggest that TGF beta may represent an autocrine mechanism of controlling the growth response to TSH in thyroid follicular cells, while allowing the continuance of differentiated function

  2. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

    Directory of Open Access Journals (Sweden)

    Mohamed I Husseiny

    Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

  3. CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice.

    Science.gov (United States)

    Jiao, Yang; Rieck, Sebastian; Le Lay, John; Kaestner, Klaus H

    2013-11-01

    Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice. We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing Cish (loxP/loxP) mice with Pdx1-Cre (Early) mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR. There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice. Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.

  4. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    DEFF Research Database (Denmark)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

    2016-01-01

    developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

  5. Pathway decision-making strategies for generating pancreatic beta-cells: systems biology or hit and miss?

    Science.gov (United States)

    Jensen, Jan

    2007-08-01

    Method selection strategies to achieve beta-cell differentiation from human embryonic stem cells are reviewed. Expectations are high for an embryonic stem cell derived cellular replacement method to cure diabetes, and recent observations indicate that mature beta-cells can be derived from human embryonic stem cells. In terms of the translational setting, however, we are not there yet; conversion of embryonic stem cells to fully mature beta-cells is not effective and does not provide an exclusive beta-cell population as the end-product. Studies emphasize that expecting development of a 'magic media bullet' that promotes endocrine fate is rather optimistic; consecutive administration of signaling inducers, carefully provided to mimic normal development, is more likely to succeed. To accomplish this task we need better understanding of the extracellular signaling pathways that drive progressive endodermal fate choices throughout development. Knowledge of cell-intrinsic control of fate assignments in pancreas is growing rapidly. Nevertheless, insufficient information regarding morphogen codes that operate in endoderm and pancreas is hindering development of better, directed differentiation schema of uncommitted embryonic stem cells toward endodermal, pancreatic, and subsequent endocrine specific fates. A systematic approach to defining morphogen codes in developing endoderm and pancreas appears timely and justified.

  6. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  7. Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α.

    Science.gov (United States)

    Ocaña, Gail J; Pérez, Liliana; Guindon, Lynette; Deffit, Sarah N; Evans-Molina, Carmella; Thurmond, Debbie C; Blum, Janice S

    2017-06-01

    A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity. © 2017 John Wiley & Sons Ltd.

  8. Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism.

    Science.gov (United States)

    During, Alexandrine; Doraiswamy, Sundari; Harrison, Earl H

    2008-08-01

    The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.

  9. Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/{beta}-catenin signaling pathway in HCT116 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); Kim, Chul Young [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); College of Pharmacy, Hanyang University, Ansan 426-791 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of)

    2012-11-16

    Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

  10. Mammary Analog Secretory Carcinoma of the Nasal Cavity

    DEFF Research Database (Denmark)

    Baneckova, Martina; Agaimy, Abbas; Andreasen, Simon

    2018-01-01

    Secretory carcinoma, originally described as mammary analog secretory carcinoma (MASC), is a low-grade salivary gland tumor characterized by a t(12;15)(p13;q25) translocation, resulting in an ETV6-NTRK3 gene fusion. Most MASCs are localized to the parotid gland and intraoral minor salivary glands...

  11. Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

    International Nuclear Information System (INIS)

    Liotta, A.S.; Yamaguchi, H.; Krieger, D.T.

    1981-01-01

    Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate

  12. Determination of Insulin Resistance and Beta Cell Function in Healthy Obese and Non-obese Individuals

    International Nuclear Information System (INIS)

    Kazmi, A.; Sattar, A.; Tariq, K. M.; Najamussahar; Hashim, R.; Almani, M. I.

    2013-01-01

    Objective: To determine insulin resistance and beta cell function in healthy obese and nonobese individuals of the local population. Study Design: Case control study. Place and Duration of Study: AFIP Rawalpindi in collaboration with department of medicine military hospital(MH) Rawalpindi, from Aug 2008 to Mar 2009. Methods: Eighty obese(n=40) and non-obese(n=40) subjects were selected by non-probability convenience sampling. Plasma insulin, glucose, and serum total cholestrol were estimated in fasting state. Insulin resistance was calculated by HOMA-IR and beta cell function by HOMA- equation. Results: Significant differences were observed between obese and non-obese individuals regarding insulin resistance, beta cell function, and BMI and serum total cholesterol. Mean insulin resistance in obese group was found to be 11.1 +- 5.1(range 7.0-16.2) and in non-obese group it was 0.9+-0.4 (range 0.5-1.3). This difference was highly significant (p=0.001). There was a highly significant difference between the two groups in term of beta cell function with mean rank 60.1 for obese group and 20.9 non obese groups (Asym sig. 2 tailed 0.000). Also the correlation (r = 0.064) between insulin resistance and beta cell function in obese group is highly significant (p = 0.000). Mean serum leptin levels were lower (6.3 ng/ml) in non-obese, and high (57.2 ng/ml) in the obese group. Conclusions: Insulin resistance is found higher in obese individuals. Beta cell function is significantly different between obese and non-obese groups. (author)

  13. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  14. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  15. THE EFFECT OF CURCUMIN ON SECRETORY ACTIVITY, PROLIFERATION AND APOPTOSIS OF THE PORCINE OVARIAN GRANULOSA CELLS

    Directory of Open Access Journals (Sweden)

    Attila Kádasi

    2012-08-01

    Full Text Available The aim of this in vitro study was to examine the effect of natural plant (Curcuma longa molecule curcumin on secretory activity, proliferation and apoptosis of porcine granulosa cells. The secretion of steroid hormones (progesterone, testosterone, accumulation of PCNA (marker of proliferation and bax (marker of apoptosis in granulosa cells of swine ovaries after curcumin treatment at the doses 0, 1, 10, 100 μg.mL-1 was determined by RIA and immunocytochemistry. It was observed that, addition of curcumin stimulated progesterone (at doses 1 and 10 μg.mL-1, but not 100 μg.mL-1 and testosterone at (100 μg.mL-1 but not 1 and 10 μg.mL-1 release. The number of cells contained PCNA was down-regulated by curcumin administration (at dose of 10 μg.mL-1, but not of 1 and 100 μg.mL-1. Bax expression was stimulated by curcumin at all doses added. Our results suggest a direct effect of curcumin on ovarian functions: steroidogenesis, proliferation and apoptosis. This could suggest antireproductive properties of curcumin in swine ovaries.

  16. Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells.

    Science.gov (United States)

    Bahk, Young Yil; Pak, Jhang Ho

    2016-10-01

    Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

  17. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    International Nuclear Information System (INIS)

    Li, Jason Z.; Ke, Yuebin; Misra, Hara P.; Trush, Michael A.; Li, Y. Robert; Zhu, Hong; Jia, Zhenquan

    2014-01-01

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  18. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jason Z. [Virginia Tech CRC, Blacksburg, VA (United States); Ke, Yuebin [Shenzhen Center for Disease Control and Prevention, Shenzhen 518055 (China); Misra, Hara P. [Virginia Tech CRC, Blacksburg, VA (United States); Trush, Michael A. [Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD (United States); Li, Y. Robert [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Virginia Tech-Wake Forest University SBES, Blacksburg, VA (United States); Department of Biology, University of North Carolina at Greensboro, NC (United States); Zhu, Hong, E-mail: zhu@campbell.edu [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Jia, Zhenquan, E-mail: z_jia@uncg.edu [Department of Biology, University of North Carolina at Greensboro, NC (United States)

    2014-12-15

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  19. Involvement of interleukin 1 and interleukin 1 antagonist in pancreatic beta-cell destruction in insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Zumsteg, U; Reimers, J

    1993-01-01

    In this review we propose that the balance between the action of interleukin 1 (IL-1) and its natural antagonist IL-1ra on the level of the insulin-producing pancreatic beta-cell may play a decisive role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We argue that IL-1...... potentiated by other cytokines (tumor necrosis factor alpha, interferon gamma) is an important effector molecule involved in both early and late events in the immune-mediated process that leads to beta-cell destruction and IDDM. We also point out that surprisingly high molar excesses of IL-1ra over IL-1...... are necessary to block the action of IL-1 on islet beta-cells compared to islet alpha-cells in vitro and in animals. We suggest that the selectivity of beta-cell destruction in IDDM may be conferred on several levels: (1) homing of beta-cell antigen specific T cells, (2) targeted delivery of cytokines...

  20. In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

    Directory of Open Access Journals (Sweden)

    Eizirik Decio L

    2007-02-01

    Full Text Available Abstract Background Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM. This is mediated in part by cytokines, such as interleukin (IL-1β and interferon (IFN-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF nuclear factor (NF-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. Results The performance of linear and quadratic discriminant analysis (LDA, QDA in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ and double stranded RNA (dsRNA, as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. Conclusion The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on

  1. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    International Nuclear Information System (INIS)

    Stevens, R.L.; Austen, K.F.; Fox, C.C.; Lichtenstein, L.M.

    1988-01-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35 S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [ 35 S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate [ 35 S]sulfate into separate heparin and chondroitin sulfate proteoglycans in an ∼2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [ 35 S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [ 35 S]sulfate E proteoglycans and the [ 35 S]heparin proteoglycans were exocytosed from the [ 35 S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35 S-labeled proteoglycans reside in the secretory granules of these human lung mast cells

  2. Ethacrynic acid exhibits selective toxicity to chronic lymphocytic leukemia cells by inhibition of the Wnt/beta-catenin pathway.

    Directory of Open Access Journals (Sweden)

    Desheng Lu

    Full Text Available BACKGROUND: Aberrant activation of Wnt/beta-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL cells, and that uncontrolled Wnt/beta-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL. METHODOLOGY/PRINCIPAL FINDINGS: The diuretic agent ethacrynic acid (EA was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/beta-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/beta-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/beta-catenin complex. N-acetyl-L-cysteine (NAC, which can react with the alpha, beta-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug's inhibition of Wnt/beta-catenin activation and its ability to induce apoptosis in CLL cells. CONCLUSIONS/SIGNIFICANCE: Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/beta-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.

  3. Effects of ultrasound on Transforming Growth Factor-beta genes in bone cells

    Directory of Open Access Journals (Sweden)

    J Harle

    2005-12-01

    Full Text Available Therapeutic ultrasound (US is a widely used form of biophysical stimulation that is increasingly applied to promote fracture healing. Transforming growth factor-beta (TGF-beta, which is encoded by three related but different genes, is known to play a major part in bone growth and repair. However, the effects of US on the expression of the TGF-beta genes and the physical acoustic mechanisms involved in initiating changes in gene expression in vitro, are not yet known. The present study demonstrates that US had a differential effect on these TGF-beta isoforms in a human osteoblast cell line, with the highest dose eliciting the most pronounced up-regulation of both TGF-beta1 and TGF-beta3 at 1 hour after treatment and thereafter declining. In contrast, US had no effect on TGF-beta2 expression. Fluid streaming rather than thermal effects or cavitation was found to be the most likely explanation for the gene responses observed in vitro.

  4. Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

    Science.gov (United States)

    Siese, A; Jaros, P P; Willig, A

    1999-02-01

    In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  6. Histone deacetylase 4 promotes TGF-beta1-induced synovium-derived stem cell chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy.

    Science.gov (United States)

    Pei, Ming; Chen, Demeng; Li, Jingting; Wei, Lei

    2009-12-01

    The transforming growth factor-beta (TGF-beta) superfamily members play diverse roles in cartilage development and maintenance. TGF-beta up-regulates chondrogenic gene expression by enhancing transcription factor SRY (sex determining region Y)-box 9 (Sox9) and inhibits osteoblast differentiation by repressing runt-related transcription factor 2 (Runx2). Recently, histone deacetylases (HDACs) were reported to act as negative regulators of chondrocyte hypertrophy. It was speculated that HDAC4 may promote TGF-beta1-induced MSC chondrogenesis. In this study, the adenovirus-mediated HDAC4 gene (Ad.HDAC4) was utilized to infect synovium-derived stem cells (SDSCs). Adenovirus-mediated LacZ (Ad.LacZ) served as a control. The infected cells were centrifuged to form SDSC pellets followed by incubation in a serum-free chondrogenic medium for 15 days with or without 10ng/mL TGF-beta1. Transfection efficiency was determined in SDSCs using Ad.LacZ. Cytotoxicity was measured using lactate dehydrogenase assay. Histology, immunostaining, biochemical analysis, and real-time polymerase chain reaction were performed to assess chondrogenesis at protein and mRNA levels in infected SDSCs. Our data demonstrated that supplementation with TGF-beta1 could initiate and promote SDSC chondrogenesis; however, TGF-beta1 alone was insufficient to fully differentiate SDSCs into chondrocytes. Ad.HDAC4 could be efficiently transfected into SDSCs. Without TGF-beta1 treatment, HDAC4 had no effect on SDSC chondrogenesis; however, in the presence of TGF-beta1, HDAC4 could speed up and maintain a high level of chondrogenesis while down-regulating the hypertrophic marker - type X collagen expression. This study is the first report showing that HDAC4 overexpression promotes TGF-beta1-induced SDSC chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy. The mechanism underlying this process needs further investigation.

  7. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    International Nuclear Information System (INIS)

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H.

    1990-01-01

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis

  8. Cyclodextrin-facilitated bioconversion of 17 beta-estradiol by a phenoloxidase from Mucuna pruriens cell cultures

    NARCIS (Netherlands)

    Woerdenbag, H.J.; Pras, N.; Frijlink, H.W.; Lerk, C.F.; Malingré, T.M.

    1990-01-01

    After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased

  9. Generation of Functional Beta-Like Cells from Human Exocrine Pancreas.

    Directory of Open Access Journals (Sweden)

    Maria J Lima

    Full Text Available Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

  10. Secretory products from thrombin-stimulated human platelets exert an inhibitory effect on NK-cytotoxic activity

    DEFF Research Database (Denmark)

    Skov Madsen, P; Hokland, P; Hokland, M

    1987-01-01

    We have investigated the interaction between human platelets and the NK-system, with special emphasis on the action of secretory products from platelets in an NK assay with 51Cr-labelled K562 as target cells. Supernatants from thrombin-stimulated platelets added to the NK assay consistently...... decreased the NK-cytotoxicity by 40% +/- 4.3%, indicating the existence of secreted products from platelets as a source of NK-inhibiting substances. In contrast, no direct cytotoxic effect of these secretory products on the target cells (K562) was seen. Thus, normal human platelets, when stimulated...... with thrombin, are capable of secreting different, yet undefined factors, which significantly inhibit NK activity in vitro. The results also suggest that the role of products from contaminating in vitro activated platelets should be borne in mind when performing conventional NK assays. Udgivelsesdato: 1986-Oct...

  11. The Secretory Response of Rat Peritoneal Mast Cells on Exposure to Mineral Fibers.

    Science.gov (United States)

    Borelli, Violetta; Trevisan, Elisa; Francesca, Vita; Zabucchi, Giuliano

    2018-01-10

    Exposure to mineral fibers is of substantial relevance to human health. A key event in exposure is the interaction with inflammatory cells and the subsequent generation of pro-inflammatory factors. Mast cells (MCs) have been shown to interact with titanium oxide (TiO₂) and asbestos fibers. In this study, we compared the response of rat peritoneal MCs challenged with the asbestos crocidolite and nanowires of TiO₂ to that induced by wollastonite employed as a control fiber. Rat peritoneal MCs (RPMCs), isolated from peritoneal lavage, were incubated in the presence of mineral fibers. The quantities of secreted enzymes were evaluated together with the activity of fiber-associated enzymes. The ultrastructural morphology of fiber-interacting RPMCs was analyzed with electron microscopy. Asbestos and TiO₂ stimulate MC secretion. Secreted enzymes bind to fibers and exhibit higher activity. TiO₂ and wollastonite bind and improve enzyme activity, but to a lesser degree than crocidolite. (1) Mineral fibers are able to stimulate the mast cell secretory process by both active (during membrane interaction) and/or passive (during membrane penetration) interaction; (2) fibers can be found to be associated with secreted enzymes-this process appears to create long-lasting pro-inflammatory environments and may represent the active contribution of MCs in maintaining the inflammatory process; (3) MCs and their enzymes should be considered as a therapeutic target in the pathogenesis of asbestos-induced lung inflammation; and (4) MCs can contribute to the inflammatory effect associated with selected engineered nanomaterials, such as TiO₂ nanoparticles.

  12. Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines

    DEFF Research Database (Denmark)

    Lundh, M; Christensen, D P; Rasmussen, D N

    2010-01-01

    Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim...

  13. Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

    International Nuclear Information System (INIS)

    Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya; Hirose, Takahisa; Kawamori, Ryuzo; Fujitani, Yoshio; Watada, Hirotaka

    2009-01-01

    Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

  14. Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

    Energy Technology Data Exchange (ETDEWEB)

    Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Hirose, Takahisa [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Kawamori, Ryuzo [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Center for Beta Cell Biology and Regeneration, Juntendo University School of Medicine, Tokyo (Japan); Sportology Center, Juntendo University School of Medicine, Tokyo (Japan); Fujitani, Yoshio, E-mail: fujitani@juntendo.ac.jp [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Watada, Hirotaka, E-mail: hwatada@juntendo.ac.jp [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Sportology Center, Juntendo University School of Medicine, Tokyo (Japan)

    2009-12-18

    Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

  15. The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H

    2000-01-01

    The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found......(2+)-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation. Udgivelsesdato: 2000-Jan-21...

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

    Science.gov (United States)

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

    2009-01-01

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

  17. Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

    Science.gov (United States)

    Fenske, Rachel J; Kimple, Michelle E

    2018-03-01

    Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

  18. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  19. Expression of beta-keratin mRNAs and proline uptake in epidermal cells of growing scales and pad lamellae of gecko lizards

    Science.gov (United States)

    Alibardi, Lorenzo; Toni, Mattia; Valle, Luisa Dalla

    2007-01-01

    Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine–proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine–proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life. PMID:17553098

  20. The time has come to test the beta cell preserving effects of exercise in patients with new onset type 1 diabetes

    DEFF Research Database (Denmark)

    Narendran, Parth; Solomon, Thomas; Kennedy, Amy

    2015-01-01

    Type 1 diabetes is characterised by immune-mediated destruction of insulin-producing beta cells. Significant beta cell function is usually present at the time of diagnosis with type 1 diabetes, and preservation of this function has important clinical benefits. The last 30 years have seen a number...... for physical exercise as a therapy for the preservation of beta cell function in patients with newly diagnosed type 1 diabetes. We highlight possible mechanisms by which exercise could preserve beta cell function and then present evidence from other models of diabetes that demonstrate that exercise preserves...... beta cell function. We conclude by proposing that there is now a need for studies to explore whether exercise can preserve beta cell in patients newly diagnosed with type 1 diabetes....

  1. Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

    Directory of Open Access Journals (Sweden)

    Moina Hasni Ebou

    Full Text Available Diabetes is a major complication of chronic Glucocorticoids (GCs treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1 and 2 (Tph2, leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

  2. Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance

    DEFF Research Database (Denmark)

    Campos, Lia S; Leone, Dino P; Relvas, Joao B

    2004-01-01

    , signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of beta1 integrin using cre/lox technology reduces the level of MAPK activity. We conclude that integrins are therefore an important part of the signalling mechanisms that control......The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression...... in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from...

  3. Analysis of Protein Localization and Secretory Pathway Function Using the Yeast "Saccharomyces Cerevisiae"

    Science.gov (United States)

    Vallen, Elizabeth

    2002-01-01

    The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a…

  4. Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

    Science.gov (United States)

    Hillman, Kristin L; Doze, Van A; Porter, James E

    2005-08-01

    Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists

  5. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

    International Nuclear Information System (INIS)

    Hicks, Sarah M.; Vassallo, Jeffrey D.; Dieter, Matthew Z.; Lewis, Cindy L.; Whiteley, Laurence O.; Fix, Andrew S.; Lehman-McKeeman, Lois D.

    2003-01-01

    Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression

  6. Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

    Science.gov (United States)

    Li, Dong-Sheng; Warnock, Garth L; Tu, Han-Jun; Ao, Ziliang; He, Zehua; Lu, Hong; Dai, Long-Jun

    2009-10-07

    Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

  7. Enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

    International Nuclear Information System (INIS)

    Castle, J.D.; Castle, A.M.; Ma, A.K.; Stukenbrok, H.

    1984-01-01

    Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24-25 degrees C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17-37 degrees C. Both radioactive linoleic and palmitic acids were incorporated selectively at the beta-position of the glycerol backbone of PC. PC labeling by [3H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced beta-acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A2 that favors PC as a substrate. Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [3H]palmitate-labeled PC. Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly

  8. Islet immunity and beta cell reserve of indigenous Black South Africans with ketoacidosis at initial diagnosis of diabetes.

    Science.gov (United States)

    Ekpebegh, Chukwuma; Longo-Mbenza, Benjamin; Blanco-Blanco, Ernesto

    2013-01-01

    Islet immunity and beta cell reserve status were utilized to classify persons with ketoacidosis as the initial manifestation of diabetes. The clinical features of the various diabetes classes were also characterized. Prospective cross sectional study. Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province, South Africa. Indigenous Black South Africans with ketoacidosis as the initial manifestation of diabetes. Islet immunity and beta cell reserve were respectively assessed using serum anti-glutamic acid decarboxylase 65 (GAD) antibody and serum C-peptide after 1 mg of intravenous glucagon. Serum anti-GAD 65 antibody > or = 5 units/L and or = 0.5 ng/mL and < 0.5 ng/mL, respectively. The proportions of patients with A+beta-, A+beta+, A-beta- and A-beta+ and their clinical characteristics were determined. Of the 38 males and 33 females who participated in the study, patients were categorized in various classes: A-beta+, 46.5% (n=33/ 71); A-beta-, 26.8% (n=19/71); A+beta-, 22.5% (n=16/71); and A+beta+, 4.2% (n=3/71). The ages of the various classes were: 41.8 +/- 13.8 years for A-beta+ (n=33); 36.5 +/- 14.6 years for A-beta- (n=19); and 20.6 +/- 7.1 years for the combination of A+beta- with A+beta+ (n=19) (P<.0001, P<.0001 for the combination of A+beta- and A+beta+ vs A-beta+, P=.001 for the combination of A+beta- and A+beta+ vs A-beta-and P=.2 for A-beta- vs A-beta+. The clinical features of type 2 diabetes were most prevalent in A-beta+ class while the A+beta- and A+beta+ groups had the clinical profile of type 1A diabetes. Most of the indigenous Black South African patients with ketoacidosis as the initial manifestation of diabetes had islet immunity, beta cell reserve status and clinical profiles of type 2 diabetes.

  9. Beta Blockers Suppress Dextrose-Induced Endoplasmic Reticulum Stress, Oxidative Stress, and Apoptosis in Human Coronary Artery Endothelial Cells.

    Science.gov (United States)

    Haas, Michael J; Kurban, William; Shah, Harshit; Onstead-Haas, Luisa; Mooradian, Arshag D

    Beta blockers are known to have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. To determine whether beta blockers can also prevent dextrose-induced endoplasmic reticulum (ER) stress in addition to their antioxidative effects, human coronary artery endothelial cells and hepatocyte-derived HepG2 cells were treated with 27.5 mM dextrose for 24 hours in the presence of carvedilol (a lipophilic beta blockers with alpha blocking activity), propranolol (a lipophilic nonselective beta blockers), and atenolol (a water-soluble selective beta blockers), and ER stress, oxidative, stress and cell death were measured. ER stress was measured using the placental alkaline phosphatase assay and Western blot analysis of glucose regulated protein 78, c-Jun-N-terminal kinase (JNK), phospho-JNK, eukaryotic initiating factor 2α (eIF2α), and phospho-eIF2α and measurement of X-box binding protein 1 (XBP1) mRNA splicing using reverse transcriptase-polymerase chain reaction. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA) chemiluminescence. Cell viability was measured by propidium iodide staining method. The ER stress, SO production, and cell death induced by 27.5 mM dextrose were inhibited by all 3 beta blockers tested. The antioxidative and ER stress reducing effects of beta blockers were also observed in HepG2 cells. The salutary effects of beta blockers on endothelial cells in reducing both ER stress and oxidative stress may contribute to the cardioprotective effects of these agents.

  10. The effect of suppressor of cytokine signaling 3 on GH signaling in beta-cells

    DEFF Research Database (Denmark)

    Rønn, Sif G; Hansen, Johnny A; Lindberg, Karen

    2002-01-01

    GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in beta-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway...... stable transfection of the beta-cell lines with plasmids expressing SOCS-3 under the control of an inducible promoter, a time- and dose-dependent expression of SOCS-3 in the cells was obtained. EMSA showed that SOCS-3 is able to inhibit GH-induced DNA binding of both STAT3 and STAT5 in RIN-5AH cells...

  11. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Bani-Yaghoub, Mahmud [Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Taylor, Rod [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Johnston, Linda J., E-mail: Linda.Johnston@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Pezacki, John Paul, E-mail: John.Pezacki@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada)

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  12. Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

    Science.gov (United States)

    Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

    1996-07-01

    Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

  13. Involvment of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPP-treated neurons.

    Directory of Open Access Journals (Sweden)

    Agnès Petit-Paitel

    Full Text Available Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD. 1-methyl-4-phenylpyridinium iodide (MPP(+, the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3beta (GSK-3beta, a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3beta in modulating MPP(+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP(+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3beta, evidenced by the increased level of the active form of the kinase, i.e. GSK-3beta phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3beta partially localized within mitochondria in both neuronal cell models. Moreover, MPP(+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3beta labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP(+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3beta activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP(+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3beta is a critical mediator of MPTP/MPP(+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3beta activity might provide protection against

  14. The zinc transporter ZNT3 co-localizes with insulin in INS-1E pancreatic beta cells and influences cell survival, insulin secretion capacity, and ZNT8 expression

    DEFF Research Database (Denmark)

    Smidt, Kamille; Larsen, Agnete; Brønden, Andreas

    2016-01-01

    Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage in the granu......Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage...

  15. TGF-{beta}1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-{kappa}B/IL-6/MMP-2

    Energy Technology Data Exchange (ETDEWEB)

    Binker, Marcelo G. [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); CBRHC Research Center, Buenos Aires (Argentina); Binker-Cosen, Andres A. [CBRHC Research Center, Buenos Aires (Argentina); Gaisano, Herbert Y. [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); Cosen, Rodica H. de [CBRHC Research Center, Buenos Aires (Argentina); Cosen-Binker, Laura I., E-mail: laura.cosen.binker@utoronto.ca [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); CBRHC Research Center, Buenos Aires (Argentina)

    2011-02-04

    Research highlights: {yields} Rac1 mediates TGF-{beta}1-induced SW1990 invasion through MMP-2 secretion and activation. {yields} NADPH-generated ROS act downstream of Rac1 in TGF-{beta}1-challenged SW1990 cells. {yields} TGF-{beta}1-stimulated ROS activate NF-{kappa}B in SW1990 cells. {yields} NF{kappa}B-induced IL-6 release is required for secretion and activation of MMP-2 in SW1990 cells. -- Abstract: Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-{beta}1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-{beta}1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-{beta}1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-{beta}1-stimulated invasion. Our results also indicate that signaling events involved in TGF-{beta}1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.

  16. Normal and abnormal secretion by haemopoietic cells

    Science.gov (United States)

    STINCHCOMBE, JANE C; GRIFFITHS, GILLIAN M

    2001-01-01

    The secretory lysosomes found in haemopoietic cells provide a very efficient mechanism for delivering the effector proteins of many immune cells in response to antigen recognition. Although secretion shows some similarities to the secretion of specialized granules in other secretory cell types, some aspects of secretory lysosome release appear to be unique to melanocytes and cells of the haemopoietic lineage. Mast cells and platelets have provided excellent models for studying secretion, but recent advances in characterizing the immunological synapse allow a very fine dissection of the secretory process in T lymphocytes. These studies show that secretory lysosomes are secreted from the centre of the talin ring at the synapse. Proper secretion requires a series of Rab and cytoskeletal elements which play critical roles in the specialized secretion of lysosomes in haemopoietic cells. PMID:11380687

  17. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Srisuttee, Ratakorn; Moon, Jeong [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Choi, Young-Whan [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Oh, Sangtaek [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1

  18. Vanadyl Sulfate Treatment Stimulates Proliferation and Regeneration of Beta Cells in Pancreatic Islets

    Directory of Open Access Journals (Sweden)

    Samira Missaoui

    2014-01-01

    Full Text Available We examined the effects of vanadium sulfate (VOSO4 treatment at 5 and 10 mg/kg for 30 days on endocrine pancreas activity and histology in nondiabetic and STZ-induced diabetic rats. In diabetic group, blood glucose levels significantly increased while insulinemia level markedly decreased. At the end of treatment, VOSO4 at a dose of 10 mg/Kg normalized blood glucose level in diabetic group, restored insulinemia, and significantly improved insulin sensitivity. VOSO4 also increased in a dose-dependent manner the number of insulin immunopositive beta cells in pancreatic islets of nondiabetic rats. Furthermore, in the STZ-diabetic group, the decrease in the number of insulin immunopositive beta cells was corrected to reach the control level mainly with the higher dose of vanadium. Therefore, VOSO4 treatment normalized plasma glucose and insulin levels and improved insulin sensitivity in STZ-experimental diabetes and induced beta cells proliferation and/or regeneration in normal or diabetic rats.

  19. Oxidative Metabolism Genes Are Not Responsive to Oxidative Stress in Rodent Beta Cell Lines

    Directory of Open Access Journals (Sweden)

    Faer Morrison

    2012-01-01

    Full Text Available Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6 mmol/L, ambient (11 mmol/L, and high (28 mmol/L glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS production was evident in INS-1 cells after 48 hours (P<0.05. TLDA analysis revealed a significant (P<0.05 upregulation of 16 of the 90 genes under hyperglycemic conditions, although these expression differences did not reflect differences in ROS. We conclude that although altered glycemia may influence the expression of some oxidative metabolism genes, this effect is probably not mediated by increased ROS production. The alterations to the expression of oxidative metabolism genes previously observed in human diabetic skeletal muscle do not appear to be mirrored in rodent pancreatic beta cells.

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

    Science.gov (United States)

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

    2006-11-17

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

  1. Lycopene and beta-carotene induce growth inhibition and proapoptotic effects on ACTH-secreting pituitary adenoma cells.

    Directory of Open Access Journals (Sweden)

    Natália F Haddad

    Full Text Available Pituitary adenomas comprise approximately 10-15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27(kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2 and increased expression of p27(kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27(kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing's disease.

  2. Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

    Science.gov (United States)

    Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

    2008-11-15

    Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

  3. Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

    Science.gov (United States)

    Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

    1998-11-01

    Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

  4. Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase

    Directory of Open Access Journals (Sweden)

    Okulicz William C

    2004-07-01

    Full Text Available Abstract Background In the endometrium the steroid hormone progesterone (P, acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators. Methods In this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21–23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays. Results Of ~12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21–23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin, histone 2A, polo-like kinase (PLK, spermidine/spermine acetyltransferase 2 (SAT2, secretory leukocyte protease inhibitor (SLPI and metallothionein 1G (MT1G, all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3, matrix metalloproteinase 11 (stromelysin 3, proenkephalin (PENK, cysteine/glycine-rich protein 2 (CSRP2, collagen type VII alpha 1 (COL7A1, secreted frizzled-related protein 4 (SFRP4, progesterone receptor membrane component 1 (PGRMC1, chemokine (C-X-C ligand 12 (CXCL12 and biglycan (BGN. In addition, many novel/unknown genes were also identified. Validation of array data

  5. [Factors causing damage and destruction of beta-cells of the islets of Langerhans in the pancreas].

    Science.gov (United States)

    Anděl, Michal; Němcová, Vlasta; Pavlíková, Nela; Urbanová, Jana; Cecháková, Marie; Havlová, Andrea; Straková, Radka; Večeřová, Livia; Mandys, Václav; Kovář, Jan; Heneberg, Petr; Trnka, Jan; Polák, Jan

    2014-09-01

    Insulin secretion in patients with manifested diabetes mellitus tends to disappear months to decades after the diagnosis, which is a clear sign of a gradual loss of pancreatic islet beta-cells. In our sample of 30 type 2 diabetic patients, whose disease manifested between 30 and 45 years of age, about a half have retained or even increased insulin secretion 30 years later, while the other half exhibit a much diminished or lost insulin secretion. Factors that can damage or destroy beta-cells can be divided into the following groups: Metabolic factors: hyperglycemia and glucotoxicity, lipotoxicity, hypoxia, reactive oxygen species; Pharmacological factors: antimicrobial medication pentamidine, SSRI antidepressants; Factors related to impaired insulin secretion: MODY type diabetes; Environmental toxic factors: rat poison Vacor, streptozotocin, polychlorinated and polybrominated hydrocarbons; Disorders of the exocrine pancreas: tumor infiltration, fibrous infiltration, chronic pancreatitis, cystic fibrosis; Infections, inflammation, autoimmunity, viral factors: Coxsackie viruses, H1N1 influenza, enteroviruses. We are currently working on finding other factors leading to beta-cell damage, studying their effect on apoptosis and necrosis and looking for possible protective factors to prevent this damage. We our increasing knowledge about the mechanisms of beta-cell damage and destruction we come ever closer to suggest measures for their prevention. In this review we offer a brief and simplified summary of some of the findings related to this area.Key words: pancreatic islet beta-cells of Langerhans - factors damaging or destroying beta-cells - insulin secretion.

  6. Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles

    Directory of Open Access Journals (Sweden)

    Ekkapongpisit M

    2012-04-01

    Full Text Available Maneerat Ekkapongpisit1,*, Antonino Giovia1,*, Giuseppina Nicotra1, Matteo Ozzano1, Giuseppe Caputo2,3, Ciro Isidoro1 1Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del Piemonte Orientale "A. Avogadro", Novara, Italy; 2Department of Chemistry, University of Turin, Turin, 3Cyanine Technology SpA, Torino, Italy *These authors contributed equally to this workBackground: For a safe ‘in vivo’ biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth.Aim and methods: In this work, mastocyte-like rat basophilic leukemia (RBL cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS nanoparticles (NPs and naked mesoporous silica (MPS 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging.Results: Our findings were that: (1 secretory granules of mastocytes are accessible by NPs via endocytosis; (2 PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3 PS NPs induce calcium-dependent exocytosis of inflammatory granules.Conclusion: These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.Keywords: secretory lysosomes, inflammation, nanoparticles, vesicular traffic

  7. Excretory/secretory products from Trichinella spiralis adult worms ameliorate DSS-induced colitis in mice.

    Directory of Open Access Journals (Sweden)

    Xiaodi Yang

    Full Text Available BACKGROUND: Many evidences show the inverse correlation between helminth infection and allergic or autoimmune diseases. Identification and characterization of the active helminth-derived products responsible for the beneficial effects on allergic or inflammatory diseases will provide another feasible approach to treat these diseases. METHODS AND FINDINGS: Colitis was induced in C57BL/6 mice by giving 3% DSS orally for 7 days. During this period, the mice were treated daily with the excretory/secretory products from T. spiralis adult worms (AES intraperitoneally. The severity of colitis was monitored by measuring body weight, stool consistency or bleeding, colon length and inflammation. To determine the T. spiralis AES product-induced immunological response, Th1, Th2, Th17 and regulatory cytokine profiles were measured in lymphocytes isolated from colon, mesenteric lymph nodes (MLN, and the spleen of treated mice. The CD4+ CD25+ FOXP3+ regulatory T cells (Tregs were also measured in the spleens and MLN of treated mice. Mice treated with AES significantly ameliorated the severity of the DSS-induced colitis indicated by the reduced disease manifestations, improved macroscopic and microscopic inflammation correlated with the up-regulation of Treg response (increased regulatory cytokines IL-10, TGF-beta and regulatory T cells and down-regulation of pro-inflammatory cytokines (IFN-gamma, IL-6 and IL-17 in the spleens, MLN and colon of treated mice. CONCLUSIONS: Our results provide direct evidences that T. spiralis AES have a therapeutic potential for alleviating inflammatory colitis in mice. This effect is possibly mediated by the immunomodulation of regulatory T cells to produce regulatory and anti-inflammatory cytokines and inhibit pro-inflammatory cytokines.

  8. High value of the radiobiological parameter Dq correlates to expression of the transforming growth factor beta type II receptor in a panel of small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Krarup, M; Nørgaard, P

    1998-01-01

    Our panel of SCLC cell lines have previously been examined for their radiobiological characteristics and sensitivity to treatment with TGF beta 1. In this study we examined the possible correlations between radiobiological parameters and the expression of the TGF beta type II receptor (TGF beta......-rII). We have, in other studies, shown that the presence of TGF beta-rII was mandatory for transmitting the growth inhibitory effect of TGF beta. The results showed a statistically significant difference in Dq, i.e. the shoulder width of the survival curve, between cell lines expressing TGF beta......-rII and cell lines which did not express the receptor (P = 0.01). Cell lines expressing TGF beta-rII had a high Dq-value. TGF beta-rII expression did not correlate with any other radiobiological parameters. We suggest that an intact growth inhibitory pathway mediated by the TGF beta-rII may have a significant...

  9. Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

    International Nuclear Information System (INIS)

    Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

    1989-01-01

    Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

  10. Impact of fetal and neonatal environment on beta cell function and development of diabetes

    DEFF Research Database (Denmark)

    Nielsen, Jens H; Haase, Tobias N; Jaksch, Caroline

    2014-01-01

    on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including...... that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy...... nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most...

  11. (-)[125I]-iodopindolol, a new highly selective radioiodinated beta-adrenergic receptor antagonist: measurement of beta-receptors on intact rat astrocytoma cells

    International Nuclear Information System (INIS)

    Barovsky, K.; Brooker, G.

    1980-01-01

    (-)-Pindolol, one of the most potent beta-adrenergic receptor antagonists, was radioiodinated using chloramine-T oxidation of carrier-free Na 125I and separated from unreacted pindolol to yield 2200 Ci/mmole (-)-[125I]-iodopindolol ((-)-[125I]-IPin). Mass and ultraviolet spectra confirmed that the iodination occurred on the indole ring, presumably at the 3 position. The binding of radiolabeled (-)-[125I]-IPin to beta-adrenergic receptors has been studied using intact C6 rat astrocytoma cells (2B subclone) grown in monolayer cultures. Binding of (-)[125IPin was saturable with time and concentration. Using 13 pM (-)-[125I]IPin, binding equilibrium was reached in 90 min at 21-22 degrees C. The reverse rate constant was 0.026 min-1 at 21 0 C. Specific binding (expressed as 1 microM(-)-propranolol displaceable counts) of (-)-[125I]-IPin was 95% of total binding. Scatchard analysis of (-)-[125I]-I]Pin binding revealed approximately 4300 receptors/cell and a dissociation constant of 30 pM. This was in excellent agreement with the kinetically determined dissociation constant of 35 pM. Displacement by propranolol and isoproterenol showed that (-)-[125I]-IPin binding sites were pharmacologically and stereospecifically selective. These results indicate that (-)-[125I]-IPin, a pure (-)-stereoisomer, high specific activity radioligand, selectively binds to beta-adrenergic receptors in whole cells with a high percentage of specific binding and should therefore be useful in the study and measurement of cellular beta-adrenergic receptors

  12. Calculation of absorbed dose of anchorage-dependent cells from internal beta-rays irradiation

    International Nuclear Information System (INIS)

    Chen Jianwei; Huang Gang; Li Shijun

    2001-01-01

    Objective: To elicit the formula of internal dosimetry in anchorage-dependent cells by beta-emitting radionuclides from uniformly distributed volume sources. Methods: By means of the definition of absorbed dose and the MIRD (Medical International Radiation Dose) scheme the formula of internal dosimetry was reasonably deduced. Firstly, studying the systems of suspension culture cells. Then, taking account of the speciality of the systems of the anchorage-dependent cells and the directions of irradiation, the absorbed dose of anchorage -dependent cells was calculated by the accumulated radioactivity, beta-ray energy, and the volume of the cultured systems. Results: The formula of internal dosimetry of suspension culture cells and anchorage-dependent cells were achieved. At the same time, the formula of internal dosimetry of suspension culture cells was compared with that of MIRD and was confirmed accurate. Conclusion: The formula of internal dosimetry is concise, reliable and accurate

  13. Generic sorting of raft lipids into secretory vesicles in yeast

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W

    2011-01-01

    Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma...... a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...

  14. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  15. The evolution of plant secretory structures and emergence of terpenoid chemical diversity.

    Science.gov (United States)

    Lange, Bernd Markus

    2015-01-01

    Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages.

  16. Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells.

    Science.gov (United States)

    Belhadj, Sahla; Hentati, Olfa; Hamdaoui, Ghaith; Fakhreddine, Khaskhoussi; Maillard, Elisa; Dal, Stéphanie; Sigrist, Séverine

    2018-03-20

    Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS) levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (-48%, p jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

  17. Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.

    Directory of Open Access Journals (Sweden)

    Juan Rodríguez-Vita

    Full Text Available BACKGROUND: The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-beta (TGF-beta in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-beta/Smad pathway in atherosclerosis and vascular cells. METHODOLOGY: In cultured vascular smooth muscle cells (VSMCs statins enhanced Smad pathway activation caused by TGF-beta. In addition, statins upregulated TGF-beta receptor type II (TRII, and increased TGF-beta synthesis and TGF-beta/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-beta induced apoptosis and increased TGF-beta-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-beta/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected. CONCLUSIONS: Statins enhance TGF-beta/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-beta/Smad pathway is essential for statins-dependent actions in VSMCs.

  18. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yong, E-mail: yongzhao@uic.edu [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Zhang, Yongkang [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Jain, Sumit [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Skidgel, Randal A. [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Prabhakar, Bellur S. [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Mazzone, Theodore [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Holterman, Mark J. [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  19. Overexpression of thyroid hormone beta1 nuclear receptor is associated with an increased proliferation of human hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, K; Lin, Y; McPhie, P [Chang-Gung College of Medicine and Technology, Graduate Institute of Clinical Medicine, Taoyuan (Taiwan, Province of China); Cheng, S [National Cancer Inst., Bethesda, MD (United States)

    1994-12-31

    It is evaluated the expression of thyroid hormone nuclear receptors (TRs) and their possible roles in the carcinogenesis of human hepatocarcinoma. The expression of TR{beta}1 and TR{alpha} genes was evaluated at both the mRNA and protein levels. The expression of TR{beta}1 and TR{alpha}1 mRNAs is similar to those found in normal liver. However, the expression of TR isoform proteins depends on the cell-type. The expression of TRaplha1 protein is low in all cell lines examined. However, TR{Beta}1 protein is overexpressed in Mahlavu, SK-Hep-1, and HA22T, moderately expressed in J5, J7, and J328 and is very low HepG2, Hep3B, and PLC/PRF/5 cells. The proliferation of cells in which TR{beta}1 is overexpressed is stimulated by the thyroid hormone, 3,3`,5- triiodo-L-thyronine. These results suggest that TR{beta}1, not TR{alpha}1, is probably involved in the prolifaration of hepatoma cells.

  20. COBRA-LIKE2, a Member of the Glycosylphosphatidylinositol-Anchored COBRA-LIKE Family, Plays a Role in Cellulose Deposition in Arabidopsis Seed Coat Mucilage Secretory Cells1,2[OPEN

    Science.gov (United States)

    Ben-Tov, Daniela; Abraham, Yael; Stav, Shira; Thompson, Kevin; Loraine, Ann; Elbaum, Rivka; de Souza, Amancio; Pauly, Markus; Kieber, Joseph J.; Harpaz-Saad, Smadar

    2015-01-01

    Differentiation of the maternally derived seed coat epidermal cells into mucilage secretory cells is a common adaptation in angiosperms. Recent studies identified cellulose as an important component of seed mucilage in various species. Cellulose is deposited as a set of rays that radiate from the seed upon mucilage extrusion, serving to anchor the pectic component of seed mucilage to the seed surface. Using transcriptome data encompassing the course of seed development, we identified COBRA-LIKE2 (COBL2), a member of the glycosylphosphatidylinositol-anchored COBRA-LIKE gene family in Arabidopsis (Arabidopsis thaliana), as coexpressed with other genes involved in cellulose deposition in mucilage secretory cells. Disruption of the COBL2 gene results in substantial reduction in the rays of cellulose present in seed mucilage, along with an increased solubility of the pectic component of the mucilage. Light birefringence demonstrates a substantial decrease in crystalline cellulose deposition into the cellulosic rays of the cobl2 mutants. Moreover, crystalline cellulose deposition into the radial cell walls and the columella appears substantially compromised, as demonstrated by scanning electron microscopy and in situ quantification of light birefringence. Overall, the cobl2 mutants display about 40% reduction in whole-seed crystalline cellulose content compared with the wild type. These data establish that COBL2 plays a role in the deposition of crystalline cellulose into various secondary cell wall structures during seed coat epidermal cell differentiation. PMID:25583925

  1. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Laura Bordone

    2006-02-01

    Full Text Available Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.

  2. MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jian-Yong [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi' an (China); Huang, Yi [Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, 710032 Xi' an (China); Li, Ji-Peng [State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi' an (China); Zhang, Xiang; Wang, Lei [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Meng, Yan-Ling [Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Yan, Bo [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Bian, Yong-Qian [State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi' an (China); Zhao, Jing [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Wang, Wei-Zhong, E-mail: weichang@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi' an (China); and others

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colon cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.

  3. MicroRNAs as regulators of beta-cell function and dysfunction

    DEFF Research Database (Denmark)

    Osmai, Mirwais; Osmai, Yama; Bang-Berthelsen, Claus Heiner

    2016-01-01

    , recent studies have demonstrated that miRNAs are important regulators of the islet transcriptome, controlling apoptosis, differentiation and proliferation, as well as regulating unique islet and beta-cell functions and pathways such as insulin expression, processing and secretion. Furthermore, a large...

  4. IL-1beta-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells

    DEFF Research Database (Denmark)

    Jacobsen, M L B; Rønn, S G; Bruun, C

    2008-01-01

    AIMS/HYPOTHESIS: Chemokines recruit activated immune cells to sites of inflammation and are important mediators of insulitis. Activation of the pro-apoptotic receptor Fas leads to apoptosis-mediated death of the Fas-expressing cell. The pro-inflammatory cytokines IL-1beta and IFN-gamma regulate...... the transcription of genes encoding the Fas receptor and several chemokines. We have previously shown that suppressor of cytokine signalling (SOCS)-3 inhibits IL-1beta- and IFN-gamma-induced nitric oxide production in a beta cell line. The aim of this study was to investigate whether SOCS-3 can influence cytokine......-induced Fas and chemokine expression in beta cells. METHODS: Using a beta cell line with inducible Socs3 expression or primary neonatal rat islet cells transduced with a Socs3-encoding adenovirus, we employed real-time RT-PCR analysis to investigate whether SOCS-3 affects cytokine-induced chemokine and Fas m...

  5. Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

  6. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miclea, Razvan L., E-mail: R.L.Miclea@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Horst, Geertje van der, E-mail: G.van_der_Horst@lumc.nl [Department of Urology, LUMC, Leiden (Netherlands); Robanus-Maandag, Els C., E-mail: E.C.Robanus@lumc.nl [Department of Human Genetics, LUMC, Leiden (Netherlands); Loewik, Clemens W.G.M., E-mail: C.W.G.M.Lowik@lumc.nl [Department of Endocrinology and Metabolic Diseases, LUMC, Leiden (Netherlands); Oostdijk, Wilma, E-mail: W.Oostdijk@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Wit, Jan M., E-mail: J.M.Wit@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Karperien, Marcel, E-mail: H.B.J.Karperien@tnw.utwente.nl [MIRA Institute for Biomedical Technology and Technical Medicine, Department of Tissue Regeneration, University of Twente, Zuidhorst Room ZH 144, Drienerlolaan 5, 7522 NB Enschede (Netherlands)

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  7. Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

    Science.gov (United States)

    Ford, Stephen P; Zhang, Liren; Zhu, Meijun; Miller, Myrna M; Smith, Derek T; Hess, Bret W; Moss, Gary E; Nathanielsz, Peter W; Nijland, Mark J

    2009-09-01

    Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

  8. Repetitive in vivo treatment with human recombinant interleukin-1 beta modifies beta-cell function in normal rats

    DEFF Research Database (Denmark)

    Wogensen, L D; Reimers, J; Nerup, J

    1992-01-01

    It is unknown whether interleukin-1 exerts a bimodal effect on Beta-cell function in vivo, and whether interleukin-1 has a diabetogenic action in normal animals. We therefore studied: (a) acute effects 2 h after an intraperitoneal bolus injection of 4 micrograms of recombinant human interleukin-1...

  9. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cleasby, Mark E. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Millard, Susan; Leong, Gary M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cooney, Gregory J. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Muscat, George E.O., E-mail: g.muscat@imb.uq.edu.au [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia)

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  10. Movement of beta-irradiated epidermal basal cells to the spinous-granular layers in the absence of cell division

    International Nuclear Information System (INIS)

    Etoh, H.; Taguchi, Y.H.; Tabachnick, J.

    1975-01-01

    Guinea-pig epidermis was irradiated with 3000 rad of beta rays 1 hr after two injections of [ 3 H]thymidine 5 hr apart (labeled cells in S phase and G 2 phase) or 18 hr after injection (labeled early G 1 cells). In nonirradiated epidermis labeled basal cells divided within 24 hr with daughter cells remaining in the basal layer, and approximately 50 percent of the labeled cells moved into the spinal layer by the 3rd day. Cell division in nonirradiated epidermis diluted the number of silver grains/nucleus, and lightly labeled cells were found in the granular layer by day 7. Beta irradiation inhibited cell division but it did not slow the rate of transit (ca 8 days) of irradiated labeled cells from basal to granular layer, some of these remaining heavily labeled. Although cell division may play some role in upward movement of basal cells in normal epidermis detachment of a basal cell from the basement membrane and its transit to the granular layer is unimpaired in the absence of cell division. These findings suggest that some radioresistant metabolic function(s), not cell division, is responsible for upward movement of basal cells. (auth)

  11. Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

    Directory of Open Access Journals (Sweden)

    Kusmiyati Tjahjono DK

    2013-04-01

    Full Text Available Background The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. Methods A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. Results After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p=0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. Conclusion TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells.

  12. Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

    Directory of Open Access Journals (Sweden)

    Kusmiyati Tjahjono DK

    2015-12-01

    Full Text Available BACKGROUND The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. METHODS A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. RESULTS After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p= 0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. CONCLUSION TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells

  13. Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

    DEFF Research Database (Denmark)

    Breau, Marie A; Pietri, Thomas; Eder, Olivier

    2006-01-01

    The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural...

  14. Patients with neuroglycopenia after gastric bypass surgery have exaggerated incretin and insulin secretory responses to a mixed meal.

    Science.gov (United States)

    Goldfine, A B; Mun, E C; Devine, E; Bernier, R; Baz-Hecht, M; Jones, D B; Schneider, B E; Holst, J J; Patti, M E

    2007-12-01

    Hyperinsulinemic hypoglycemia is newly recognized as a rare but important complication after Roux-en-Y gastric bypass (GB). The etiology of the syndrome and metabolic characteristics remain incompletely understood. Recent studies suggest that levels of incretin hormones are increased after GB and may promote excessive beta-cell function and/or growth. We performed a cross-sectional analysis of metabolic variables, in both the fasting state and after a liquid mixed-meal challenge, in four subject groups: 1) with clinically significant hypoglycemia [neuroglycopenia (NG)] after GB surgery, 2) with no symptoms of hypoglycemia at similar duration after GB surgery, 3) without GB similar to preoperative body mass index of the surgical cohorts, and 4) without GB similar to current body mass index of the surgical cohorts. Insulin and C-peptide after the liquid mixed meal were both higher relative to the glucose level achieved in persons after GB with NG compared with asymptomatic individuals. Glucagon, glucagon-like peptide 1, and glucose-dependent insulinotropic peptide levels were higher in both post-GB surgical groups compared with both overweight and morbidly obese persons, and glucagon-like peptide 1 was markedly higher in the group with NG. Insulin resistance, assessed by homeostasis model assessment of insulin resistance, the composite insulin sensitivity index, or adiponectin, was similar in both post-GB groups. Dumping score was also higher in both GB groups but did not discriminate between asymptomatic and symptomatic patients. Notably, the frequency of asymptomatic hypoglycemia after a liquid mixed meal was high in post-GB patients. A robust insulin secretory response was associated with postprandial hypoglycemia in patients after GB presenting with NG. Increased incretin levels may contribute to the increased insulin secretory response.

  15. Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

    Directory of Open Access Journals (Sweden)

    Lisa Cadavez

    Full Text Available In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP. The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR, perturbing endoplasmic reticulum (ER homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP or protein disulfite isomerase (PDI, and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA or 4-phenylbutyrate (PBA, alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

  16. Rac1 Regulates Endometrial Secretory Function to Control Placental Development

    Science.gov (United States)

    Davila, Juanmahel; Laws, Mary J.; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N.; Bagchi, Milan K.; Bagchi, Indrani C.

    2015-01-01

    During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

  17. Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

    Directory of Open Access Journals (Sweden)

    Juanmahel Davila

    2015-08-01

    Full Text Available During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions

  18. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    Energy Technology Data Exchange (ETDEWEB)

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  19. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different

  20. A two-hybrid assay to study protein interactions within the secretory pathway.

    Directory of Open Access Journals (Sweden)

    Danielle H Dube

    Full Text Available Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H, a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.

  1. Lycopene and Beta-Carotene Induce Growth Inhibition and Proapoptotic Effects on ACTH-Secreting Pituitary Adenoma Cells

    Science.gov (United States)

    Leite de Oliveira, Felipe; Soares, Nathália; de Mattos, Rômulo Medina; Hecht, Fábio; Dezonne, Rômulo Sperduto; Vairo, Leandro; Goldenberg, Regina Coeli dos Santos; Gomes, Flávia Carvalho Alcântara; de Carvalho, Denise Pires; Gadelha, Mônica R.; Nasciutti, Luiz Eurico; Miranda-Alves, Leandro

    2013-01-01

    Pituitary adenomas comprise approximately 10–15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing’s disease. PMID:23667519

  2. Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

    Science.gov (United States)

    Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

    1998-12-01

    Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

  3. beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor.

    Directory of Open Access Journals (Sweden)

    Udi Gluschnaider

    2010-02-01

    Full Text Available Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. beta-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.Here we show that beta-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against beta-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that beta-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining beta-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.

  4. Cytokines and beta-cell biology: from concept to clinical translation

    DEFF Research Database (Denmark)

    Donath, M.Y.; Storling, J.; Berchtold, L.A.

    2007-01-01

    The tale of cytokines and the beta-cell is a long story, starting with in vitro discovery in 1984, evolving via descriptive and phenomenological studies to detailed mapping of the signalling pathways, gene- and protein expression patterns, molecular and biochemical effector mechanisms to in vivo...

  5. Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

    Science.gov (United States)

    Rochereau, Nicolas; Pavot, Vincent; Verrier, Bernard; Jospin, Fabienne; Ensinas, Agathe; Genin, Christian; Corthésy, Blaise; Paul, Stéphane

    2016-01-01

    Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Ionizing Radiation Promotes Migration and Invasion of Cancer Cells Through Transforming Growth Factor-Beta-Mediated Epithelial-Mesenchymal Transition

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Yongchun [Department of Radiation Oncology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Liu Junye; Li Jing; Zhang Jie [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Xu Yuqiao [Department of Pathology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Zhang Huawei; Qiu Lianbo; Ding Guirong [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China); Su Xiaoming [Department of Radiation Oncology, 306th Hospital of PLA, Beijing (China); Mei Shi [Department of Radiation Oncology, Xijing Hospital Fourth Military Medical University, Xi' an (China); Guo Guozhen, E-mail: guozhenguo@hotmail.com [Department of Radiation Medicine, College of Preventive Medicine, Xijing Hospital Fourth Military Medical University, Xi' an (China)

    2011-12-01

    Purpose: To examine whether ionizing radiation enhances the migratory and invasive abilities of cancer cells through transforming growth factor (TGF-{beta})-mediated epithelial-mesenchymal transition (EMT). Methods and Materials: Six cancer cell lines originating from different human organs were irradiated by {sup 60}Co {gamma}-ray at a total dose of 2 Gy, and the changes associated with EMT, including morphology, EMT markers, migration and invasion, were observed by microscope, Western blot, immunofluorescence, scratch assay, and transwell chamber assay, respectively. Then the protein levels of TGF-{beta} in these cancer cells were detected by enzyme-linked immunosorbent assay, and the role of TGF-{beta} signaling pathway in the effect of ionizing radiation on EMT was investigate by using the specific inhibitor SB431542. Results: After irradiation with {gamma}-ray at a total dose of 2 Gy, cancer cells presented the mesenchymal phenotype, and compared with the sham-irradiation group the expression of epithelial markers was decreased and of mesenchymal markers was increased, the migratory and invasive capabilities were strengthened, and the protein levels of TGF-{beta} were enhanced. Furthermore, events associated with EMT induced by IR in A549 could be reversed through inhibition of TGF-{beta} signaling. Conclusions: These results suggest that EMT mediated by TGF-{beta} plays a critical role in IR-induced enhancing of migratory and invasive capabilities in cancer cells.

  7. Association of NEFA composition with insulin sensitivity and beta cell function in the Prospective Metabolism and Islet Cell Evaluation (PROMISE) cohort.

    Science.gov (United States)

    Johnston, Luke W; Harris, Stewart B; Retnakaran, Ravi; Giacca, Adria; Liu, Zhen; Bazinet, Richard P; Hanley, Anthony J

    2018-04-01

    Our aim was to determine the longitudinal associations of individual NEFA with the pathogenesis of diabetes, specifically with differences in insulin sensitivity and beta cell function over 6 years in a cohort of individuals who are at risk for diabetes. In the Prospective Metabolism and Islet Cell Evaluation (PROMISE) longitudinal cohort, 477 participants had serum NEFA measured at the baseline visit and completed an OGTT at three time points over 6 years. Outcome variables were calculated using the OGTT values. At each visit, insulin sensitivity was assessed using the HOMA2 of insulin sensitivity (HOMA2-%S) and the Matsuda index, while beta cell function was assessed using the insulinogenic index over HOMA-IR (IGI/IR) and the insulin secretion-sensitivity index-2 (ISSI-2). Generalised estimating equations were used, adjusting for time, waist, sex, ethnicity, baseline age, alanine aminotransferase (ALT) and physical activity. NEFA were analysed as both concentrations (nmol/ml) and proportions (mol%) of the total fraction. Participants' (73% female, 70% with European ancestry) insulin sensitivity and beta cell function declined by 14-21% over 6 years of follow-up. In unadjusted models, several NEFA (e.g. 18:1 n-7, 22:4 n-6) were associated with lower insulin sensitivity, however, nearly all of these associations were attenuated in fully adjusted models. In adjusted models, total NEFA, 16:0, 18:1 n-9 and 18:2 n-6 (as concentrations) were associated with 3.7-8.0% lower IGI/IR and ISSI-2, while only 20:5 n-3 (as mol%) was associated with 7.7% higher HOMA2-%S. Total NEFA concentration was a strong predictor of lower beta cell function over 6 years. Our results suggest that the association with beta cell function is due to the absolute size of the serum NEFA fraction, rather than the specific fatty acid composition.

  8. Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

    Energy Technology Data Exchange (ETDEWEB)

    Scherbakov, Alexander M., E-mail: alex.scherbakov@gmail.com [Laboratory of Clinical Biochemistry, Institute of Clinical Oncology, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation); Stefanova, Lidia B.; Sorokin, Danila V.; Semina, Svetlana E. [Laboratory of Molecular Endocrinology, Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation); Berstein, Lev M. [Laboratory of Oncoendocrinology, N.N. Petrov Research Institute of Oncology, St. Petersburg 197758 (Russian Federation); Krasil’nikov, Mikhail A. [Laboratory of Molecular Endocrinology, Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow 115478 (Russian Federation)

    2013-12-10

    The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O{sub 2} atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling. Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well

  9. Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Myoung Sook Kim

    2010-02-01

    Full Text Available Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH in a significant proportion of primary esophageal squamous cell carcinoma (ESCC samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

  10. Phenotypical and functional characterization of double-negative (CD4-CD8-) alpha beta T-cell receptor positive cells from an immunodeficient patient

    DEFF Research Database (Denmark)

    Illum, N; Ralfkiaer, E; Pallesen, G

    1991-01-01

    We have characterized CD4-CD8- double-negative (DN) alpha beta TCR+ T cells from a patient with immunodeficiency, lymphocytosis, lymphadenopathy, and hepatosplenomegaly. The majority of peripheral blood lymphocytes were DN alpha beta TCR+ T cells as evaluated by FACS and biochemical analysis...... (MoAbs) indicated a polyclonal T-cell expansion. Thymic biopsy showed normal histology, whereas lymph node biopsy samples showed altered histological and immunohistological patterns with markedly expanded paracortical areas containing the DN T cells of the same phenotype as found in peripheral blood T...

  11. Glucose-induced lipogenesis in pancreatic beta-cells is dependent on SREBP-1

    DEFF Research Database (Denmark)

    Sandberg, Maria B; Fridriksson, Jakob; Madsen, Lise

    2005-01-01

    High concentrations of glucose induce de novo fatty acid synthesis in pancreatic beta-cells and chronic exposure of elevated glucose and fatty acids synergize to induce accumulation of triglycerides, a phenomenon termed glucolipotoxicity. Here we investigate the role of sterol-regulatory element......, de novo fatty acid synthesis and lipid accumulation are induced primarily through sterol-regulatory elements (SREs) and not E-Boxes. Adenoviral expression of a dominant negative SREBP compromises glucose induction of some lipogenic genes and significantly reduces glucose-induction of de novo fatty...... acid synthesis. Thus, we demonstrate for the first time that SREBP activity is necessary for full glucose induction of de novo fatty acid synthesis in pancreatic beta-cells....

  12. The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

    Science.gov (United States)

    Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

  13. Characterization of beta-adrenergic receptors through the replicative life span of IMR-90 cells

    International Nuclear Information System (INIS)

    Scarpace, P.J.

    1987-01-01

    Beta-adrenergic receptor number and receptor affinity for isoproterenol were assessed at various in vitro ages of the human diploid fibroblast cell line IMR-90. From population doubling level (PDL) 33 to 44, there was a positive correlation between beta-adrenergic receptor density and PDL. Beta-adrenergic receptors, assessed by Scatchard analysis of [ 125 I]-iodocyanopindolol (ICYP) binding, increased from 15 fmol/mg protein at PDL 33 to 36 fmol/mg protein at PDL 44. In contrast, from PDL 44 to 59, there was a negative correlation between beta-adrenergic receptor density and PDL. Receptor density declined to 12 fmol/mg protein at PDL 59. When the density of beta-adrenergic receptors was expressed as receptor per cell, the findings were similar. Receptor agonist affinity for isoproterenol was determined from Hill plots of [ 125 I]-ICYP competition with isoproterenol. There was no change in the dissociation constant for isoproterenol with in vitro age. In humans, serum norepinephrine concentrations increase with age. This increase in serum norepinephrine may be partially responsible for the decreased beta-adrenergic receptor-agonist affinity observed with age in human lymphocytes and rat heart and lung. The present findings are consistent with the hypothesis that the decreases in receptor agonist affinity in rat and man with age are secondary to increases in catecholamine concentrations

  14. Lactogenic differentiation of HC11 cells is not accompanied by downregulation of AP-2 transcription factor genes

    Directory of Open Access Journals (Sweden)

    Schorle Hubert

    2008-06-01

    Full Text Available Abstract Background During pregnancy the mammary epithelium undergoes a complex developmental process which culminates in the generation of the milk-secreting epithelium. Secretory epithelial cells display lactogenic differentiation which is characterized by the expression of milk protein genes, such as beta-casein or whey acidic protein (WAP. Transcription factors AP-2alpha and AP-2gamma are downregulated during lactation, and their overexpression in transgenic mice impaired the secretory differentiation of the mammary epithelium, resulting in lactation failure. To explore whether the downregulation of AP-2alpha and AP-2gamma is of functional significance for lactogenic differentiation, we analyzed the expression of the AP-2 family members during the lactogenic differentiation of HC11 mammary epithelial cells in vitro. Differentiation of HC11 cells was induced following established protocols by applying the lactogenic hormones prolactin, dexamethasone and insulin. Findings HC11 cells express all AP-2 family members except AP-2delta. Using RT-PCR we could not detect a downregulation of any of these genes during the lactogenic differentiation of HC11 cells in vitro. This finding was confirmed for AP-2alpha and AP-2gamma using Northern analysis. Differentiating HC11 cells displayed lower expression levels of milk protein genes than mammary glands of mid-pregnant or lactating mice. Conclusion The extent of lactogenic differentiation of HC11 cells in vitro is limited compared to mammary epithelium undergoing secretory differentiation in vivo. Downregulation of AP-2 transcription factor genes is not required for lactogenic differentiation of HC11 cells but may functionally be involved in aspects of lactogenic differentiation in vivo that are not reflected by the HC11 system.

  15. Trichostatin A inhibits beta-casein expression in mammary epithelial cells

    International Nuclear Information System (INIS)

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2002-01-01

    Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types

  16. Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells

    OpenAIRE

    Arrio-Dupont, M.; Foucault, G.; Vacher, M.; Douhou, A.; Cribier, S.

    1997-01-01

    The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A frac...

  17. Clinical significance of the radioimmunological determination of C-peptide

    International Nuclear Information System (INIS)

    Dudczak, R.; Waldhaeusl, W.

    1979-01-01

    The aim of this study was to investigate the relationship of metabolic control to insulinogenic reserve in insulin-dependent diabetics. Thus, the secretory reserve of the pancreatic beta cell was estimated in insulin-dependent diabetics by measuring changes in peripheral serum immunoreactive C-peptide (IRCP) concentrations in response to intravenous arginine (n = 19; 0.5 g/kg,t = 30 min) or glibenclamide-glucose (n = 6; 2 mg HB 419 - 0.33 g/kg intravenously). In the majority of ''stable'' diabetics a small secretory reserve of the beta cell was demonstrated, but both the absolute and relative increase in IRCP was reduced after intravenous arginine or glibenclamide-glucose in comparison with normal controls. In ''unstable'' diabetics a decreased basal concentration of IRCP, significantly smaller than that seen in ''stable'' diabetics (p < 0.01), was accompanied by a complete lack of IRCP release on intravenous arginine administration. Thus, we conclude that the radioimmunological determination of IRCP is of clinical interest in assessing the residual secretory capacity of the beta cell in insulin-dependent diabetics. In revealing a lack of insulin this diagnostic tool seems to detect a group of potentially ''unstable'' diabetics in need of strict observation, which would minimize the risks of bad metabolic control. (author)

  18. Clinical significance of the radioimmunological determination of C-peptide

    Energy Technology Data Exchange (ETDEWEB)

    Dudczak, R; Waldhaeusl, W [Vienna Univ. (Austria). 1. Medizinische Klinik

    1979-10-12

    The aim of this study was to investigate the relationship of metabolic control to insulinogenic reserve in insulin-dependent diabetics. Thus, the secretory reserve of the pancreatic beta cell was estimated in insulin-dependent diabetics by measuring changes in peripheral serum immunoreactive C-peptide (IRCP) concentrations in response to intravenous arginine (n = 19; 0.5 g/kg,t = 30 min) or glibenclamide-glucose (n = 6; 2 mg HB 419 - 0.33 g/kg intravenously). In the majority of stable diabetics a small secretory reserve of the beta cell was demonstrated, but both the absolute and relative increase in IRCP was reduced after intravenous arginine or glibenclamide-glucose in comparison with normal controls. In unstable diabetics a decreased basal concentration of IRCP, significantly smaller than that seen in stable diabetics (p < 0.01), was accompanied by a complete lack of IRCP release on intravenous arginine administration. Thus, we conclude that the radioimmunological determination of IRCP is of clinical interest in assessing the residual secretory capacity of the beta cell in insulin-dependent diabetics. In revealing a lack of insulin this diagnostic tool seems to detect a group of potentially unstable diabetics in need of strict observation, which would minimize the risks of bad metabolic control.

  19. CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

    Science.gov (United States)

    Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

    2009-02-15

    Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

  20. Beta-Poisson model for single-cell RNA-seq data analyses.

    Science.gov (United States)

    Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Rantalainen, Mattias; Pawitan, Yudi

    2016-07-15

    Single-cell RNA-sequencing technology allows detection of gene expression at the single-cell level. One typical feature of the data is a bimodality in the cellular distribution even for highly expressed genes, primarily caused by a proportion of non-expressing cells. The standard and the over-dispersed gamma-Poisson models that are commonly used in bulk-cell RNA-sequencing are not able to capture this property. We introduce a beta-Poisson mixture model that can capture the bimodality of the single-cell gene expression distribution. We further integrate the model into the generalized linear model framework in order to perform differential expression analyses. The whole analytical procedure is called BPSC. The results from several real single-cell RNA-seq datasets indicate that ∼90% of the transcripts are well characterized by the beta-Poisson model; the model-fit from BPSC is better than the fit of the standard gamma-Poisson model in > 80% of the transcripts. Moreover, in differential expression analyses of simulated and real datasets, BPSC performs well against edgeR, a conventional method widely used in bulk-cell RNA-sequencing data, and against scde and MAST, two recent methods specifically designed for single-cell RNA-seq data. An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq data is available under GPL-3 license at https://github.com/nghiavtr/BPSC CONTACT: yudi.pawitan@ki.se or mattias.rantalainen@ki.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

    Science.gov (United States)

    Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

    2004-07-01

    The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

  2. Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.

    Science.gov (United States)

    Chamayou, S; Alecci, C; Ragolia, C; Giambona, A; Siciliano, S; Maggio, A; Fichera, M; Guglielmino, A

    2002-05-01

    In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.

  3. Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

    Science.gov (United States)

    Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

    2009-02-01

    PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

  4. Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

    Science.gov (United States)

    De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

    2014-01-01

    Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

  5. The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

    DEFF Research Database (Denmark)

    Iba, K; Albrechtsen, R; Gilpin, B

    2000-01-01

    The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments......-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading......, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did...

  6. Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

    Science.gov (United States)

    Rabbitt, E H; Ayuk, J; Boelaert, K; Sheppard, M C; Hewison, M; Stewart, P M; Gittoes, N J L

    2003-03-20

    The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, Pprotein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

  7. Cell secretion machinery: Studies using the AFM

    International Nuclear Information System (INIS)

    Jena, Bhanu P.

    2006-01-01

    A new field in biology, 'nano-cell biology', has emerged from the successful use of force microscopy in understanding the structure and dynamics of cells and biomolecules, at nm resolution and in real time. Atomic force microscopy, in combination with conventional tools and approaches (electron microscopy, electrophysiology, X-ray diffraction, photon correlation spectroscopy, mass spectroscopy, biochemistry, and molecular biology), has revealed for the first time, the universal molecular machinery and mechanism of secretion in cells. Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them, dock and fuse at plasma membrane-associated supramolecular structures called Porosome, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle swelling, and the fusion of opposing bilayers, i.e., the fusion of secretory vesicle membrane at the base of the porosome membrane, has also been resolved

  8. Lipomatous secretory meningioma: case report and review of the literature

    International Nuclear Information System (INIS)

    Liebig, T.; Hoffmann, T.; Hosten, N.; Sander, B.; Lanksch, W.R.

    1998-01-01

    Secretory meningioma is a rare entity which may be characterised by imaging features unusual for other subtypes of meningoma, such as low attenuation on CT, high (fat-tissue equivalent) signal intensity on T1-weighted MRI, marked surrounding oedema, and irregular contrast enhancement. We report a case of secretory meningioma and review the literature. (orig.) (orig.)

  9. Unprotected daily sun exposure is differently associated with central adiposity and beta-cell dysfunction by gender: The Korean national health and nutrition examination survey (KNHANES) V

    Energy Technology Data Exchange (ETDEWEB)

    Ohn, Jung Hun [Division of Endocrinology and Metabolism, Department of Internal Medicine, Hallym University College of Medicine, Chuncheon (Korea, Republic of); Kwon, In Ho [Department of Dermatology, Hallym University Dongtan Sacred Heart Hospital, Hwaseong (Korea, Republic of); Park, Juri; Ryu, Ohk Hyun; Lee, Seong Jin; Kim, Doo-Man; Ihm, Sung-Hee; Choi, Moon-Gi; Yoo, Hyung Joon [Division of Endocrinology and Metabolism, Department of Internal Medicine, Hallym University College of Medicine, Chuncheon (Korea, Republic of); Hong, Eun-Gyoung, E-mail: hegletter@hallym.or.kr [Division of Endocrinology and Metabolism, Department of Internal Medicine, Hallym University College of Medicine, Chuncheon (Korea, Republic of)

    2014-08-15

    Background: Ultraviolet irradiation by sun exposure has been associated with both harms and benefits to metabolic health. Objective: The objective of this study was to determine whether unprotected daily sun exposure is associated with the prevalence of diabetes and explore the underlying mechanism. Methods: We analyzed the Korean National Health and Nutrition Survey V from 2010 to 2011. Participants 19–60 years of age were asked about the average amount of time they had been exposed to direct sunlight per day since the age of 19. We categorized participants into three groups with different levels of lifetime daily sun exposure and explored the association of sun exposure with the prevalence of diabetes. Results: The risk of diabetes was higher in subjects with more than 5 h of unprotected sun exposure per day, with an odds ratio of 2.39 (95% CI 1.75–3.25), compared to those with less than 2 h of sun exposure, and the association remained significant after adjusting for diabetes risk factors. Long-term sun exposure was associated with increased central obesity and the possibility of an increase in visceral adiposity, especially among women, and with decrease in beta cell function and peripheral adiposity or percent body fat in men. Conclusions: Our study provides a cutoff for upper limit of sun exposure and suggests unprotected daily sun exposure for more than 5 h should be avoided to prevent diabetes. Increased central adiposity and decreased beta cell function were observed in women and men, respectively, who had long-term unprotected daily sun exposure. - Highlights: • Sun exposure for more than 5 h per day is associated with diabetes risk. • Insulin resistance associated with visceral adiposity may play a role in women. • Insulin secretory defect may explain diabetes risk in men.

  10. Unprotected daily sun exposure is differently associated with central adiposity and beta-cell dysfunction by gender: The Korean national health and nutrition examination survey (KNHANES) V

    International Nuclear Information System (INIS)

    Ohn, Jung Hun; Kwon, In Ho; Park, Juri; Ryu, Ohk Hyun; Lee, Seong Jin; Kim, Doo-Man; Ihm, Sung-Hee; Choi, Moon-Gi; Yoo, Hyung Joon; Hong, Eun-Gyoung

    2014-01-01

    Background: Ultraviolet irradiation by sun exposure has been associated with both harms and benefits to metabolic health. Objective: The objective of this study was to determine whether unprotected daily sun exposure is associated with the prevalence of diabetes and explore the underlying mechanism. Methods: We analyzed the Korean National Health and Nutrition Survey V from 2010 to 2011. Participants 19–60 years of age were asked about the average amount of time they had been exposed to direct sunlight per day since the age of 19. We categorized participants into three groups with different levels of lifetime daily sun exposure and explored the association of sun exposure with the prevalence of diabetes. Results: The risk of diabetes was higher in subjects with more than 5 h of unprotected sun exposure per day, with an odds ratio of 2.39 (95% CI 1.75–3.25), compared to those with less than 2 h of sun exposure, and the association remained significant after adjusting for diabetes risk factors. Long-term sun exposure was associated with increased central obesity and the possibility of an increase in visceral adiposity, especially among women, and with decrease in beta cell function and peripheral adiposity or percent body fat in men. Conclusions: Our study provides a cutoff for upper limit of sun exposure and suggests unprotected daily sun exposure for more than 5 h should be avoided to prevent diabetes. Increased central adiposity and decreased beta cell function were observed in women and men, respectively, who had long-term unprotected daily sun exposure. - Highlights: • Sun exposure for more than 5 h per day is associated with diabetes risk. • Insulin resistance associated with visceral adiposity may play a role in women. • Insulin secretory defect may explain diabetes risk in men

  11. Rapid agonist-induced loss of sup 125 I-. beta. -endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Cone, R.I.; Lameh, J.; Sadee, W. (Univ. of California, San Francisco (United States))

    1991-01-01

    The authors have measured {mu} and {delta} opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of {sup 125}I-{beta}-endorphin ({beta}E) as a tracer, together with {beta}E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface {mu} and {delta} opioid receptor sites. Labeling was at {delta} sites in NG108-15 cells and predominantly at {mu} sites in SK-N-SH cells. Pretreatment with the {mu} and {delta} agonist, DADLE, caused a rapid loss of cell surface {delta} receptor sites in NG108-15 cells, but failed to reduce significantly {mu} receptor density in SK-N-SH cells.

  12. Proinsulin maturation disorder is a contributor to the defect of subsequent conversion to insulin in {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jie, E-mail: jie.wang2@osumc.edu [Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, The Ohio State University, Columbus, OH (United States); Osei, Kwame [Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, The Ohio State University, Columbus, OH (United States)

    2011-07-22

    Highlights: {yields} Primary proinsulin maturation disorder is inherent in Ins2{sup +/Akita} islets/{beta}-cells. {yields} A consequence is the inefficient conversion of proinsulin to insulin. {yields} Post-translational defects occur as well in the involved PC1/3 and PC2 convertases. {yields} Proinsulin maturation chaos results in defects in the following conversion process. {yields} A link of the proinsulin maturation disorder and hyperproinsulinemia is suggested. -- Abstract: Disproportionate hyperproinsulinemia is an indicator of {beta}-cell dysfunction in diabetes and the basis underlying this abnormality remains obscure. Recently, we have found proinsulin is an aggregation-prone molecule inherent with a low relative folding rate and maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) in normal {beta}-cells as a result of the integration of maturation and disposal processes. PIHO is susceptible to environmental and genetic influences. Perturbation of PIHO produces a number of toxic consequences with known association to {beta}-cell failure in diabetes. To explore whether the perturbation of PIHO has a link to disproportionate hyperproinsulinemia, we investigated proinsulin conversion and the involved prohormone convertase 1/3 (PC1/3) and 2 (PC2) in mouse Ins2{sup +/Akita} islets/{beta}-cells that preserve a primary PIHO disorder due to a mutation (C96Y) in the insulin 2 (Ins2) gene. Our metabolic-labeling studies found an increased ratio of proinsulin to insulin in the cellular or released proteins of Ins2{sup +/Akita} islets. Histological, metabolic-labeling, and RT-PCR analyses revealed decreases of the PC1/3 and PC2 immunoreactivities in the {beta}-cells of Ins2{sup +/Akita} islets in spite of no declines of these two convertases at the transcriptional and translational levels. Immunoblot analyses in cloned Ins2{sup +/Akita} {beta}-cells further confirmed the increased ratio of proinsulin

  13. Effects of short-hairpin RNA-inhibited {beta}-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Wenqing, E-mail: liangwenqing_1234@126.com [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Yang, Chengwei [Department of Spinal Surgery, Lanzhou General Hospital, Lanzhou Military Area Command, 333 Nanbinhe Road, Lanzhou 730050 (China); Qian, Yu [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Fu, Qiang, E-mail: chyygklwq@hotmail.com [Department of Orthopaedics, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.

  14. Localization of thymosin beta-4 in tumors

    DEFF Research Database (Denmark)

    Larsson, L. -I.; Holck, Susanne

    2007-01-01

    in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured......Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker...... breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide...

  15. A gut-homing, oligoclonal CD4+ T cell population in severe-combined immunodeficient mice expressing a rearranged, transgenic class I-restricted alpha beta T cell receptor

    DEFF Research Database (Denmark)

    Reimann, J; Rudolphi, A; Spiess, S

    1995-01-01

    We studied the peripheral T cell compartment of H-2b severe combined immunodeficient (scid) mice that express a transgenic (tg) alpha beta T cell receptor (TcR) specific for the H-Y (male) epitope presented by the H-2 class I Db molecule. Large populations of CD3+ NK1.1-TCR beta T+ T cells were...

  16. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

    Science.gov (United States)

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

    2006-10-01

    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

  17. A standardized method for in vivo mouse pancreas imaging and semiquantitative beta cell mass measurement by dual isotope SPECT

    NARCIS (Netherlands)

    Mathijs, I.; Xavier, C.; Peleman, C.; Caveliers, V.; Brom, M.; Gotthardt, M.; Herrera, P.L.; Lahoutte, T.; Bouwens, L.

    2015-01-01

    PURPOSE: In order to evaluate future beta cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective beta cell tracer within the pancreas. PROCEDURES: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and

  18. Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

    Science.gov (United States)

    Pinho, Andreia V; Bensellam, Mohammed; Wauters, Elke; Rees, Maxine; Giry-Laterriere, Marc; Mawson, Amanda; Ly, Le Quan; Biankin, Andrew V; Wu, Jianmin; Laybutt, D Ross; Rooman, Ilse

    2015-01-01

    Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

  19. Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

    Directory of Open Access Journals (Sweden)

    Andreia V Pinho

    Full Text Available Sirtuin 1 (Sirt1 has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r as well as a marked down regulation of endoplasmic reticulum (ER chaperones that participate in the Unfolded Protein Response (UPR pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

  20. Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop.

    Directory of Open Access Journals (Sweden)

    Gediminas Matulis

    2010-06-01

    Full Text Available Invariant Natural Killer T cells (iNKT are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

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