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Sample records for cell secretes microparticles

  1. IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components.

    Science.gov (United States)

    Kovach, Melissa A; Singer, Benjamin H; Newstead, Michael W; Zeng, Xianying; Moore, Thomas A; White, Eric S; Kunkel, Steven L; Peters-Golden, Marc; Standiford, Theodore J

    2016-08-01

    Interleukin-36 is a family of novel interleukin-1-like proinflammatory cytokines that are highly expressed in epithelial tissues and several myeloid-derived cell types. Like those of classic interleukin-1 cytokines, the secretion mechanisms of interleukin-36 are not well understood. Interleukin-36γ secretion in dermal epithelial cells requires adenosine 5'-triphosphate, which suggests a nonclassical mechanism of secretion. In this study, murine pulmonary macrophages and human alveolar macrophages were treated with recombinant pathogen-associated molecular patterns (intact bacteria: Klebsiella pneumoniae or Streptococcus pneumoniae). Cell lysates were analyzed for messenger ribonucleic acid by quantitative real-time polymerase chain reaction, and conditioned medium was analyzed for interleukin-36γ by enzyme-linked immunosorbent assay, with or without sonication. In addition, conditioned medium was ultracentrifuged at 25,000 g and 100,000 g, to isolate microparticles and exosomes, respectively, and interleukin-36γ protein was assessed in each fraction by Western blot analysis. Interleukin-36γ mRNA was induced in both murine and human lung macrophages by a variety of pathogen-associated molecular patterns, as well as heat-killed and live Klebsiella pneumoniae and Streptococcus pneumoniae, and induction occurred in a myeloid differentiation response gene 88-dependent manner. Secretion of interleukin-36γ protein was enhanced by adenosine 5'-triphosphate. Furthermore, extracellular interleukin-36γ protein detection was markedly enhanced by sonication to disrupt membrane-bound structures. Interleukin-36γ protein was detected by Western blot in microparticles and exosome fractions isolated by ultracentrifugation. Interleukin-36γ was induced and secreted from lung macrophages in response to Gram-negative and -positive bacterial stimulation. The results suggest that interleukin-36γ is secreted in a non-Golgi-dependent manner by lung macrophages in response to Gram

  2. Red blood cell-derived microparticles: An overview.

    Science.gov (United States)

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). PMID:27282583

  3. Microparticles as biomarkers of osteonecrosis of the hip in sickle cell disease

    NARCIS (Netherlands)

    Marsh, Anne; Schiffelers, Raymond; Kuypers, Frans; Larkin, Sandra; Gildengorin, Ginny; van Solinge, Wouter; Hoppe, Carolyn

    2015-01-01

    Osteonecrosis of the femoral head (ONFH) is a common complication of sickle cell disease (SCD). To examine the association between microparticles and ONFH in SCD, we compared plasma microparticle levels in 20 patients with and without ONFH. Microparticles were quantified using nanoparticle tracking

  4. Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles

    OpenAIRE

    Pitsillides, Costas M; Joe, Edwin K.; Wei, Xunbin; Anderson, R. Rox; Lin, Charles P.

    2003-01-01

    We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond ...

  5. Microparticles in sickle cell anaemia: promise and pitfalls.

    Science.gov (United States)

    Hebbel, Robert P; Key, Nigel S

    2016-07-01

    Blood from patients with sickle cell disease contains microparticles (MP) derived from multiple cell sources, including red cells, platelets, monocytes and endothelial cells. MPs are of great interest because of their disease associations, their status as promising biomarkers, and the intercellular communications they mediate. To illustrate the likelihood of their relevance in sickle cell disease, we discuss the nature of MP, their profiling in sickle disease, some caveats relevant to their detection, their roles in supporting coagulation and the disparate influences they may exert upon the pathobiology of sickle cell disease. PMID:27136195

  6. Effect of Irradiation on Microparticles in Red Blood Cell Concentrates

    OpenAIRE

    Cho, Chi Hyun; Yun, Seung Gyu; Koh, Young Eun; Lim, Chae Seung

    2016-01-01

    Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The...

  7. The preservation of living cells with biocompatible microparticles

    Science.gov (United States)

    Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

    2016-07-01

    Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

  8. Stimulation of incretin secreting cells

    OpenAIRE

    Pais, Ramona; Gribble, Fiona M.; Reimann, Frank

    2016-01-01

    The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1) are secreted from enteroendocrine cells in the gut and regulate physiological and homeostatic functions related to glucose control, metabolism and food intake. This review provides a systematic summary of the molecular mechanisms underlying secretion from incretin cells, and an understanding of how they sense and interact with lumen and vascular factors and the enteric nervous system t...

  9. Manipulation of microparticles and red blood cells using optoelectronic tweezers

    Indian Academy of Sciences (India)

    R S Verma; R Dasgupta; N Kumar; S Ahlawat; A Uppal; P K Gupta

    2014-02-01

    We report the development of an optoelectronic tweezers set-up which works by lightinduced dielectrophoresis mechanism to manipulate microparticles. We used thermal evaporation technique for coating the organic polymer, titanium oxide phthalocyanine (TiOPc), as a photoconductive layer on ITO-coated glass slide. Compare to the conventional optical tweezers, the technique requires optical power in W range and provides a manipulation area of a few mm2. The set-up was used to manipulate the polystyrene microspheres and red blood cells (RBCs). The RBCs could be attracted or repelled by varying the frequency of the applied AC bias.

  10. Stimulation of incretin secreting cells

    Science.gov (United States)

    Pais, Ramona; Gribble, Fiona M.; Reimann, Frank

    2016-01-01

    The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1) are secreted from enteroendocrine cells in the gut and regulate physiological and homeostatic functions related to glucose control, metabolism and food intake. This review provides a systematic summary of the molecular mechanisms underlying secretion from incretin cells, and an understanding of how they sense and interact with lumen and vascular factors and the enteric nervous system through transporters and G-protein coupled receptors (GPCRs) present on their surface to ultimately culminate in hormone release. Some of the molecules described below such as sodium coupled glucose transporter 1 (SGLT1), G-protein coupled receptor (GPR) 119 and GPR40 are targets of novel therapeutics designed to enhance endogenous gut hormone release. Synthetic ligands at these receptors aimed at treating obesity and type 2 diabetes are currently under investigation. PMID:26885360

  11. Stimulation of incretin secreting cells.

    Science.gov (United States)

    Pais, Ramona; Gribble, Fiona M; Reimann, Frank

    2016-02-01

    The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1) are secreted from enteroendocrine cells in the gut and regulate physiological and homeostatic functions related to glucose control, metabolism and food intake. This review provides a systematic summary of the molecular mechanisms underlying secretion from incretin cells, and an understanding of how they sense and interact with lumen and vascular factors and the enteric nervous system through transporters and G-protein coupled receptors (GPCRs) present on their surface to ultimately culminate in hormone release. Some of the molecules described below such as sodium coupled glucose transporter 1 (SGLT1), G-protein coupled receptor (GPR) 119 and GPR40 are targets of novel therapeutics designed to enhance endogenous gut hormone release. Synthetic ligands at these receptors aimed at treating obesity and type 2 diabetes are currently under investigation. PMID:26885360

  12. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.

    Science.gov (United States)

    Abbasian, Nima; Burton, James O; Herbert, Karl E; Tregunna, Barbara-Emily; Brown, Jeremy R; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J; Goodall, Alison H; Bevington, Alan

    2015-09-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  13. Effect of Irradiation on Microparticles in Red Blood Cell Concentrates.

    Science.gov (United States)

    Cho, Chi Hyun; Yun, Seung Gyu; Koh, Young Eun; Lim, Chae Seung

    2016-07-01

    Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×10⁹/L (±22.7×10⁹/L), and the total number of MPs ranged from 2.6×10⁹/L to 96.9×10⁹/L. The mean number of MPs increased to 22.6×10⁹/L (±31.6×10⁹/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×10⁹/L) and 2.2% (263×10⁶/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×10⁹/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×10⁶/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change. PMID:27139610

  14. Enhancing microparticle internalization by nonphagocytic cells through the use of noncovalently conjugated polyethyleneimine

    Directory of Open Access Journals (Sweden)

    Patiño T

    2012-11-01

    Full Text Available Tania Patiño, Carme Nogués, Elena Ibáñez, Leonardo BarriosUnitat de Biologia Cel lular, Departament de Biologia Cel lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, SpainAbstract: Development of micro- and nanotechnology for the study of living cells, especially in the field of drug delivery, has gained interest in recent years. Although several studies have reported successful results in the internalization of micro- and nanoparticles in phagocytic cells, when nonphagocytic cells are used, the low internalization efficiency represents a limitation that needs to be overcome. It has been reported that covalent surface modification of micro- and nanoparticles increases their internalization rate. However, this surface modification represents an obstacle for their use as drug-delivery carriers. For this reason, the aim of the present study was to increase the capability for microparticle internalization of HeLa cells through the use of noncovalently bound transfection reagents: polyethyleneimine (PEI Lipofectamine™ 2000 and FuGENE 6®. Both confocal microscopy and flow cytometry techniques allowed us to precisely quantify the efficiency of microparticle internalization by HeLa cells, yielding similar results. In addition, intracellular location of microparticles was analyzed through transmission electron microscopy and confocal microscopy procedures. Our results showed that free PEI at a concentration of 0.05 mM significantly increased microparticle uptake by cells, with a low cytotoxic effect. As determined by transmission electron and confocal microscopy analyses, microparticles were engulfed by plasma-membrane projections during internalization, and 24 hours later they were trapped in a lysosomal compartment. These results show the potential use of noncovalently conjugated PEI in microparticle internalization assays.Keywords: HeLa cells, internalization efficiency, endocytosis, drug

  15. Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells

    International Nuclear Information System (INIS)

    Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells

  16. Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Schock, Sarah C. [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Edrissi, Hamidreza [University of Ottawa, Neuroscience Graduate Program, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Burger, Dylan [Ottawa Hospital Research Institute, Kidney Centre, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Cadonic, Robert; Hakim, Antoine [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada); Thompson, Charlie, E-mail: charliet@uottawa.ca [Ottawa Hospital Research Institute, Neuroscience, 451 Smyth Road, Ottawa, ON K1H 8M5 (Canada)

    2014-07-18

    Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.

  17. Analysis of Cell-Derived Microparticles with Highly Precise Nanotechnological Methods

    DEFF Research Database (Denmark)

    Cherré, Solène; Østergaard, Ole; Heegaard, Niels H.H.;

    2014-01-01

    Cell-derived microparticles have gained a broad interest in the past years. Being released by blood cells upon activation or induction of apoptosis, they have a great potential as novel diagnostic markers and their investigation can bring new knowledge into the pathogenesis of various diseases. H...

  18. Circulating endothelial progenitor cell and platelet microparticle impact on platelet activation in hypertension associated with hypercholesterolemia.

    Directory of Open Access Journals (Sweden)

    Nicoleta Alexandru

    Full Text Available AIM: The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs and platelet microparticles (PMPs on blood platelet function in experimental hypertension associated with hypercholesterolemia. METHODS: Golden Syrian hamsters were divided in six groups: (i control, C; (ii hypertensive-hypercholesterolemic, HH; (iii 'prevention', HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv 'regression', HHfin-EPCs, HH treated with EPCs after HH feeding; (v HH treated with PMPs, HH-PMPs, and (vi HH treated with EPCs and PMPs, HH-EPCs-PMPs. RESULTS: Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied. CONCLUSION: EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.

  19. Cell Secretion: Current Structural and Biochemical Insights

    Directory of Open Access Journals (Sweden)

    Saurabh Trikha

    2010-01-01

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

  20. Macrophage and osteoblast responses to biphasic calcium phosphate microparticles.

    Science.gov (United States)

    Fellah, Borhane Hakim; Delorme, Bruno; Sohier, Jérôme; Magne, David; Hardouin, Pierre; Layrolle, Pierre

    2010-06-15

    The aim of this work was to investigate in vitro the biological events leading to ectopic bone formation in contact with microporous biphasic calcium phosphate (BCP) ceramics. After implantation, microparticles may arise from their degradation and induce an inflammatory response involving macrophages. The secretion of pro-inflammatory cytokines may affect the differentiation of osteoblasts. Mouse macrophage-like (J774) and osteoblast-like (MC3T3-E1) cells were cultured in the presence of BCP microparticles of different sizes (response initiated by BCP microparticles may have both detrimental and beneficial effects on osteogenesis. PMID:20014296

  1. Enhanced cell adhesion to the dimpled surfaces of golf-ball-shaped microparticles.

    Science.gov (United States)

    Lee, Joo Hyuk; Lee, Chang-Soo; Cho, Kuk Young

    2014-10-01

    Engineering surface morphology as in honeycomb-structured planar films is of great importance for providing new potential application and improved performance in biomedical fields. We demonstrate potential new applications for the uniform biocompatible golf-ball-shaped microparticles that resembles 3D feature of honeycomb-structured film. Dimple size controllable golf-ball-shaped microparticles were fabricated by microfluidic device. Surface dimples not only can act as picoliter beaker but also enhance cell adhesion without any chemical modification of the surface. PMID:25265359

  2. Sustained release of matrix metalloproteinase-3 to trabecular meshwork cells using biodegradable PLGA microparticles.

    Science.gov (United States)

    Turturro, Sanja; Sunoqrot, Suhair; Ying, Hongyu; Hong, Seungpyo; Yue, Beatrice Y J T

    2013-08-01

    Accumulation of extracellular matrix (ECM) materials in the trabecular meshwork (TM) is believed to be a contributing factor to intraocular pressure (IOP) elevation, a risk factor/cause of primary open angle glaucoma, a major blinding disease. Matrix metalloproteinase-3 (MMP-3) is one of the proteinases that can effectively degrade ECM elements such as fibronectin, and MMP-3 delivery to the TM represents a promising approach for IOP reduction and treatment of glaucoma. In this study, we tested the feasibility of using polymeric microparticles to achieve a slow and sustained release of active MMP-3 to cultured human TM cells. β-Casein, with molecular weight (24 kDa) and hydrophobicity similar to those of the active MMP-3 fragment (19.2 kDa), was first employed as a model for initial testing. β-casein was encapsulated into poly(lactic-co-glycolic acid) (PLGA) microparticles using a double emulsion procedure at an encapsulation efficiency of approximately 45%. The PLGA microparticles were chosen given their biocompatibility and the proven capacity of sustained release of encapsulated molecules. The release test conducted in the culture medium showed a slow and sustained release of the protein over 20 days without a significant initial burst release. Active MMP-3 was subsequently encapsulated into PLGA microparticles with an encapsulation efficiency of approximately 50%. A biofunctional assay utilizing human TM cells was set up in which the reduction of fibronectin was used as an indicator of enzyme activity. It was observed that fibronectin staining was markedly reduced by the medium collected from MMP-3-microparticle-treated cultures compared to that from blank- and β-casein-microparticle controls, which was validated using a direct MMP-3 activity assay. The controlled release of MMP-3 from the microparticles resulted in sustained degradation of fibronectin up to 10 days. This proof-of-concept undertaking represents the first study on the controlled and sustained

  3. Stiffening of Human Mesenchymal Stem Cell Spheroid Microenvironments Induced by Incorporation of Gelatin Microparticles

    OpenAIRE

    Baraniak, Priya R.; Cooke, Marissa T; Saeed, Rabbia; Kinney, Melissa A.; Krista M Fridley; McDevitt, Todd C.

    2012-01-01

    Culturing multipotent adult mesenchymal stem cells as 3D aggregates augments their differentiation potential and paracrine activity. One caveat of stem cell spheroids, though, can be the limited diffusional transport barriers posed by the inherent 3D structure of the multicellular aggregates. In order to circumvent such limitations, polymeric microparticles have been incorporated into stem cell aggregates as a means to locally control the biochemical and physical properties of the 3D microenv...

  4. Facile moldless fabrication of disk-shaped and reed blood cell-like microparticles using photopolymerization of tripropylene glycol diacrylate

    Science.gov (United States)

    Choi, Jongchul; Won, June; Song, Simon

    2014-12-01

    A facile method for the moldless fabrication of 2- or 3-dimensional microparticles is proposed by using a photopolymerization technique. Using only a monomer solution of tripropylene glycol diacrylate, a film mask and standard UV lithography equipment, we were able to fabricate microparticles of various shapes, such as disks, dimpled disks similar in shape to red blood cells, and slender gourd shapes, unlike previous moldless fabrication techniques requiring expensive and/or sophisticated equipment. The simple method could produce more than one million particles in a single batch, indicating that it can be applied to the mass production of polymer microparticles. Analyses of scanning electron micrographs and optical micrographs of the microparticles indicated that their size distribution was highly monodisperse. Detailed fabrication processes and statistics on the microparticle sizes are given in this paper.

  5. Facile moldless fabrication of disk-shaped and reed blood cell-like microparticles using photopolymerization of tripropylene glycol diacrylate

    International Nuclear Information System (INIS)

    A facile method for the moldless fabrication of 2- or 3-dimensional microparticles is proposed by using a photopolymerization technique. Using only a monomer solution of tripropylene glycol diacrylate, a film mask and standard UV lithography equipment, we were able to fabricate microparticles of various shapes, such as disks, dimpled disks similar in shape to red blood cells, and slender gourd shapes, unlike previous moldless fabrication techniques requiring expensive and/or sophisticated equipment. The simple method could produce more than one million particles in a single batch, indicating that it can be applied to the mass production of polymer microparticles. Analyses of scanning electron micrographs and optical micrographs of the microparticles indicated that their size distribution was highly monodisperse. Detailed fabrication processes and statistics on the microparticle sizes are given in this paper. (technical note)

  6. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    Science.gov (United States)

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds. PMID:26652045

  7. Stiffening of Human Mesenchymal Stem Cell Spheroid Microenvironments Induced by Incorporation of Gelatin Microparticles

    Science.gov (United States)

    Baraniak, Priya R.; Cooke, Marissa T.; Saeed, Rabbia; Kinney, Melissa A.; Fridley, Krista M.; McDevitt, Todd C.

    2012-01-01

    Culturing multipotent adult mesenchymal stem cells as 3D aggregates augments their differentiation potential and paracrine activity. One caveat of stem cell spheroids, though, can be the limited diffusional transport barriers posed by the inherent 3D structure of the multicellular aggregates. In order to circumvent such limitations, polymeric microparticles have been incorporated into stem cell aggregates as a means to locally control the biochemical and physical properties of the 3D microenvironment. However, the introduction of biomaterials to the 3D stem cell microenvironment could alter the mechanical forces sensed by cells within aggregates, which in turn could impact various cell behaviors and overall spheroid mechanics. Therefore, the objective of this study was to determine the acute effects of biomaterial incorporation within mesenchymal stem cell spheroids on aggregate structure and mechanical properties. The results of this study demonstrate that although gelatin microparticle incorporation results in similar multi-cellular organization within human mesenchymal stem cell spheroids, the introduction of gelatin materials significantly impacts spheroid mechanical properties. The marked differences in spheroid mechanics induced by microparticle incorporation may hold major implications for in vitro directed differentiation strategies and offer a novel route to engineer the mechanical properties of tissue constructs ex vivo. PMID:22658155

  8. One-step magnetic modification of yeast cells by microwave-synthesized iron oxide microparticles

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Procházková, G.; Šafařík, Ivo

    2013-01-01

    Roč. 56, č. 6 (2013), s. 456-461. ISSN 0266-8254 R&D Projects: GA MŠk(CZ) LD13023; GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : invert sugar formation * magnetic iron oxide microparticles * magnetic modification * magnetic separation * microwave-assisted synthesis * whole-cell biocatalyst * yeast cells Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.749, year: 2013

  9. Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells.

    Science.gov (United States)

    Safarik, Ivo; Pospiskova, Kristyna; Maderova, Zdenka; Baldikova, Eva; Horska, Katerina; Safarikova, Mirka

    2015-01-01

    An extremely simple procedure has been developed for the immobilization of Saccharomyces cerevisiae cells on magnetic chitosan microparticles. The magnetic carrier was prepared using an inexpensive, simple, rapid, one-pot process, based on the microwave irradiation of chitosan and ferrous sulphate at high pH. Immobilized yeast cells have been used for sucrose hydrolysis, hydrogen peroxide decomposition and the adsorption of selected dyes. PMID:24753015

  10. On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves

    OpenAIRE

    Ding, Xiaoyun; Lin, Sz-Chin Steven; Kiraly, Brian; Yue, Hongjun; Li, Sixing; Chiang, I-Kao; Shi, Jinjie; Benkovic, Stephen J.; Huang, Tony Jun

    2012-01-01

    Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based “acoustic tweezers” that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve re...

  11. Circulating Endothelial Progenitor Cell and Platelet Microparticle Impact on Platelet Activation in Hypertension Associated with Hypercholesterolemia

    OpenAIRE

    Nicoleta Alexandru; Doina Popov; Emanuel Dragan; Eugen Andrei; Adriana Georgescu

    2013-01-01

    AIM: The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia. METHODS: Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) 'prevention', HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) 'regression', HHfin-EPCs, HH treated with EPCs aft...

  12. Injectable PEGylated fibrinogen cell-laden microparticles made with a continuous solvent- and oil-free preparation method.

    Science.gov (United States)

    Oliveira, Mariana B; Kossover, Olga; Mano, João F; Seliktar, Dror

    2015-02-01

    A new methodology is reported for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photoreactive poly(ethylene glycol) (PEG)-fibrinogen (PF) polymer was transported through a transparent injector exposed to light irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data revealed the crosslinking kinetics of the PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture just prior to atomization. The partially polymerized drops of PF/cells fell into a gelation bath for further crosslinking until fully polymerized hydrogel microparticles were formed. As the drops of solution exited the air-in-jet nozzle, their viscosity was designed to be sufficiently high so as to prevent rapid mixing and/or dilution in the gelation bath, but without undergoing complete gelation in the nozzle. Several parameters of this system were varied to control the size and polydispersity of the microparticles, including the cell density, the flow rate and the air pressure in the nozzle. The system was capable of producing cell-laden microparticles with an average diameter of between 88.1 to 347.1 μm, and a dispersity of between 1.1 and 2.4, depending on the parameters chosen. Varying the precursor flow rate and/or cell density was beneficial in controlling the size and polydispersity of the microparticles; all microparticles exhibited very high cell viability, which was not affected by these parameters. In conclusion, this dropwise photopolymerization methodology for preparing cell-laden microparticles is an attractive alternative to existing techniques that use harsh solvents/oils and offer limited control over particle size and polydispersity. PMID:25462849

  13. Regulated Mucin Secretion from Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    BurtonFDickey

    2013-09-01

    Full Text Available Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3x10^6 D per monomer whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ~1 um in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among MARCKS, cysteine string protein (CSP, HSP70 and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG. Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the

  14. The SNARE machinery in mast cell secretion

    Directory of Open Access Journals (Sweden)

    Axel eLorentz

    2012-06-01

    Full Text Available Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators – stored in granules – as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-Sensitive Factor (NSF Attachment Protein (SNAP Receptors (SNARE proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, SCAMPs, complexins or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

  15. Membrane Properties Involved in Calcium-Stimulated Microparticle Release from the Plasma Membranes of S49 Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Lauryl E. Campbell

    2014-01-01

    Full Text Available This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32–42°C. A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.

  16. IA-2 modulates dopamine secretion in PC12 cells

    OpenAIRE

    Nishimura, Takuya; Harashima, Shin-ichi; Yafang, Hu; Notkins, Abner Louis

    2009-01-01

    The secretion of the hormone insulin from beta cells is modulated by the expression of the dense core vesicle transmembrane protein IA-2. Since IA-2 is found in neuroendocrine cells throughout the body, the present experiments were initiated to determine whether the expression of IA-2 also modulates the secretion of neurotransmitters. Using the dopamine-secreting pheochromocytoma cell line PC12, we found that the overexpressions of IA-2 increased the cellular content and secretion of dopamine...

  17. Myeloma cells suppress osteoblasts through sclerostin secretion

    International Nuclear Information System (INIS)

    Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM

  18. Galvanic zinc-copper microparticles inhibit melanogenesis via multiple pigmentary pathways.

    Science.gov (United States)

    Won, Yen-Kim; Lin, Connie B; Seiberg, Miri; Chen, Nannan; Hu, Yaping; Rossetti, Dianne; Saliou, Claude; Loy, Chong-Jin

    2014-01-01

    The endogenous electrical field of human skin plays an important role in many skin functions. However, the biological effects and mechanism of action of externally applied electrical stimulation on skin remain unclear. Recent study showed that galvanic zinc-copper microparticles produce electrical stimulation and reduce inflammatory and immune responses in intact skin, suggesting the important role of electrical stimulation in non-wounded skin. The objective of this study is to investigate the biological effect of galvanic zinc-copper microparticles on skin pigmentation. Our findings showed that galvanic zinc-copper microparticles inhibited melanogenesis in a human melanoma cell line (MNT-1), human keratinocytes and melanoma cells co-cultures, and in pigmented epidermal equivalents. Treatment of galvanic zinc-copper microparticles inhibited melanogenesis by reducing the promoter transactivation of tyrosinase and tyrosinase-related protein-1 in human melanoma cells. In a co-culture Transwell system of keratinocytes and melanoma cells, galvanic zinc-copper microparticles reduced melanin production via downregulation of endothelin-1 secretion from keratinocytes and reduced tyrosinase gene expression in melanoma cells. In addition, exposure of pigmented epidermal equivalents to galvanic zinc-copper microparticles resulted in reduced melanin deposition. In conclusion, our data demonstrated for the first time that galvanic zinc-copper microparticles reduced melanogenesis in melanoma cells and melanin deposition in pigmented epidermal equivalents by affecting multiple pigmentary pathways. PMID:23700242

  19. On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves

    Science.gov (United States)

    Ding, Xiaoyun; Lin, Sz-Chin Steven; Kiraly, Brian; Yue, Hongjun; Li, Sixing; Chiang, I-Kao; Shi, Jinjie; Benkovic, Stephen J.; Huang, Tony Jun

    2012-01-01

    Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based “acoustic tweezers” that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve real-time control of a standing surface acoustic wave field, which enables flexible manipulation of most known microparticles. The power density required by our acoustic device is significantly lower than its optical counterparts (10,000,000 times less than optical tweezers and 100 times less than optoelectronic tweezers), which renders the technique more biocompatible and amenable to miniaturization. Cell-viability tests were conducted to verify the tweezers’ compatibility with biological objects. With its advantages in biocompatibility, miniaturization, and versatility, the acoustic tweezers presented here will become a powerful tool for many disciplines of science and engineering. PMID:22733731

  20. Differentiation of human embryonic stem cells into insulin- secreting cells

    OpenAIRE

    S Mollamohammadi; Massumi, M.; H Jafary; Baharvand, H.

    2006-01-01

    Introduction: Type I diabetes mellitus is caused by autoimmune destruction of the insulin-producing β-cells. A new potential method for curing the disease is transplantation of differentiated insulin- secreting cells from human embryonic stem cells. Methods: Human embryonic stem cell lines (Royan H1) were used to produce embryoid bodies. Differentiation carried out by growth factor-mediated selection of nestin positive cells. In final stage, these cells were expanded in the presence of bFGF, ...

  1. Phospholipid Binding Protein C Inhibitor (PCI Is Present on Microparticles Generated In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Katrin Einfinger

    Full Text Available Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.

  2. γ-aminobutyric acid secreted from islet β-cells modulates exocrine secretion in rat pancreas

    Institute of Scientific and Technical Information of China (English)

    Yong-Deuk Park; Zheng-Yun Cui; Guang Wu; Hyung-Seo Park; Hyoung-Jin Park

    2006-01-01

    AIM: To investigate the role of endogenous γ-aminobutyric acid (GABA) in pancreatic exocrine secretion.METHODS: The isolated, vascularly perfused rat pancreas was employed in this study to eliminate the possible influences of extrinsic nerves and hormones.Cholecystokinin (CCK; 10 pmol/L) was intra-arterially given to stimulate exocrine secretion of the pancreas.RESULTS: Glutamine, a major precursor of GABA, which was given intra-arterially at concentrations of 1, 4 and 10 mmol/L, dose-dependently elevated the CCK-stimulated secretions of fluid and amylase in the normal pancreas.Bicuculline (10 μmol/L), a GABAA receptor antagonist,blocked the enhancing effect of glutamine (4 mmol/L) on the CCK-stimulated exocrine secretions. Glutamine, at concentrations of 1, 4 and 10 mmol/L, dose-dependently increased the GABA concentration in portal effluent of the normal pancreas. The effects of glutamine on the CCK-stimulated exocrine secretion as well as the GABA secretion were markedly reduced in the streptozotocintreated pancreas.CONCLUSION: GABA could be secreted from β-cells into the islet-acinar portal system after administration of glutainine, and could enhance the CCK-stimulated exocrine secretion through GABAA receptors. Thus,GABA in islet β-cells is a hormone modulating pancreatic exocrine secretion.

  3. Delivery of oncolytic adenovirus into the nucleus of tumorigenic cells by tumor microparticles for virotherapy.

    Science.gov (United States)

    Ran, Li; Tan, Xiaohua; Li, Yanchun; Zhang, Huafeng; Ma, Ruihua; Ji, Tiantian; Dong, Wenqian; Tong, Tong; Liu, Yuying; Chen, Degao; Yin, Xiaonan; Liang, Xiaoyu; Tang, Ke; Ma, Jingwei; Zhang, Yi; Cao, Xuetao; Hu, Zhuowei; Qin, Xiaofeng; Huang, Bo

    2016-05-01

    Oncolytic viruses have been utilized for the treatment of various cancers. However, delivery of the viral particles to tumor cells remains a major challenge. Microparticles (MP) are vesicle forms of plasma membrane fragments of 0.1-1 μm in size that are shed by cells. We have previously shown the delivery of chemotherapeutic drugs using tumor cell-derived MPs (T-MP). Here we report that T-MPs can be utilized as a unique carrier system to deliver oncolytic adenoviruses to human tumors, leading to highly efficient cytolysis of tumor cells needed for in vivo treatment efficacy. This T-MP-mediated oncolytic virotherapy approach holds multiple advantages, including: 1) delivery of oncolytic adenovirus by T-MPs is able to avoid the antiviral effect of host antibodies; 2) delivery of oncolytic adenovirus by T-MPs is not limited by virus-specific receptor that mediates the entry of virus into tumor cells; 3) T-MPs are apt at delivering oncolytic adenoviruses to the nucleus of tumor cells as well as to stem-like tumor-repopulating cells for the desired purpose of killing them. These findings highlight a novel oncolytic adenovirus delivery system with highly promising clinical applications. PMID:26950165

  4. Evaluation of Gelatin Microparticles as Adherent-Substrates for Mesenchymal Stem Cells in a Hydrogel Composite.

    Science.gov (United States)

    Lu, Steven; Lee, Esther J; Lam, Johnny; Tabata, Yasuhiko; Mikos, Antonios G

    2016-06-01

    Due to the lack of cell-adhesive moieties in traditional synthetic hydrogels, the present work investigated the use of degradable gelatin microparticles (GMPs) as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs of varying crosslinking densities and sizes to investigate their role on influencing MSC differentiation and aggregation. The MSC-seeded GMPs were then encapsulated in poly(ethylene glycol)-based hydrogels and cultured in serum-free, growth factor-free osteochondral medium. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Over the course of 35 days, MSCs seeded on GMPs exhibited more cell-cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Although the factors of GMP crosslinking and size had nominal influence on MSC differentiation and aggregation, GMPs demonstrate potential as an adherent-substrate for improving cell delivery from hydrogel scaffolds by facilitating cell-cell contacts and improving MSC differentiation. PMID:26935924

  5. Self-Locking Optoelectronic Tweezers for Single-Cell and Microparticle Manipulation across a Large Area in High Conductivity Media

    Science.gov (United States)

    Yang, Yajia; Mao, Yufei; Shin, Kyeong-Sik; Chui, Chi On; Chiou, Pei-Yu

    2016-03-01

    Optoelectronic tweezers (OET) has advanced within the past decade to become a promising tool for cell and microparticle manipulation. Its incompatibility with high conductivity media and limited throughput remain two major technical challenges. Here a novel manipulation concept and corresponding platform called Self-Locking Optoelectronic Tweezers (SLOT) are proposed and demonstrated to tackle these challenges concurrently. The SLOT platform comprises a periodic array of optically tunable phototransistor traps above which randomly dispersed single cells and microparticles are self-aligned to and retained without light illumination. Light beam illumination on a phototransistor turns off the trap and releases the trapped cell, which is then transported downstream via a background flow. The cell trapping and releasing functions in SLOT are decoupled, which is a unique feature that enables SLOT’s stepper-mode function to overcome the small field-of-view issue that all prior OET technologies encountered in manipulation with single-cell resolution across a large area. Massively parallel trapping of more than 100,000 microparticles has been demonstrated in high conductivity media. Even larger scale trapping and manipulation can be achieved by linearly scaling up the number of phototransistors and device area. Cells after manipulation on the SLOT platform maintain high cell viability and normal multi-day divisibility.

  6. Flow bioreactor design for quantitative measurements over endothelial cells using micro-particle image velocimetry

    Science.gov (United States)

    Leong, Chia Min; Voorhees, Abram; Nackman, Gary B.; Wei, Timothy

    2013-04-01

    Mechanotransduction in endothelial cells (ECs) is a highly complex process through which cells respond to changes in hemodynamic loading by generating biochemical signals involving gene and protein expression. To study the effects of mechanical loading on ECs in a controlled fashion, different in vitro devices have been designed to simulate or replicate various aspects of these physiological phenomena. This paper describes the design, use, and validation of a flow chamber which allows for spatially and temporally resolved micro-particle image velocimetry measurements of endothelial surface topography and stresses over living ECs immersed in pulsatile flow. This flow chamber also allows the study of co-cultures (i.e., ECs and smooth muscle cells) and the effect of different substrates (i.e., coverslip and/or polyethylene terepthalate (PET) membrane) on cellular response. In this report, the results of steady and pulsatile flow on fixed endothelial cells seeded on PET membrane and coverslip, respectively, are presented. Surface topography of ECs is computed from multiple two-dimensional flow measurements. The distributions of shear stress and wall pressure on each individual cell are also determined and the importance of both types of stress in cell remodeling is highlighted.

  7. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T; Østergaard, Ole; Stener, Line;

    2012-01-01

    To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters.......To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters....

  8. Argonaute 2 in cell-secreted microvesicles guides the function of secreted miRNAs in recipient cells.

    Directory of Open Access Journals (Sweden)

    Zhiyuan Lv

    Full Text Available MicroRNAs (miRNAs secreted by cells into microvesicles (MVs form a novel class of signal molecules that mediate intercellular communication. However, several fundamental aspects of secreted miRNAs remain unknown, particularly the mechanism that governs the function or fate of exogenous miRNAs in recipient cells. In the present study, we provide evidence indicating that Argonaute 2 (Ago2 plays a role in stabilizing miRNAs and facilitating the packaging of secreted miRNAs into MVs. More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells directs the function of secreted miRNAs. First, Ago2 overexpression clearly increased the level of miR-16 in cells transfected with a miR-16 mimic by protecting the miRNAs from degradation in lysosomes. Second, Ago2 overexpression increased the level of miR-16 in cell-secreted MVs, suggesting that Ago2 may facilitate the packaging of secreted miRNAs into MVs. Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2. Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.

  9. Real-time imaging of microparticles and living cells with CMOS nanocapacitor arrays

    Science.gov (United States)

    Laborde, C.; Pittino, F.; Verhoeven, H. A.; Lemay, S. G.; Selmi, L.; Jongsma, M. A.; Widdershoven, F. P.

    2015-09-01

    Platforms that offer massively parallel, label-free biosensing can, in principle, be created by combining all-electrical detection with low-cost integrated circuits. Examples include field-effect transistor arrays, which are used for mapping neuronal signals and sequencing DNA. Despite these successes, however, bioelectronics has so far failed to deliver a broadly applicable biosensing platform. This is due, in part, to the fact that d.c. or low-frequency signals cannot be used to probe beyond the electrical double layer formed by screening salt ions, which means that under physiological conditions the sensing of a target analyte located even a short distance from the sensor (∼1 nm) is severely hampered. Here, we show that high-frequency impedance spectroscopy can be used to detect and image microparticles and living cells under physiological salt conditions. Our assay employs a large-scale, high-density array of nanoelectrodes integrated with CMOS electronics on a single chip and the sensor response depends on the electrical properties of the analyte, allowing impedance-based fingerprinting. With our platform, we image the dynamic attachment and micromotion of BEAS, THP1 and MCF7 cancer cell lines in real time at submicrometre resolution in growth medium, demonstrating the potential of the platform for label/tracer-free high-throughput screening of anti-tumour drug candidates.

  10. Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

    Science.gov (United States)

    Gao, Chunyan; Ji, Shuting; Dong, Weijun; Qi, Yushan; Song, Wen; Cui, Debin; Shi, Jialan

    2015-11-01

    Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process. PMID:26516916

  11. Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

    Directory of Open Access Journals (Sweden)

    Chunyan Gao

    2015-10-01

    Full Text Available Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs, the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS and indole-3-acetic acid (IAA on procoagulant activity (PCA of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients. Phosphatidylserine (PS exposure of RBCs and their microparticles (MPs release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+] with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

  12. "Kill" the messenger: Targeting of cell-derived microparticles in lupus nephritis.

    Science.gov (United States)

    Nielsen, Christoffer T; Rasmussen, Niclas S; Heegaard, Niels H H; Jacobsen, Søren

    2016-07-01

    Immune complex (IC) deposition in the glomerular basement membrane (GBM) is a key early pathogenic event in lupus nephritis (LN). The clarification of the mechanisms behind IC deposition will enable targeted therapy in the future. Circulating cell-derived microparticles (MPs) have been proposed as major sources of extracellular autoantigens and ICs and triggers of autoimmunity in LN. The overabundance of galectin-3-binding protein (G3BP) along with immunoglobulins and a few other proteins specifically distinguish circulating MPs in patients with systemic lupus erythematosus (SLE), and this is most pronounced in patients with active LN. G3BP co-localizes with deposited ICs in renal biopsies from LN patients supporting a significant presence of MPs in the IC deposits. G3BP binds strongly to glomerular basement membrane proteins and integrins. Accordingly, MP surface proteins, especially G3BP, may be essential for the deposition of ICs in kidneys and thus for the ensuing formation of MP-derived electron dense structures in the GBM, and immune activation in LN. This review focuses on the notion of targeting surface molecules on MPs as an entirely novel treatment strategy in LN. By targeting MPs, a double hit may be achieved by attenuating both the autoantigenic fueling of immune complexes and the triggering of the adaptive immune system. Thereby, early pathogenic events may be blocked in contrast to current treatment strategies that primarily target and modulate later events in the cellular and humoral immune response. PMID:26970484

  13. Peroxicretion : a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, Cees M.J.; Haaft, Peter J. ten; Minneboo, Ingeborg M.; Hartog, Miranda P.; Damveld, Robbert A.; Laan, Jan Metske van der; Akeroyd, Michiel; Wenzel, Thibaut J.; Luesken, Francisca A.; Veenhuis, Marten; Klei, Ida van der; Winde, Johannes H. de

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  14. Peroxicretion: a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, C.M.J.; Ten Haaft, P.J.T; Minneboo, I.M.; Hartog, M.P.; Damveld, R.A.; Van der Laan, J.M.; Akeroyd, M; Wenzel, T.J.; Luesken, F.A.; Veenhuis, M.; Van der Klei, I.; De Winde, J.H.

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  15. Secret Chambers: The Insider Story of Cells and Complex Life

    OpenAIRE

    Farid Pazhoohi

    2014-01-01

    Review of Secret Chambers: The Insider Story of Cells and Complex Life. Martin Brasier. 2012. Oxford University Press, UK. Pp. 320 with 15 black and white illustrations and 8 pages of color plates. £16.99 (hardcover). ISBN 9780199644001.

  16. EFFECTS OF SECRETABLE PLACENTAL FACTORS UPON SECRETION OF CYTOKINES BY THP-1 MONOCYTE-LIKE CELLS

    Directory of Open Access Journals (Sweden)

    Ya. S. Onokhina

    2013-01-01

    Full Text Available Abstract. Мonocytes in feto-placental circulation are exposed to factors secreted by placental tissue. These factors influence monocyte functions in pregnancy. In present study, an in vitro model (monocyte-like THP-1 cells was used for assessing effects of soluble placental factors obtained from women with physiological pregnancies, or preeclampsia cases. The following effects of placental factors were revealed: increased secretion of VEGF by THP-1 cells along with decreased secretion of IL-6, IL-8 and MCP-1 under the influence of placental factors from the I. trimester of pregnancy in comparison with III. trimester. Secretion of IL-6 and MCP-1 by THP-1 cells was increased, and secretion of soluble TNFRII was decreased upon co-cultivation with soluble placental factors from the women with preeclampsia, as compared with placental products from physiological pregnancies.The work is supported by grants ГК № 02.740.11.0711 from Ministry of Education and Science, and НШ-3594.2010.7 grant from the President of Russian Federation.

  17. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway.

    Science.gov (United States)

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang; McCrae, Keith R

    2013-11-28

    The antiphospholipid syndrome is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLAs). Most pathogenic APLAs are directed against β2-glycoprotein I (β2GPI), a plasma phospholipid binding protein. One mechanism by which circulating antiphospholipid/anti-β2GPI antibodies may promote thrombosis is by inducing the release of procoagulant microparticles from endothelial cells. However, there is no information available concerning the mechanisms by which anti-β2GPI antibodies induce microparticle release. In seeking to identify proteins phosphorylated during anti-β2GPI antibody-induced endothelial activation, we observed phosphorylation of nonmuscle myosin II regulatory light chain (RLC), which regulates cytoskeletal assembly. In parallel, we observed a dramatic increase in the formation of filamentous actin, a two- to fivefold increase in the release of endothelial cell microparticles, and a 10- to 15-fold increase in the expression of E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and tissue factor messenger RNA. Microparticle release, but not endothelial cell surface E-selectin expression, was blocked by inhibiting RLC phosphorylation or nonmuscle myosin II motor activity. These results suggest that distinct pathways, some of which mediate cytoskeletal assembly, regulate the endothelial cell response to anti-β2GPI antibodies. Inhibition of nonmuscle myosin II activation may provide a novel approach for inhibiting microparticle release by endothelial cells in response to anti-β2GPI antibodies. PMID:23954892

  18. Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells

    OpenAIRE

    1988-01-01

    We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate- containing molecules (HS[PG]) were the major sulfated products synthesized and secreted...

  19. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  20. Model for glucagon secretion by pancreatic α-cells.

    Directory of Open Access Journals (Sweden)

    Virginia González-Vélez

    Full Text Available Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia. However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic Ca2+, as in other neuroendocrine and endocrine cells, but also by the level of extracellular glucose. We found that, although calcium oscillations are highly variable, the average secretion rates predicted by the model fall into the range of values reported in the literature, for both stimulated and non-stimulated conditions. For low glucose levels, the model predicts that there would be a well-controlled number of releasable granules refilled slowly from a large reserve pool, probably to ensure a secretion rate that could last for several minutes. Studying the α-cell response to the addition of insulin at low glucose, we observe that the presence of insulin reduces glucagon release by decreasing the islet Ca2+ level. This observation is in line with previous work reporting that Ca2+ dynamics, mainly frequency, is altered by insulin. Thus, the present results emphasize the main role played by Ca2+ and glucose in the control of glucagon secretion by α-cells. Our modeling approach also shows that calcium oscillations potentiate glucagon secretion as compared to constant levels of this cellular messenger. Altogether, the model sheds new light on the subcellular mechanisms involved in α-cell exocytosis, and provides a quantitative predictive tool for studying glucagon secretion modulators in physiological and pathological

  1. Naringenin stimulates cholecystokinin secretion in STC-1 cells

    OpenAIRE

    Park, Min; Kim, Kyong; Lee, Yu Mi; Rhyu, Mee Ra; Kim, Hye Young

    2014-01-01

    BACKGROUND/OBJECTIVES Cholecystokinin (CCK), a hormone or neuropeptide, is secreted in response to intraluminal nutrients by enteroendocrine I-cells of the intestine and has important physiological actions related to appetite regulation and satiety. The stimulation on CCK secretion from the intestine is of potential relevance for body weight management. Naringenin (4',5,7-trihydroxyflavanone) and its glycoside naringin (naringenin 7-rhamnoglucoside) have been reported to have many biological ...

  2. Release and intercellular transfer of cell surface CD81 via microparticles

    Czech Academy of Sciences Publication Activity Database

    Fritsching, B.; Schwer, B.; Kartenbeck, J.; Pedal, A.; Hořejší, Václav; Ott, M.

    2002-01-01

    Roč. 169, č. 10 (2002), s. 5531-5537. ISSN 0022-1767 R&D Projects: GA MŠk LN00A026 Keywords : CD81 * microparticles * hepatitis C Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.014, year: 2002

  3. Microparticles as Potential Biomarkers of Cardiovascular Disease

    Energy Technology Data Exchange (ETDEWEB)

    França, Carolina Nunes, E-mail: carolufscar24@gmail.com [Universidade Federal de São Paulo - UNIFESP - UNISA, SP, São Paulo (Brazil); Universidade de Santo Amaro - UNISA, SP, São Paulo (Brazil); Izar, Maria Cristina de Oliveira; Amaral, Jônatas Bussador do; Tegani, Daniela Melo; Fonseca, Francisco Antonio Helfenstein [Universidade Federal de São Paulo - UNIFESP - UNISA, SP, São Paulo (Brazil)

    2015-02-15

    Primary prevention of cardiovascular disease is a choice of great relevance because of its impact on health. Some biomarkers, such as microparticles derived from different cell populations, have been considered useful in the assessment of cardiovascular disease. Microparticles are released by the membrane structures of different cell types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered physiological) and in patients with different pathologies. Many studies have suggested an association between microparticles and different pathological conditions, mainly the relationship with the development of cardiovascular diseases. Moreover, the effects of different lipid-lowering therapies have been described in regard to measurement of microparticles. The studies are still controversial regarding the levels of microparticles that can be considered pathological. In addition, the methodologies used still vary, suggesting the need for standardization of the different protocols applied, aiming at using microparticles as biomarkers in clinical practice.

  4. Acetylation modification regulates GRP78 secretion in colon cancer cells.

    Science.gov (United States)

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  5. Acetylation modification regulates GRP78 secretion in colon cancer cells

    Science.gov (United States)

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  6. Placental Microparticles and MicroRNAs in Pregnant Women with Plasmodium falciparum or HIV Infection.

    Directory of Open Access Journals (Sweden)

    Laura Moro

    Full Text Available During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles.Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement.Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively. miR-517c was overexpressed in mothers with placental malaria (P = 0.034, compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021 and lower production of MCP1 (P = 0.008 than microparticles from non-infected women.In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.

  7. Unstimulated secretion of protein from rat exocrine pancreas cells.

    Science.gov (United States)

    Kramer, M F; Poort, C

    1972-01-01

    Our earlier work demonstrated that the rate of protein synthesis in the exocrine cells of the rat pancreas is constant in different physiological states, including prolonged fasting. In this study we have followed the fate of the protein in the pancreatic cells of the fasting animal in vivo as well as in vitro. The data were obtained by quantitative radioautography and by biochemical determinations. In nonanesthesized, fasting rats, without cannulated pancreatic duct, some 80% of the proteins synthesized at a given time leaves the cell within 12 hr by way of secretion, intracellular breakdown not being important. Two mechanisms of fasting secretion exist. The first, starting at a slow rate after 20 min, is inferred to result from fortuitous contacts of young secretory granules with the apical cell membrane. The rate of secretion is the same in vivo as in vitro, at least during the first 4 hr after pulse labeling. Within 7 hr about 20% of the total amount of newly synthesized protein has left the cell. The second mechanism consists of an orderly movement of the mass of secretory granules towards the apical cell membrane as caused by the continuous assembly of new granules. The granules that come into contact with the cell membrane are discharged. It takes about 7-12 hr for secretory protein transported in this way to reach the cell membrane. The addition of new secretory granules to those present is essential for the second mechanism, for the blockade of protein synthesis by cycloheximide decreases the rate of this phase of secretion without interfering with the secretory process proper. Atropin does not inhibit the fasting secretion in vitro, nor does extensive washing of the tissue slices, excluding possible secretagogues as important factors in fasting secretion. PMID:4331296

  8. Renin secretion from permeabilized juxtaglomerular cells requires a permeant cation

    DEFF Research Database (Denmark)

    Jensen, B L; Ellekvist, Peter; Skøtt, O

    1999-01-01

    The cytosolic concentration of chloride correlates directly with renin secretion from renal juxtaglomerular granular (JG) cells. In the present study, the mechanism by which chloride stimulates renin release was investigated in a preparation of permeabilized rat glomeruli with attached JG cells. ...

  9. Characterisation of BHK-21 cells engineered to secrete human insulin

    OpenAIRE

    Gammell, Patrick; O'Driscoll, Lorraine; Clynes, Martin

    2003-01-01

    Autoimmune destruction of β cells in the pancreas leads to type I, or insulin dependent diabetes mellitus (IDDM), through the loss of endogenous insulin production capacity. This paper describes an attempt to generate ‘artificial’β cells using the fibroblast cell line BHK21. Stable transfectants expressing the human preproinsulin (PPI) gene were isolated and characterised. The resulting clone selected for further analysis (BHK-PPI-C16) was capable of secreting 0.12 pmol proinsulin/hr/105 cell...

  10. Secretion of immunoregulatory cytokines by mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Dobroslav; Kyurkchiev; Ivan; Bochev; Ekaterina; Ivanova-Todorova; Milena; Mourdjeva; Tsvetelina; Oreshkova; Kalina; Belemezova; Stanimir; Kyurkchiev

    2014-01-01

    According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells(MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system.

  11. Chaos evidence in catecholamine secretion at chromaffin cells

    International Nuclear Information System (INIS)

    Highlights: ► Nonlinear behavior in protein secretion. ► Nonlinear characterization of complex behavior in catecholamine secretion. ► Chaos in chromaffin cells activity at the adrenaline secretion. ► Preliminary evidences on the chaotic behavior in catecholamine secretion. - Abstract: Chromaffin cells secrete catecholamine molecules via exocytosis process. Each exocytotic event is characterized by a current spike, which corresponds to the amount of released catecholamine from secretory vesicles after fusing to plasma membrane. The current spike might be measured by the oxidation of catecholamine molecules and can be experimentally detected through amperometry technique. In this contribution, the secretion of catecholamine, namely adrenaline, of a set of bovine chromaffin cells is measured individually at each single cell. The aim is to study quantitative results of chaotic behavior in catecholamine secretion. For analysis, time series were obtained from amperometric measurements of each single chromaffin cell. Three analysis techniques were exploited: (i) A low-order attractor was generated by means of phase space reconstruction, Average Mutual Information (AMI) and False Nearest Neighbors (FNN) were used to compute embedding lag and embedding dimension, respectively. (ii) The properties of power spectrum density of time series were studied by Fast Fourier Transform (FFT) looking for possible dominant frequencies in power spectrum. (iii) Maximun Lyapunov Exponent (MLE) analysis was done to study the divergence of trajectories of the time series. Nevertheless, in order to dismiss the possibility of positiveness of MLE are due to the inherent noise in experiments, seven surrogate data sets computed using the Amplitude Adjusted Fourier Transform (AAFT) algorithm was computed. The phase space reconstruction showed that, in all cases, the trajectories lie in an embedding subspace suggesting oscillatory nature. The FFT analysis showed high dispersion of the power

  12. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway

    OpenAIRE

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang; McCrae, Keith R

    2013-01-01

    Activation of endothelial cells by anti-β2GPI antibodies causes myosin RLC phosphorylation, leading to actin-myosin association.In response to anti-β2GPI antibodies, release of endothelial microparticles, but not E-selectin expression, requires actomyosin assembly.

  13. Regulation of renin secretion by renal juxtaglomerular cells

    DEFF Research Database (Denmark)

    Friis, Ulla G; Madsen, Kirsten; Stubbe, Jane;

    2013-01-01

    calcium paradoxically inhibits renin secretion likely through attenuated formation and enhanced degradation of cAMP; by activation of chloride currents and interaction with calcineurin. Connexin 40 is necessary for localization of JG cells in the vascular wall and for pressure- and macula densa...

  14. Proteomic analysis of exosomes secreted by human mesothelioma cells

    NARCIS (Netherlands)

    J.P.J.J. Hegmans (Joost); A. Hemmes (Annabrita); T.M. Luider (Theo); M.J. Kleijmeer (Monique); J-B. Prins (Jan-Bas); L. Zitvogel; S.A. Burgers (Sjaak); H.C. Hoogsteden (Henk); B.N.M. Lambrecht (Bart); M.P.L. Bard (Martin)

    2004-01-01

    textabstractExosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins pre

  15. Detecting Secreted Analytes from Immune Cells: An Overview of Technologies.

    Science.gov (United States)

    Pike, Kelly A; Hui, Caitlyn; Krawczyk, Connie M

    2016-01-01

    The tumor microenvironment is largely shaped by secreted factors and infiltrating immune cells and the nature of this environment can profoundly influence tumor growth and progression. As such, there is an increasing need to identify and quantify secreted factors by tumor cells, tumor-associated cells, and infiltrating immune cells. To meet this need, the dynamic range of immunoassays such as ELISAs and ELISpots have been improved and the scope of reagents commercially available has been expanded. In addition, new bead-based and membrane-based screening arrays have been developed to allow for the simultaneous detection of multiple analytes in one sample. Similarly, the optimization of intracellular staining for flow cytometry now allows for the quantitation of multiple cytokines from either a purified cell population or a complex mixed cell suspension. Herein, we review the rapidly evolving technologies that are currently available to detect secreted analytes. Emphasis is placed on discussing the advantages and disadvantages of these assays and their applications. PMID:27581018

  16. Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.

    OpenAIRE

    Le Doux, J M; Morgan, J.R.; Snow, R G; Yarmush, M. L.

    1996-01-01

    Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited ampho...

  17. Hsp60 is actively secreted by human tumor cells.

    Directory of Open Access Journals (Sweden)

    Anna M Merendino

    Full Text Available BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. METHODOLOGY/PRINCIPAL FINDINGS: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

  18. Design and characterization of core-shell mPEG-PLGA composite microparticles for development of cell-scaffold constructs

    DEFF Research Database (Denmark)

    Wen, Yanhong; Gallego, Monica Ramos; Nielsen, Lene Feldskov;

    2013-01-01

    combined drug delivery/scaffold system. Composite microparticles were produced by incorporating either alginate/dermatan sulfate (Alg/DS) or alginate/chitosan/dermatan sulfate (Alg/CS/DS) particles in mPEG-PLGA microparticles using coaxial ultrasonic atomization. The encapsulation and distribution of Alg...

  19. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1979-01-01

    Efforts were directed towards maintenance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro. The production of human growth hormone (hGH) by this means would be of benefit for the treatment of certain human hypopituitary diseases such as dwarfism. One of the primary approaches was the testing of agents which may logically be expected to increase hGH release. The progress towards this goal is summarized. Results from preliminary experiments dealing with electrophoresis of pituitary cell for the purpose of somatotroph separation are described.

  20. Putative interaction of brush cells with bicarbonate secreting cells in the proximal corpus mucosa

    Directory of Open Access Journals (Sweden)

    Julia Anna-Maria Eberle

    2013-07-01

    Full Text Available The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the gastric groove, the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII, the cystic fibrosis transmembrane conductance regulator (CFTR and the Na+/H+ exchanger (NHE1. However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracrine interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1 and the neighbouring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution.

  1. Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells

    DEFF Research Database (Denmark)

    Vogel, L K; Suske, G; Beato, M;

    1993-01-01

    A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK and ...... the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type....

  2. Rapamycin promotes Schwann cell migration and nerve growth factor secretion

    Institute of Scientific and Technical Information of China (English)

    Fang Liu; Haiwei Zhang; Kaiming Zhang; Xinyu Wang; Shipu Li; Yixia Yin

    2014-01-01

    Rapamycin, similar to FK506, can promote neural regeneration in vitro. We assumed that the mechanisms of action of rapamycin and FK506 in promoting peripheral nerve regeneration were similar. This study compared the effects of different concentrations of rapamycin and FK506 on Sc hwann cells and investigated effects and mechanisms of rapamycin on improving peripheral nerve regeneration. Results demonstrated that the lowest rapamycin concentration (1.53 nmol/L) more signiifcantly promoted Schwann cell migration than the highest FK506 concentration (100μmol/L). Rapamycin promoted the secretion of nerve growth factors and upregulated growth-associated protein 43 expression in Schwann cells, but did not signiifcantly affect Schwann cell proliferation. Therefore, rapamycin has potential application in peripheral nerve regeneration therapy.

  3. Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN.

    Directory of Open Access Journals (Sweden)

    Jasmina S Redzic

    Full Text Available Extracellular vesicles (EVs are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.

  4. Cre Reporter System To Monitor the Translocation of Type III Secreted Proteins into Host Cells

    OpenAIRE

    Briones, Gabriel; Hofreuter, Dirk; Galán, Jorge E.

    2006-01-01

    Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III s...

  5. Exosomes from B cells and Dendritic cells: mechanisms of formation, secretion and targeting

    NARCIS (Netherlands)

    Buschow, S.I.

    2006-01-01

    Many cell types, including dendritic cells (DC) and B cells, secrete small vesicles called exosomes. Exosomes from immune cells are thought to have immuno-regulatory functions but their precise role remains unresolved. The aim of the studies presented in this thesis was to get more insight into the

  6. Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma

    Science.gov (United States)

    Ko, Sheung-Fat; Hsu, Shu-Yuan; Chen, Chih-Hung; Sung, Pei-Hsun; Zhen, Meng-Shen TongYen-Yi; Chen, Yi-Ling; Huang, Tien-Hung; Chen, Sheng-Yi; Kao, Gour-Shenq; Chen, Hong-Hwa; Chang, Chia-Lo; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Yip, Hon-Kan

    2016-01-01

    This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 106 cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 106, IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34+, CXCR4+, c-Kit+, CK19+, VEGF+ and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-β), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma.

  7. Adipocyte secreted factors enhance aggressiveness of prostate carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Ângela Moreira

    Full Text Available Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1 and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01. Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01. Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05. Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

  8. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

    International Nuclear Information System (INIS)

    It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation

  9. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

    Energy Technology Data Exchange (ETDEWEB)

    Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, São Paulo, SP, Brasil, Departamento de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2014-04-15

    It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation.

  10. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

    Science.gov (United States)

    Sousa, Cristovão M; Biancur, Douglas E; Wang, Xiaoxu; Halbrook, Christopher J; Sherman, Mara H; Zhang, Li; Kremer, Daniel; Hwang, Rosa F; Witkiewicz, Agnes K; Ying, Haoqiang; Asara, John M; Evans, Ronald M; Cantley, Lewis C; Lyssiotis, Costas A; Kimmelman, Alec C

    2016-08-25

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment. PMID:27509858

  11. PLGA-based microparticles loaded with bacterial-synthesized prodigiosin for anticancer drug release: Effects of particle size on drug release kinetics and cell viability.

    Science.gov (United States)

    Obayemi, J D; Danyuo, Y; Dozie-Nwachukwu, S; Odusanya, O S; Anuku, N; Malatesta, K; Yu, W; Uhrich, K E; Soboyejo, W O

    2016-09-01

    This paper presents the synthesis and physicochemical characterization of biodegradable poly (d,l-lactide-co-glycolide) (PLGA)-based microparticles that are loaded with bacterial-synthesized prodigiosin drug obtained from Serratia marcescens subsp. Marcescens bacteria for controlled anticancer drug delivery. The micron-sized particles were loaded with anticancer drugs [prodigiosin (PG) and paclitaxel (PTX) control] using a single-emulsion solvent evaporation technique. The encapsulation was done in the presence of PLGA (as a polymer matrix) and poly-(vinyl alcohol) (PVA) (as an emulsifier). The effects of processing conditions (on the particle size and morphology) are investigated along with the drug release kinetics and drug-loaded microparticle degradation kinetics. The localization and apoptosis induction by prodigiosin in breast cancer cells is also elucidated along with the reduction in cell viability due to prodigiosin release. The implication of this study is for the potential application of prodigiosin PLGA-loaded microparticles for controlled delivery of cancer drug and treatment to prevent the regrowth or locoregional recurrence, following surgical resection of triple negative breast tumor. PMID:27207038

  12. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hogan, Niamh M. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Joyce, Myles R. [Department of Colorectal Surgery, University College Hospital, Galway (Ireland); Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy [Regenerative Medicine Institute, National University of Ireland, Galway (Ireland); Kerin, Michael J. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Dwyer, Roisin M., E-mail: roisin.dwyer@nuigalway.ie [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)

    2013-06-14

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  13. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    International Nuclear Information System (INIS)

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  14. Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

    OpenAIRE

    1992-01-01

    Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine ...

  15. Steroid hormone secretion in inflammatory breast cancer cell lines.

    Science.gov (United States)

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were 90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  16. Role of microparticles in endothelial dysfunction and arterial hypertension

    Institute of Scientific and Technical Information of China (English)

    Thomas; Helbing; Christoph; Olivier; Christoph; Bode; Martin; Moser; Philipp; Diehl

    2014-01-01

    Microparticles are small cell vesicles that can be released by almost all eukaryotic cells during cellular stress and cell activation. Within the last 1-2 decades it has been shown that microparticles are useful blood surrogate markers for different pathological conditions, such as vascular inflammation, coagulation and tumour diseases. Several studies have investigated the abundance of microparticles of different cellular origins in multiple cardiovascular diseases. It thereby has been shown that microparticles released by platelets, leukocytes and endothelial cells can be found in conditions of endothelial dysfunction, acute and chronic vascular inflammation and hypercoagulation. In addition to their function as surrogate markers, several studies indicate that circulating microparticles can fuse with distinct target cells, such as endothelial cells or leukocyte, and thereby deliver cellular components of their parental cells to the target cells. Hence, microparticles are a novel entity of circulating, paracrine, biological vectors which can influence the phenotype, the function and presumably even the transcriptome of their target cells.This review article aims to give a brief overview about the microparticle biology with a focus on endothelial activation and arterial hypertension. More detailed information about the role of microparticles in pathophysiology and disease can be found in already published work.

  17. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1.

    NARCIS (Netherlands)

    Nolte-'t Hoen, E.N.; Buschow, S.I.; Anderton, S.M.; Stoorvogel, W.; Wauben, M.H.M.

    2009-01-01

    Dendritic cells (DCs) are known to secrete exosomes that transfer membrane proteins, like major histocompatibility complex class II, to other DCs. Intercellular transfer of membrane proteins is also observed during cognate interactions between DCs and CD4(+) T cells. The acquired proteins are functi

  18. Activated Human T Cells Secrete Exosomes That Participate in IL-2 Mediated Immune Response Signaling

    OpenAIRE

    Wahlgren, Jessica; Tanya De L Karlson; Glader, Pernilla; Telemo, Esbjörn; Valadi, Hadi

    2012-01-01

    It has previously been shown that nano-meter sized vesicles (30–100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3+ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3+ T cells have on resting CD3+ T cells by studying T cell proliferation, c...

  19. Secretion mechanisms of volatile organic compounds in specialized cells of aromatic plants

    OpenAIRE

    Caissard, Jean-Claude; Joly, Caroline; Bergougnoux, Véronique; Hugueney, Philippe; Mauriat, Mélanie; Baudino, Sylvie

    2004-01-01

    The present review focuses on cells secreting volatile odorant compounds. This cell type is found in a wide variety of plants, grouped under the term aromatic plants. Such secreting cells are very diverse in morphology, from highly specialized trichomes to nonspecialized cells, including the secretory epidermal cells of petals and osmophores. In these various types of cell, the biosynthetic pathways of three main groups of volatile organic compounds are recognized: isoprenoids, fatty acid der...

  20. Induction of insulin secretion in engineered liver cells by nitric oxide

    Directory of Open Access Journals (Sweden)

    Özcan Sabire

    2007-10-01

    Full Text Available Abstract Background Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. Results Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. Conclusion Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.

  1. The study of exosomes and microvesicles secreted from breast cancer cell lines

    OpenAIRE

    Zheng, Ying

    2012-01-01

    Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD...

  2. Tracking Mesenchymal Stem Cells with Iron Oxide Nanoparticle Loaded Poly(lactide-co-glycolide) Microparticles

    OpenAIRE

    Xu, Chenjie; Miranda-Nieves, David; Ankrum, James A.; Matthiesen, Mads Emil; Phillips, Joseph A.; Roes, Isaac; Wojtkiewicz, Gregory R.; Juneja, Vikram; Kultima, Jens Roat; Zhao, Weian; Vemula, Praveen Kumar; Lin, Charles P.; Nahrendorf, Matthias; Karp, Jeffrey M.

    2012-01-01

    Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its non-invasiveness, deep penetration, and high spatial resolution. While tracking cells in pre-clinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron conte...

  3. Tumor cell secretion of soluble factor(s) for specific immunosuppression

    OpenAIRE

    Kano, Arihiro

    2015-01-01

    Studies of tumor models using syngeneic transplantation have advanced our understanding of tumor immunity, including both immune surveillance and evasion. Murine mammary carcinoma 4T1 cells secrete immunosuppressive soluble factors as demonstrated in splenocyte culture. Cultured primary splenocytes secrete IFN-γ, which was strikingly elevated when the cells were isolated from 4T1 tumor-bearing mice. The secretion of IFN-γ peaked a week after 4T1 inoculation and then declined. This reduction m...

  4. Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically.

    OpenAIRE

    Love, Kerry Routenberg; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J. Christopher

    2010-01-01

    The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single-cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell-cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner.

  5. Effect of Ureaplasma Urealyticum Infection on the Interleukin-1 Secretion in Rat Sertoli Cells

    Institute of Scientific and Technical Information of China (English)

    李伟毅; 周葵; 陈广洁; 席晔斌; 王保国

    2001-01-01

    Objective To investigate the effect of Ureaplasma Urealyticum (UU) on the expression of IL-1 secretion in rat Sertoli cells Material & Methods Isolated rat Sertoli cells were infected by living UU , UU super natants, inactivated UU of different dosage respectively. IL- 1 secretion by rat Sertoli cells is determined by using group t-test and F-test.Results UU infection, both living UU and supernatant without UU, could inhibit the IL-1 secretion in rats Sertoli cells (P< 0. 01).Conclusion The infection of Sertoli cells by UU inhibit the biological function of the Sertoli cells in rat.

  6. Secretion of mucus proteinase inhibitor and elafin by Clara cell and type II pneumocyte cell lines.

    Science.gov (United States)

    Sallenave, J M; Silva, A; Marsden, M E; Ryle, A P

    1993-02-01

    The regulation of proteinases secreted by neutrophils is very important for the prevention of tissue injury. We recently described the isolation of elafin from bronchial secretions, a new elastase-specific inhibitor that is also found in the skin of patients with psoriasis. In this study, we investigated the secretion of elafin and mucus proteinase inhibitor (MPI), another inhibitor showing sequence similarity with elafin, in two lung carcinoma cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. The results presented show that the two inhibitors are produced when the cells are cultured either in serum-free or in serum-containing media. MPI was detected immunologically as a unique molecule of M(r) 14 kD, in accordance with previous studies. Conversely, one or two elafin-immunoreactive species were detected depending on the cell line: a 12- to 14-kD species was observed in the A549 cell line, regardless of the culture conditions, whereas in the NCI-H322 cell line we detected a 6-kD species in serum-containing (10% fetal calf serum) conditions and a 12- to 14-kD species in serum-free conditions. The 12- to 14-kD molecule probably represents an active precursor of elafin. Whether the cleavage of the 12- to 14-kD precursor giving rise to the elafin molecule is of any physiologic significance is not known. In showing for the first time that MPI and elafin (and its precursor) are secreted by the A549 cell line, this report implicates the type II alveolar cell in the defense of the peripheral lung against the neutrophil elastase secreted during inflammation. PMID:8427705

  7. The flow of red blood cells in stenosed microvessels and the influence of red blood cells on wall-bounded rolling motion of microparticles

    Science.gov (United States)

    Vahidkhah, Koohyar; Balogh, Peter; Bagchi, Prosenjit

    2015-11-01

    In the first part of this work, we consider a 3D computational study of the flow of deformable red blood cells in stenosed microvessels. We observe that the apparent viscosity of blood increases by several folds, and the rate of increase with increasing vessel diameter is also higher than that in non-stenosed vessels, implying an enhancement of the well-known Fahraeus-Lindqvist effect. The flow of the red blood cells causes time-dependent fluctuations in the blood flow rate. The RMS of the flow rate oscillations in the stenosed vessel is observed to be significantly higher than that in the non-stenosed vessel. Furthermore, several folds increase in the Eulerian velocity fluctuations and a transient flow reversal upstream the stenosed region are also observed, which would not occur in absence of the cells. In the second part, we consider the adhesive rolling motion of wall-bounded microparticles in presence of flowing red blood cells in microvessels. We observe two contradictory role of the red blood cells: On one hand, the cells facilitate the establishment of the particle-wall contact, and, thereby, initiation of adhesion. On the other hand, they augment the rolling velocity of the particles. Implications of these results on the optimal design of drug carriers are discussed.

  8. Physics of microparticle acoustophoresis

    DEFF Research Database (Denmark)

    Barnkob, Rune

    2012-01-01

    of microparticle acoustophoresis and to develop methods for future advancement of its use. Throughout the work on this thesis the author and co-workers1 have studied the physics of microparticle acoustophoresis by comparing quantitative measurements to a theoretical framework consisting of existing hydrodynamic...

  9. A Rare Corticotroph-Secreting Tumor with Coexisting Prolactin and Growth Hormone Staining Cells

    OpenAIRE

    Subramanian Kannan; Staugaitis, Susan M.; Weil, Robert J.; Betul Hatipoglu

    2012-01-01

    Pituitary adenomas can express and secrete different hormones. Expression of pituitary hormones in nonneoplastic pituitary cells is regulated by different transcription factors. Some pituitary adenomas show plurihormonal expression. The most commonly reported plurihormonal adenomas are composed of somatotrophs, lactotrophs, thyrotrophs and gonadotrophs. Pituitary adenomas composed of both corticotroph and somatolactotroph secreting cells are not common because transcription factors regulating...

  10. Oxygen-Purged Microfluidic Device to Enhance Cell Viability in Photopolymerized PEG Hydrogel Microparticles.

    Science.gov (United States)

    Xia, Bingzhao; Krutkramelis, Kaspars; Oakey, John

    2016-07-11

    Encapsulating cells within biocompatible materials is a widely used strategy for cell delivery and tissue engineering. While cells are commonly suspended within bulk hydrogel-forming solutions during gelation, substantial interest in the microfluidic fabrication of miniaturized cell encapsulation vehicles has more recently emerged. Here, we utilize multiphase microfluidics to encapsulate cells within photopolymerized picoliter-volume water-in-oil droplets at high production rates. The photoinitiated polymerization of polyethylene glycol diacrylate (PEGDA) is used to continuously produce solid particles from aqueous liquid drops containing cells and hydrogel forming solution. It is well understood that this photoinitiated addition reaction is inhibited by oxygen. In contrast to bulk polymerization in which ambient oxygen is rapidly and harmlessly consumed, allowing the polymerization reaction to proceed, photopolymerization within air permeable polydimethylsiloxane (PDMS) microfluidic devices allows oxygen to be replenished by diffusion as it is depleted. This sustained presence of oxygen and the consequential accumulation of peroxy radicals produce a dramatic effect upon both droplet polymerization and post-encapsulation cell viability. In this work we employ a nitrogen microjacketed microfluidic device to purge oxygen from flowing fluids during photopolymerization. By increasing the purging nitrogen pressure, oxygen concentration was attenuated, and increased post-encapsulation cell viability was achieved. A reaction-diffusion model was used to predict the cumulative intradroplet concentration of peroxy radicals, which corresponded directly to post-encapsulation cell viability. The nitrogen-jacketed microfluidic device presented here allows the droplet oxygen concentration to be finely tuned during cell encapsulation, leading to high post-encapsulation cell viability. PMID:27285343

  11. Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells

    International Nuclear Information System (INIS)

    Pulmonary surfactant is synthesized and secreted by alveolar type II cells. Radioactive phosphatidylcholine has been used as a marker for surfactant secretion. The authors report findings that suggest that surfactant inhibits secretion of 3H-labeled phosphatidylcholine by cultured rat type II cells. The lipid components and the surfactant protein group of M/sub r/ 26,000-36,000 (SP 26-36) inhibit secretion to different extents. Surfactant lipids do not completely inhibit release; in concentrations of 100 μg/ml, lipids inhibit stimulated secretion by 40%. SP 26-36 inhibits release with an EC50 of 0.1 μg/ml. At concentrations of 1.0 μg/ml, SP 26-36 inhibits basal secretion and reduces to basal levels secretion stimulated by terbutaline, phorbol 12-myristate 13-acetate, and the ionophore A23187. The inhibitory effect of SP 26-36 can be blocked by washing type II cells after adding SP 26-36, by heating the proteins to 1000C for 10 min, by adding antiserum specific to SP 26-36, or by incubating cells in the presence of 0.2 mM EGTA. SP 26-36 isolated from canine and human sources also inhibits phosphatidylcholine release from rat type II cells. Neither type I collagen nor serum apolipoprotein A-1 inhibits secretion. These findings are compatible with the hypothesis that surfactant secretion is under feedback regulatory control

  12. Characterization of secreted proteins in HepG2 and LO2 cells by Raman spectroscopy

    Science.gov (United States)

    Lin, Juqiang; Ruan, Qiuyong; Liao, Fadian; Lin, Jinyong; Huang, Zufang; Liu, Nenrong; Chen, Rong

    2014-11-01

    Secreted proteins, the promising source of biomarkers for early detection and diagnosis of cancer, have received considerable attention. Raman spectroscopy and principal component analysis (PCA) were used to characterize the secreted proteins collected from the cell cultures of human hepatoma cell line HepG2 and normal human liver cell line LO2 in this paper. We found the major difference of secreted proteins Raman spectra between HepG2 and LO2 cells were in the range of 1200cm-1-1800cm-1. Compared with LO2 cells, some significant changes based on secondary structure of secreted proteins in HepG2 cells were observed, including the increase in the relative intensity of the band at 1004cm-1, 1445cm-1, 1674cm-1 and the decrease at 1074cm-1. These variations of Raman bands indicated that the species and conformation of secreted proteins in HepG2 cells changed. The measured Raman spectra of the two groups were separated into two distinct clusters with no overlap and high specificity and sensitivity by PCA. These results show that the combination of Raman spectroscopy and PCA analysis may be a powerful tool for distinguishing the secreted proteins between human hepatoma cells and normal human liver cells, provide a new thought to analyze the secreted proteins from cancer cells and find a novel cancer biomarker.

  13. Synthesis and functionalization of nano- and micro-particles for sensing and therapy in living cells

    OpenAIRE

    Penon Esteva, Oriol

    2013-01-01

    In the present thesis Supramolecular chemistry is exploited to approach applications in the area of Nanomedicine, and it is, focused on the design and preparation of different micro and nanotools for sensing and therapy, in living cells. Initially, the combination of silicon surface chemistry with the incorporation of bioactive molecules has been investigated in order to obtain a potentially microtool suitable for cell tagging. Furthermore, the design and synthesis of organic compounds as int...

  14. Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

    OpenAIRE

    Kidd, M.; Tang, L. H.; Miu, K.; Lawton, G. P.; Sandor, A.; Modlin, I. M.

    1996-01-01

    INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of...

  15. Glucose and acute exercise influence factors secreted by circulating angiogenic cells in vitro

    OpenAIRE

    Witkowski, Sarah; Guhanarayan, Gayatri; Burgess, Rachel

    2016-01-01

    Abstract Circulating angiogenic cells (CAC) influence vascular repair through the secretion of proangiogenic factors and cytokines. While CAC are deficient in patients with diabetes and exercise has a beneficial effect on CACs, the impact of these factors on paracrine secretion from CAC is unknown. We aimed to determine whether the in vitro secretion of selected cytokines and nitric oxide (NO) from CAC is influenced by hyperglycemia and acute exercise. Colony‐forming unit CAC (CFU‐CAC) were c...

  16. Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells

    OpenAIRE

    Cakmak, Hakan; Franciosi, Federica; Zamah, A. Musa; Cedars, Marcelle I; Conti, Marco

    2016-01-01

    Oocyte fitness to support embryo development and pregnancy is dependent on an elaborate cross-talk with the surrounding environment of the ovarian follicle. Here, we show that this cross-talk continues during the periovulatory period when a new set of bioactive molecules is secreted by the oocyte in mice and humans. This shift in pattern of secretion is dependent on oocyte maturation and on paracrine factors secreted by somatic cells at the time of ovulation. These changes in pattern of secre...

  17. Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

    OpenAIRE

    Shapiro, M.; Matthews, J.; Hecht, G; Delp, C; Madara, J. L.

    1991-01-01

    T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with h...

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

    Directory of Open Access Journals (Sweden)

    Sylvain J Le Marchand

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

  19. Habituation in the single cell: diminished secretion of norepinephrine with repetitive depolarization of PC12 cells.

    OpenAIRE

    McFadden, P N; Koshland, D. E.

    1990-01-01

    Neuronally differentiated PC12 cells secrete decreasing amounts of [3H]norepinephrine when repetitively stimulated by depolarizing concentrations of potassium ion. The decreasing response shows attributes that have been classically ascribed to response habituation, a behavior commonly observed in nervous systems but found here in a homogeneous cell type. Alteration of the habituation pattern was caused by activators of the protein kinase C pathway and of voltage-gated calcium channels.

  20. Habituation in the Single Cell: Diminished Secretion of Norepinephrine with Repetitive Depolarization of PC12 Cells

    Science.gov (United States)

    McFadden, Philip N.; Koshland, Daniel E., Jr.

    1990-03-01

    Neuronally differentiated PC12 cells secrete decreasing amounts of [^3H]norepinephrine when repetitively stimulated by depolarizing concentrations of potassium ion. The decreasing response shows attributes that have been classically ascribed to response habituation, a behavior commonly observed in nervous systems but found here in a homogeneous cell type. Alteration of the habituation pattern was caused by activators of the protein kinase C pathway and of voltage-gated calcium channels.

  1. Habituation in the single cell: Diminished secretion of norepinephrine with repetitive depolarization of PC12 cells

    International Nuclear Information System (INIS)

    Neuronally differentiated PC12 cells secrete decreasing amount of [3H]norepinephrine when repetitively stimulated by depolarizing concentrations of potassium ion. The decreasing response shows attributes that have been classically ascribed to response habituation, a behavior commonly observed in nervous systems but found here in a homogeneous cell type. Alteration of the habituation pattern was caused by activators of the protein kinase C pathway and of voltage-gated calcium channels

  2. Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

    International Nuclear Information System (INIS)

    The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells

  3. Decellularized extracellular matrix microparticles as a vehicle for cellular delivery in a model of anastomosis healing.

    Science.gov (United States)

    Hoganson, David M; Owens, Gwen E; Meppelink, Amanda M; Bassett, Erik K; Bowley, Chris M; Hinkel, Cameron J; Finkelstein, Eric B; Goldman, Scott M; Vacanti, Joseph P

    2016-07-01

    Extracellular matrix (ECM) materials from animal and human sources have become important materials for soft tissue repair. Microparticles of ECM materials have increased surface area and exposed binding sites compared to sheet materials. Decellularized porcine peritoneum was mechanically dissociated into 200 µm microparticles, seeded with fibroblasts and cultured in a low gravity rotating bioreactor. The cells avidly attached and maintained excellent viability on the microparticles. When the seeded microparticles were placed in a collagen gel, the cells quickly migrated off the microparticles and through the gel. Cells from seeded microparticles migrated to and across an in vitro anastomosis model, increasing the tensile strength of the model. Cell seeded microparticles of ECM material have potential for paracrine and cellular delivery therapies when delivered in a gel carrier. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1728-1735, 2016. PMID:26946064

  4. Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix

    OpenAIRE

    Luecke, N; Templin, C; Muetzelburg, M V; Neumann, D.; Just, I; Pich, A.(IFIC, Universitat de València, CSIC, Apt. Correus 22085, 46071 , València, Spain)

    2010-01-01

    Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. ...

  5. Inhibition of Salmonella typhimurium Invasion by Host Cell Expression of Secreted Bacterial Invasion Proteins

    OpenAIRE

    Carlson, Steve A.; Jones, Bradley D.

    1998-01-01

    Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the ro...

  6. Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)

    OpenAIRE

    Dagur, Pradeep K; Biancotto, Angélique; Stansky, Elena; Sen, H. Nida; Nussenblatt, Robert B.; McCoy, J. Philip

    2014-01-01

    Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults...

  7. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    Science.gov (United States)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

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

    International Nuclear Information System (INIS)

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl2-induced VEGF secretion in mast cells occurs by a Ca2+-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl2) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl2-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl2-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-15

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

  10. Optimized method for identification of the proteomes secreted by cardiac cells

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Van Eyk, J.E.

    2013-01-01

    Roč. 1005, č. 1005 (2013), s. 225-235. ISSN 1940-6029 Institutional support : RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

  11. Ionizing radiation induces tumor cell lysyl oxidase secretion

    DEFF Research Database (Denmark)

    Shen, Colette J; Sharma, Ashish; Vuong, Dinh-Van;

    2014-01-01

    BACKGROUND: Ionizing radiation (IR) is a mainstay of cancer therapy, but irradiation can at times also lead to stress responses, which counteract IR-induced cytotoxicity. IR also triggers cellular secretion of vascular endothelial growth factor, transforming growth factor beta and matrix metallop......BACKGROUND: Ionizing radiation (IR) is a mainstay of cancer therapy, but irradiation can at times also lead to stress responses, which counteract IR-induced cytotoxicity. IR also triggers cellular secretion of vascular endothelial growth factor, transforming growth factor beta and matrix...

  12. In vitro impact of pegvisomant on growth hormone-secreting pituitary adenoma cells.

    Science.gov (United States)

    Cuny, Thomas; Zeiller, Caroline; Bidlingmaier, Martin; Défilles, Céline; Roche, Catherine; Blanchard, Marie-Pierre; Theodoropoulou, Marily; Graillon, Thomas; Pertuit, Morgane; Figarella-Branger, Dominique; Enjalbert, Alain; Brue, Thierry; Barlier, Anne

    2016-07-01

    Pegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy β-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2±17% at 1μg/mL (Phuman primary tumors or GH4C1 cell line was observed. We conclude that PEG inhibits the secretion of GH and PRL in primary cultures of human GH(/PRL)-secreting pituitary adenomas without effect on cell viability or cell proliferation. PMID:27267119

  13. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

    OpenAIRE

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-01-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns w...

  14. CELL-WALL GROWTH AND PROTEIN SECRETION IN FUNGI

    NARCIS (Netherlands)

    SIETSMA, JH; WOSTEN, HAB; WESSELS, JGH

    1995-01-01

    Secretion of proteins is a vital process in fungi. Because hyphal walls form a diffusion barrier for proteins, a mechanism different from diffusion probably exist to transport proteins across the wall. In Schizophyllum commune, evidence has been obtained for synthesis at the hyphal apex of wall comp

  15. Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

    Directory of Open Access Journals (Sweden)

    Julie Sahler

    Full Text Available Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ. In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles

  16. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

    Directory of Open Access Journals (Sweden)

    Jonathan A Rose

    Full Text Available Pulmonary arterial hypertension (PAH is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  17. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension

    Science.gov (United States)

    Rose, Jonathan A.; Wanner, Nicholas; Cheong, Hoi I.; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V.; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

  18. Microparticles and Exosomes in Gynecologic Neoplasias

    NARCIS (Netherlands)

    R. Nieuwland; J.A.M. van der Post; C.A.R. Lok Gemma; G. Kenter; A. Sturk

    2010-01-01

    This review presents an overview of the functions of microparticles and exosomes in gynecologic neoplasias. Growing evidence suggests that vesicles released from cancer cells in gynecologic malignancies contribute to the hypercoagulable state of these patients and contribute to tumor progression by

  19. Harvesting microalgae with microwave synthesized magnetic microparticles

    Czech Academy of Sciences Publication Activity Database

    Procházková, G.; Šafařík, Ivo; Brányik, T.

    2013-01-01

    Roč. 130, FEB (2013), s. 472-477. ISSN 0960-8524 R&D Projects: GA MŠk LH12190 Institutional support: RVO:67179843 Keywords : harvesting microalgae * iron oxide magnetic microparticles * non-covalent interactions * microwave treatment * cell demagnetization Subject RIV: EI - Biotechnology ; Bionics Impact factor: 5.039, year: 2013

  20. High speed versus pulsed images for micro-particle image velocimetry: a direct comparison of red blood cells versus fluorescing tracers as tracking particles

    International Nuclear Information System (INIS)

    High speed photography in micro-particle image velocimetry (μPIV) using red blood cells as tracer particles and the use of fluorescing tracer particles (in conjunction with pulsed images) are directly compared by using both methods simultaneously. Measurements are taken on the same blood sample in the same microchip using both methods. This work directly and statistically compares the two methods of μPIV measurement in a controlled in vitro environment for the first time in literature. The pulsed method using fluorescing tracer particles is found to decrease the depth of correlation as expected, and to better represent the shape of the velocity profile. Two methods of velocity characterization are used (single and double parameter) and the pulsed images provide better shape representation in both cases. (paper)

  1. Sodium arsenite impairs insulin secretion and transcription in pancreatic β-cells

    International Nuclear Information System (INIS)

    Human studies have shown that chronic inorganic arsenic (iAs) exposure is associated with a high prevalence and incidence of type 2 diabetes. However, the mechanism(s) underlying this effect are not well understood, and practically, there is no information available on the effects of arsenic on pancreatic β-cells functions. Thus, since insulin secreted by the pancreas plays a crucial role in maintaining glucose homeostasis, our aim was to determine if sodium arsenite impairs insulin secretion and mRNA expression in single adult rat pancreatic β-cells. Cells were treated with 0.5, 1, 2, 5 and 10 μM sodium arsenite and incubated for 72 and 144 h. The highest dose tested (10 μM) decreased β-cell viability, by 33% and 83%, respectively. Insulin secretion and mRNA expression were evaluated in the presence of 1 and 5 μM sodium arsenite. Basal insulin secretion, in 5.6 mM glucose, was not significantly affected by 1 or 5 μM treatment for 72 h, but basal secretion was reduced when cells were exposed to 5 μM sodium arsenite for 144 h. On the other hand, insulin secretion in response to 15.6 mM glucose decreased with sodium arsenite in a dose-dependent manner in such a way that cells were no longer able to distinguish between different glucose concentrations. We also showed a significant decrease in insulin mRNA expression of cells exposed to 5 μM sodium arsenite during 72 h. Our data suggest that arsenic may contribute to the development of diabetes mellitus by impairing pancreatic β-cell functions, particularly insulin synthesis and secretion

  2. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  3. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Science.gov (United States)

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin

    2010-01-01

    Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H(+) into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+) chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca(2+) release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+) pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+) mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion. PMID:20169059

  4. Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

    Science.gov (United States)

    Hare, Rebekah F; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. PMID:25158041

  5. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  6. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Science.gov (United States)

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel

  7. Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

    Directory of Open Access Journals (Sweden)

    Lucio Barile

    2012-01-01

    Full Text Available The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  8. Distinct linkage between post-translational processing and differential secretion of progastrin derivatives in endocrine cells

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Rehfeld, Jens Frederik

    2008-01-01

    Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the...... secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated...... in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C...

  9. Syntaxin-4 is essential for IgE secretion by plasma cells

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Arman; DeCourcey, Joseph; Larbi, Nadia Ben [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland); Loughran, Sinéad T.; Walls, Dermot [School of Biotechnology and National Centre for Sensor Research, Dublin City University (Ireland); Loscher, Christine E., E-mail: christine.loscher@dcu.ie [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland)

    2013-10-11

    Highlights: •Knock-down of syntaxin-4 in U266 plasma cells resulted in reduction of IgE secretion. •Knock-down of syntaxin-4 also leads to the accumulation of IgE in the cell. •Immuno-fluorescence staining shows co-localisation of IgE and syntaxin-4 in U266 cells. •Findings suggest a critical requirement for syntaxin-4 in IgE secretion from plasma cells. -- Abstract: The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

  10. Syntaxin-4 is essential for IgE secretion by plasma cells

    International Nuclear Information System (INIS)

    Highlights: •Knock-down of syntaxin-4 in U266 plasma cells resulted in reduction of IgE secretion. •Knock-down of syntaxin-4 also leads to the accumulation of IgE in the cell. •Immuno-fluorescence staining shows co-localisation of IgE and syntaxin-4 in U266 cells. •Findings suggest a critical requirement for syntaxin-4 in IgE secretion from plasma cells. -- Abstract: The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients

  11. Resolvins Decrease Oxidative Stress Mediated Macrophage and Epithelial Cell Interaction through Decreased Cytokine Secretion

    OpenAIRE

    Cox, Ruan; Phillips, Oluwakemi; Fukumoto, Jutaro; Fukumoto, Itsuko; Tamarapu Parthasarathy, Prasanna; Mandry, Maria; Cho, Young; Lockey, Richard; Kolliputi, Narasaiah

    2015-01-01

    Background Inflammation is a key hallmark of ALI and is mediated through ungoverned cytokine signaling. One such cytokine, interleukin-1beta (IL-1β) has been demonstrated to be the most bioactive cytokine in ALI patients. Macrophages are the key players responsible for IL-1β secretion into the alveolar space. Following the binding of IL-1β to its receptor, “activated” alveolar epithelial cells show enhanced barrier dysfunction, adhesion molecule expression, cytokine secretion, and leukocyte a...

  12. Altered protein secretions during interactions between adipose tissue- or bone marrow-derived stromal cells and inflammatory cells

    OpenAIRE

    Hattori, Hidemi; Ishihara, Masayuki

    2015-01-01

    Introduction Paracrine effects can be exploited in cell-based therapies that secrete factors, such as chemokines and cytokines, and can recruit inflammatory cells to transplants. In this study, mouse adipose tissue-derived stromal cells (ASCs) and bone marrow-derived stromal cells (ST2 cells) were used to examine changes in paracrine interactions with inflammation cells. Methods Green fluorescent protein positive (GFP+) bone marrow cells (BMCs) were injected into an irradiated mouse via the f...

  13. Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells

    Directory of Open Access Journals (Sweden)

    Valero Antonio

    2011-02-01

    Full Text Available Abstract Background Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps. Methods Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10-11M-10-5M with/without desloratadine (10-5M. Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10-11M-10-5M and/or desloratadine (10-5M and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM compared to control. Results Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10-5M desloratadine and 10-9M mometasone reduced IL-6 secretion (48 ± 11%, p -11M and desloratadine (10-5M provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p Conclusions These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.

  14. Polarization of calcium signaling and fluid secretion in salivary gland cells.

    Science.gov (United States)

    Ambudkar, I S

    2012-01-01

    The secretion of fluid, electrolytes, and protein by exocrine gland acinar cells is a vectorial process that requires the coordinated regulation of multiple channel and transporter proteins, signaling components, as well as mechanisms involved in vesicular fusion and water transport. Most critical in this is the regulation of cytosolic free [Ca(2+)] ([Ca(2+)](i)) in response to neurotransmitter stimulation. Control of [Ca(2+)](i) increase in specific regions of the cell is the main determinant of fluid and electrolyte secretion in salivary gland acinar cells as it regulates several major ion flux mechanisms as well as the water channel that are required for this process. Polarized [Ca(2+)](i) signals are also essential for protein secretion in pancreatic acinar cells. Thus, the mechanisms that generate and modulate these compartmentalized [Ca(2+)](i) signals are central to the regulation of exocrine secretion. These mechanisms include membrane receptors for neurotransmitters, intracellular Ca(2+) release channels, Ca(2+) entry channels, as well Ca(2+) as pumps and mitochondria. The spatial arrangement of proteins involved in Ca(2+) signaling is of primary significance in the generation of specific compartmentalized [Ca(2+)](i) signals. Within these domains, both local and global [Ca(2+)](i) changes are tightly controlled. Control of secretion is also dependent on the targeting of ion channels and transporters to specific domains in the cell where their regulation by [Ca(2+)](i) signals is facilitated. Together, the polarized localization of Ca(2+) signaling and secretory components drive vectorial secretion of fluid, electrolytes, and proteins in the exocrine salivary glands and pancreas. This review will discuss recent findings which have led to resolution of the molecular components underlying the spatio-temporal control of [Ca(2+)](i) signals in exocrine gland cells and their role in secretion. PMID:23061636

  15. Zinc directly stimulates cholecystokinin secretion from enteroendocrine cells and reduces gastric emptying in rats.

    Science.gov (United States)

    Nakajima, Shingo; Hira, Tohru; Iwaya, Hitoshi; Hara, Hiroshi

    2016-07-15

    Zinc, an essential mineral element, regulates various physiological functions such as immune responses and hormone secretion. Cholecystokinin (CCK), a gut hormone, has a role in protective immunity through the regulation of gastrointestinal motility, appetite, and inflammatory response. Here, we examined the effect of zinc on CCK secretion in STC-1 cells, an enteroendocrine cell line derived from murine duodenum, and in rats. Extracellular zinc triggered CCK secretion accompanied with increased intracellular Ca(2+) and Zn(2+) mobilization in STC-1 cells. Zinc-induced CCK secretion was abolished in the absence of intracellular Zn(2+) or extracellular calcium. Upon inhibition of transient receptor potential ankyrin 1 (TRPA1), extracellular zinc failed to increase intracellular Ca(2+) and subsequent CCK secretion. In rats, oral zinc administration decreased gastric emptying through the activation of CCK signaling. These results suggest that zinc is a novel stimulant for CCK secretion through the activation of TRPA1 related to intracellular Zn(2+) and Ca(2+) mobilization. PMID:27107934

  16. Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

    International Nuclear Information System (INIS)

    Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients

  17. The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis.

    LENUS (Irish Health Repository)

    Matlawska-Wasowska, Ksenia

    2010-12-01

    Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.

  18. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    OpenAIRE

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and ar...

  19. Effects of Ovarian Steroids on Immunoglobulin-Secreting Cell Function in Healthy Women

    OpenAIRE

    Lü, Fabien X.; Ma, Zhongmin; Moser, Susie; Evans, Thomas G.; Christopher J. Miller

    2003-01-01

    To determine the effect of the ovarian hormone cycle on immunity, immunoglobulin-secreting cell (ISC) frequency and lymphocyte subsets were examined in the blood of healthy women. We found that immunoglobulin A (IgA)-secreting cells (IgA-ISC) were fourfold more frequent than IgG-ISC in peripheral blood mononuclear cells (PBMC). Further, the ISC frequency in PBMC was highest (P < 0.05) during the periovulatory stage of the menstrual cycle. Thus, endogenous ovarian steroids regulate the ISC fre...

  20. Interleukin 1 (IL1)-induced secretion of proteins from human synovial cells and fibroblasts

    International Nuclear Information System (INIS)

    The acidic species of human IL1 induces three neutral metalloproteinases in human synovial cells and fibroblasts with kinetics of secretion which suggest co-ordinate regulation. These proteinases, which degrade collagen, proteoglycan and gelatin, respectively, are secreted in a latent form which can be activated by aminophenylmercuric acetate. IL1-induction is concomitant with an overall decrease in protein synthesis. To determine if other proteins were induced by IL1, synovial cells and fibroblasts were incubated with 14C-leucine in the presence or absence of IL1. Synthesized proteins were analyzed by SDS-PAGE. IL1-induction altered the pattern of proteins secreted from target cells with no appreciable change in cell-associated proteins. In synovial cells, IL1 induced secretion of, most prominently, a 55kDa band, as well as bands of 56kDa, 45kDa, 12kDa, 20kDa, 17kDa and two small peptides of approximately 7 and 8kDa. In fibroblasts, IL1-induction most prominently stimulated secretion of a 60kDa band as well as 52kDa, 51kDa and 44kDa proteins. The identity of these bands, their relationship to the metalloproteinases and the kinetics of their induction will be discussed

  1. Stimulated human mast cells secrete mitochondrial components that have autocrine and paracrine inflammatory actions.

    Directory of Open Access Journals (Sweden)

    Bodi Zhang

    Full Text Available Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP. Inflammation is critical in the pathogenesis of many diseases, but the trigger(s is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD2 mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt mtDNA and ATP without cell death. Mitochondria purified from LAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(2, IL-8, TNF, and IL-1β. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial growth factor (VEGF release from cultured human keratinocytes, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted in vivo, we injected rats intraperiotoneally (ip with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD2 cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as "autopathogens" contributing to the pathogenesis of inflammatory

  2. Ca²⁺ signaling and regulation of fluid secretion in salivary gland acinar cells.

    Science.gov (United States)

    Ambudkar, Indu S

    2014-06-01

    Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca(2+) signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca(2+) is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca(2+)] ([Ca(2+)]i) triggered by IP3-induced release of Ca(2+) from ER via the IP3R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP3Rs determine the site of initiation and the pattern of [Ca(2+)]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+)]i and fluid secretion. This Ca(2+) influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca(2+) entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca(2+) signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca(2+) signal can be ascribed to the polarized arrangement of the Ca(2+) channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca(2+) signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca(2+) signals in the regulation of fluid secretion. PMID:24646566

  3. Molecular mechanisms of lipoapoptosis and metformin protection in GLP-1 secreting cells

    DEFF Research Database (Denmark)

    Kappe, Camilla; Holst, Jens Juul; Zhang, Qimin; Sjöholm, Ake

    2012-01-01

    Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where t...

  4. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin; Bangsbo, Jens; Hellsten, Ylva

    2010-01-01

    and during knee extensor exercise. The dialysate was analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion from muscle cells in culture was examined in resting and electro stimulated cells, and in response to the adenosine analogue NECA, and the adenosine A(2A) receptor...

  5. Nanosized blood microparticles

    NARCIS (Netherlands)

    Yuana, Yuana

    2011-01-01

    Microparticles (MPs) have important physiological and pathological roles in blood coagulation, inflammation and tumor progression. In recent years MPs also have been recognized to participate in important biological processes, such as in signaling and in the horizontal transfer of their specific pro

  6. Numerical Simulations of the Digital Microfluidic Manipulation of Single Microparticles.

    Science.gov (United States)

    Lan, Chuanjin; Pal, Souvik; Li, Zhen; Ma, Yanbao

    2015-09-01

    Single-cell analysis techniques have been developed as a valuable bioanalytical tool for elucidating cellular heterogeneity at genomic, proteomic, and cellular levels. Cell manipulation is an indispensable process for single-cell analysis. Digital microfluidics (DMF) is an important platform for conducting cell manipulation and single-cell analysis in a high-throughput fashion. However, the manipulation of single cells in DMF has not been quantitatively studied so far. In this article, we investigate the interaction of a single microparticle with a liquid droplet on a flat substrate using numerical simulations. The droplet is driven by capillary force generated from the wettability gradient of the substrate. Considering the Brownian motion of microparticles, we utilize many-body dissipative particle dynamics (MDPD), an off-lattice mesoscopic simulation technique, in this numerical study. The manipulation processes (including pickup, transport, and drop-off) of a single microparticle with a liquid droplet are simulated. Parametric studies are conducted to investigate the effects on the manipulation processes from the droplet size, wettability gradient, wetting properties of the microparticle, and particle-substrate friction coefficients. The numerical results show that the pickup, transport, and drop-off processes can be precisely controlled by these parameters. On the basis of the numerical results, a trap-free delivery of a hydrophobic microparticle to a destination on the substrate is demonstrated in the numerical simulations. The numerical results not only provide a fundamental understanding of interactions among the microparticle, the droplet, and the substrate but also demonstrate a new technique for the trap-free immobilization of single hydrophobic microparticles in the DMF design. Finally, our numerical method also provides a powerful design and optimization tool for the manipulation of microparticles in DMF systems. PMID:26241832

  7. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

    Science.gov (United States)

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-01-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle. PMID:27180572

  8. Cortactin Controls Cell Motility and Lamellipodial Dynamics by Regulating ECM Secretion

    Science.gov (United States)

    Sung, Bong Hwan; Zhu, Xiaodong; Kaverina, Irina; Weaver, Alissa

    2011-01-01

    Background Branched actin assembly is critical for both cell motility and membrane trafficking. The branched actin regulator, cortactin, is generally considered to promote cell migration by controlling leading edge lamellipodial dynamics. However, recent reports indicate that lamellipodia are not required for cell movement, suggesting an alternate mechanism. Results Since cortactin also regulates membrane trafficking and adhesion dynamics, we hypothesized that altered secretion of extracellular matrix (ECM) and/or integrin trafficking might underlie motility defects of cortactin-knockdown (KD) cells. Consistent with a primary defect in ECM secretion, both motility and lamellipodial defects of cortactin-KD cells were fully rescued by plating on increasing concentrations of exogenous ECM. Furthermore, cortactin-KD cell speed defects were rescued on cell-free autocrine ECM produced by control cells but not on ECM produced by cortactin-KD cells. Investigation of the mechanism revealed that whereas endocytosed FN is redeposited at the basal cell surface by control cells, cortactin-KD cells exhibit defective FN secretion and abnormal FN retention in a late endocytic/lysosomal compartment. Cortactin-KD motility and FN deposition defects were phenocopied by KD in control cells of the lysosomal fusion regulator Synaptotagmin-7. Rescue of cortactin-KD cells by expression of cortactin binding domain mutants revealed that interaction with Arp2/3 complex and actin filaments is essential for rescue of both cell motility and autocrine ECM secretion phenotypes whereas binding of SH3 domain partners is not required. Conclusions Efficient cell motility, promoted by cortactin regulation of branched actin networks, involves processing and resecretion of internalized ECM from a late endosomal/lysosomal compartment. PMID:21856159

  9. Retinoschisin, a photoreceptor-secreted protein, and its interaction with bipolar and muller cells

    OpenAIRE

    Reid, SNM; Yamashita, C; Farber, D B

    2003-01-01

    Usually, photoreceptors interact with other retinal cells through the neurotransmitter glutamate. Here we describe a nonsynaptic interaction via a secreted protein, retinoschisin. Using in situ hybridization, we found that from early postnatal life retinoschisin mRNA is present only in the outer retina of the mouse, and with single-cell RT-PCR we demonstrated its localization in rod and cone photoreceptor cells but not in Muller cells. Western blot analyses of proteins from cultured ocular ti...

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

    DEFF Research Database (Denmark)

    Owens, T

    1991-01-01

    23.1+ TH1 clone E9.D4 in F23.1 (anti-T cell receptor V-beta 8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did...... not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-gamma mAb partially inhibited Ig secretion, without affecting entry into...

  11. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells.

    OpenAIRE

    Ramasharma, K; Li, C. H.

    1987-01-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generation and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, w...

  12. Auto-stimulatory action of secreted caveolin-1 on the proliferation of Ewing’s sarcoma cells

    OpenAIRE

    Sengupta, Aniruddha; Mateo-Lozano, Silvia; Tirado, Oscar M.; Notario, Vicente

    2011-01-01

    Caveolin-1 (CAV1) is highly expressed in Ewing’s sarcoma (EWS). We previously showed that increased cellular CAV1 is associated with the regulation of the tumorigenicity, drug resistance and metastatic ability of EWS cells. Because several studies reported that melanoma and prostate cancer cells, which express relatively high CAV1 levels, secrete CAV1, and that secreted CAV1 is associated with tumor progression, our study explored the possibility that EWS cells also secreted CAV1 and that sec...

  13. Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells.

    Science.gov (United States)

    Pedersen, Morten Gram; Ahlstedt, Ingela; El Hachmane, Mickaël F; Göpel, Sven O

    2016-01-01

    Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na(+)/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications. PMID:27535321

  14. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

    LENUS (Irish Health Repository)

    Hong, Ying

    2012-01-01

    The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

  15. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1984-01-01

    A multiphase study was conducted to examine the properties of growth hormone cells. Topics investigated included: (1) to determine if growth hormone (GH) cells contained within the rat pituitary gland can be separated from the other hormone producing cell types by continuous flow electrophoresis (CFE); (2) to determine what role, if any, gravity plays in the electrophoretic separation of GH cells; (3) to compare in vitro GH release from rat pituitary cells previously exposed to microgravity conditions vs release from cells not exposed to microgravity; (4) to determine if the frequency of different hormone producing pituitary cell types contained in cell suspensions can be quantitated by flow cytometry; and (5) to determine if GH contained within the human post mortem pituitary gland can be purified by CFE. Specific experimental procedures and results are included.

  16. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Directory of Open Access Journals (Sweden)

    Ellen Melaleuca Menkhorst

    Full Text Available Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT invade through the differentiated uterine endometrium (the decidua to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8 M, medroxyprogesterone acetate (10(-7 M and cAMP (0.5 mM for 14 days. Conditioned media (CM was collected on day 2 (non-decidualized CM and 14 (decidualized CM of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1, dipeptidyl peptidase 1 (DPP1/cathepsin C and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro

  17. [Differentiation of human amniotic mesenchymal stem cells into insulin-secreting cells induced by regenerating pancreatic extract].

    Science.gov (United States)

    Zhang, Yanmei; Wang, Dianliang; Zeng, Hongyan; Wang, Lieming; Sun, Jinwei; Zhang, Zhen; Dong, Shasha

    2012-02-01

    In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 microg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro. PMID:22667123

  18. EEN regulates the proliferation and survival of multiple myeloma cells by potentiating IGF-1 secretion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Er-Wen [Guangzhou Institute of Forensic Science, Guangzhou (China); Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Xue, Sheng-Jiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Li, Xiao-Yan [Department of Pharmacy, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Xu, Suo-Wen [Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou (China); Cheng, Jian-Ding; Zheng, Jin-Xiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Shi, He; Lv, Guo-Li; Li, Zhi-Gang; Li, Yue; Liu, Chang-Hui; Chen, Xiao-Hui; Liu, Hong [Guangzhou Institute of Forensic Science, Guangzhou (China); Li, Jie, E-mail: mdlijie@sina.com [Department of Anaesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou (China); Liu, Chao, E-mail: liuchaogaj@21cn.com [Guangzhou Institute of Forensic Science, Guangzhou (China)

    2014-05-02

    Highlights: • Levels of EEN expression paralleled with the rate of cell proliferation. • EEN was involved in the proliferation and survival of multiple myeloma (MM) cells. • EEN regulated the activity of IGF-1-Akt/mTOR pathway. • EEN regulated proliferation and survival of MM cells by enhancing IGF-1 secretion. - Abstract: The molecular mechanisms of multiple myeloma are not well defined. EEN is an endocytosis-regulating molecule. Here we report that EEN regulates the proliferation and survival of multiple myeloma cells, by regulating IGF-1 secretion. In the present study, we observed that EEN expression paralleled with cell proliferation, EEN accelerated cell proliferation, facilitated cell cycle transition from G1 to S phase by regulating cyclin-dependent kinases (CDKs) pathway, and delayed cell apoptosis via Bcl2/Bax-mitochondrial pathway. Mechanistically, we found that EEN was indispensable for insulin-like growth factor-1 (IGF-1) secretion and the activation of protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. Exogenous IGF-1 overcame the phenotype of EEN depletion, while IGF-1 neutralization overcame that of EEN over-expression. Collectively, these data suggest that EEN may play a pivotal role in excessive cell proliferation and insufficient cell apoptosis of bone marrow plasma cells in multiple myeloma. Therefore, EEN may represent a potential diagnostic marker or therapeutic target for multiple myeloma.

  19. EEN regulates the proliferation and survival of multiple myeloma cells by potentiating IGF-1 secretion

    International Nuclear Information System (INIS)

    Highlights: • Levels of EEN expression paralleled with the rate of cell proliferation. • EEN was involved in the proliferation and survival of multiple myeloma (MM) cells. • EEN regulated the activity of IGF-1-Akt/mTOR pathway. • EEN regulated proliferation and survival of MM cells by enhancing IGF-1 secretion. - Abstract: The molecular mechanisms of multiple myeloma are not well defined. EEN is an endocytosis-regulating molecule. Here we report that EEN regulates the proliferation and survival of multiple myeloma cells, by regulating IGF-1 secretion. In the present study, we observed that EEN expression paralleled with cell proliferation, EEN accelerated cell proliferation, facilitated cell cycle transition from G1 to S phase by regulating cyclin-dependent kinases (CDKs) pathway, and delayed cell apoptosis via Bcl2/Bax-mitochondrial pathway. Mechanistically, we found that EEN was indispensable for insulin-like growth factor-1 (IGF-1) secretion and the activation of protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. Exogenous IGF-1 overcame the phenotype of EEN depletion, while IGF-1 neutralization overcame that of EEN over-expression. Collectively, these data suggest that EEN may play a pivotal role in excessive cell proliferation and insufficient cell apoptosis of bone marrow plasma cells in multiple myeloma. Therefore, EEN may represent a potential diagnostic marker or therapeutic target for multiple myeloma

  20. Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

    International Nuclear Information System (INIS)

    Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 ± 4.74) than in RH10Gy-SnonR (30.6 ± 3.85) cells, and lowest in McA-RH7777 (11.4 ± 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 ± 5.38), RH10Gy-SnonR (22.17 ± 4.26), and McA-RH7777 (8.3 ± 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-α and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of metastasis

  1. Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Leyuan [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Zhiming [Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Gao Yabo [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Lingyan [Experimental Research Center, Zhongshan Hospital, Fudan University, Shanghai (China); Zeng Zhaochong, E-mail: zeng.zhaochong@zs-hospital.sh.cn [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China)

    2012-11-01

    Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 {+-} 4.74) than in RH10Gy-SnonR (30.6 {+-} 3.85) cells, and lowest in McA-RH7777 (11.4 {+-} 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 {+-} 5.38), RH10Gy-SnonR (22.17 {+-} 4.26), and McA-RH7777 (8.3 {+-} 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-{alpha} and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of

  2. Optogenetic control of insulin secretion by pancreatic β-cells in vitro and in vivo.

    Science.gov (United States)

    Kushibiki, T; Okawa, S; Hirasawa, T; Ishihara, M

    2015-07-01

    The present study assessed the ability of optogenetics techniques to provide a better understanding of the control of insulin secretion, particularly regarding pancreatic β-cell function in homeostasis and pathological conditions such as diabetes mellitus (DM). We used optogenetics to investigate whether insulin secretion and blood glucose homeostasis could be controlled by regulating intracellular calcium ion concentrations ([Ca(2+)]i) in a mouse pancreatic β-cell line (MIN6) transfected with the optogenetic protein channelrhodopsin-2 (ChR2). The ChR2-transfected MIN6 (ChR2-MIN6) cells secreted insulin following irradiation with a laser (470 nm). The increase in [Ca(2+)]i was accompanied by elevated levels of messenger RNAs that encode calcium/calmodulin-dependent protein kinase II delta and adenylate cyclase 1. ChR2-MIN6 cells suspended in matrigel were inoculated into streptozotocin-induced diabetic mice that were then subjected to a glucose tolerance test. Laser irradiation of these mice caused a significant decrease in blood glucose, and the irradiated implanted cells expressed insulin. These findings demonstrate the power of optogenetics to precisely and efficiently controlled insulin secretion by pancreatic β-cells 'on demand', in contrast to techniques using growth factors or chemical inducers. Optogenetic technology shows great promise for understanding the mechanisms of glucose homeostasis and for developing treatments for metabolic diseases such as DM. PMID:25809465

  3. Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells

    International Nuclear Information System (INIS)

    Purified populations of rat peritoneal mast cells were used to study the effect of ouabain on compound 48/80-induced histamine secretion and on 86Rb+ uptake. 86Rb+ was used as a tracer for extracellular K+. The calculated value of the ouabain-sensitive uptake of K+ and 86Rb+ was considered a measure of the Na+-K+ pump activity of the cells. Ouabain caused an immediate inhibition of the pump activity and a time-dependent increase in histamine secretion in the absence of extracellular calcium. No effect on the secretion was observed in the presence of calcium. The effect of ouabain on the secretion occurs in the presence of sodium but not when sodium was replaced by lithium. Preservation by ouabain of a high intracellular sodium content in sodium-loaded cells was associated with preservation of the secretory response in a calcium-free medium. In the presence of lanthanum in a calcium-free medium, the pump activity was inhibited and the enhancement by ouabain of the secretion of histamine was blocked. A less marked inhibition of the pump was found in a calcium-free medium containing magnesium. The inhibition exerted by magnesium was concentration-dependent (0-5 mM) as was the counteraction of magnesium of the enhancement of ouabain of the secretion of histamine. These observations indicate that the echancement by ouabain of the secretory response of mast cells preincubated in a calcium-free medium is associated with accumulation of sodium inside the cell. In addition to a decreased rate of sodium-calcium exchange caused by a decreased inward directed sodium gradient, the mechanism by which ouabain enhances the secretory response is likely to involve an increased binding of calcium to membrane binding sites. (au)

  4. Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

    Science.gov (United States)

    Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

    1985-01-01

    The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

  5. Tendon synovial cells secrete fibronectin in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Banes, A.J.; Link, G.W.; Bevin, A.G.; Peterson, H.D.; Gillespie, Y.; Bynum, D.; Watts, S.; Dahners, L.

    1988-01-01

    The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of /sup 35/S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

  6. Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

    OpenAIRE

    Escrevente, Cristina; Grammel, Nicolas; Kandzia, Sebastian; Zeiser, Johannes; Tranfield, Erin M; Conradt, Harald S.; Costa, Júlia

    2013-01-01

    Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abun...

  7. Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion

    OpenAIRE

    Ilegems, E.; van Krieken, P. P.; Edlund, P. K.; Dicker, A.; Alanentalo, T.; Eriksson, Maria; Mandic, S.; Ahlgren, Ulf; Berggren, P.-O.

    2015-01-01

    The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulat...

  8. Intrinsic optical signal imaging of glucose-stimulated insulin secreting β-cells

    OpenAIRE

    Li, Yi-Chao; Cui, Wan-Xing; Wang, Xu-Jing; Amthor, Franklin; Lu, Rong-Wen; Thompson, Anthony; Yao, Xin-Cheng

    2010-01-01

    Simultaneous monitoring of many functioning β-cells is essential for understanding β-cell dysfunction as an early event in the progression to diabetes. Intrinsic optical signal (IOS) imaging has been shown to allow high resolution detection of stimulus-evoked physiological responses in the retina and other neural tissues. In this paper, we demonstrate the feasibility of using IOS imaging for functional examination of insulin secreting INS-1 cells, a popular model for investigating diabetes as...

  9. SALMON SOFT ROE DNA ON BLOOD CELLS SECRETION OF CYTOKINES IN HEALTHY DONORS

    Directory of Open Access Journals (Sweden)

    L. N. Fedjanina

    2005-01-01

    Full Text Available Abstract. Salmon soft roe DNA influence on healthy donors blood cells secretion of early hemopoietic factors (IL-3, GM-CSF, TNFα as well as biologically active substance influence on cytokine balance of Тh1 and Тh2 responses (IFNγ, IL-10 in vitro was studied. It is established, that DNA has modulatory effect on secretion of all investigated cytokines - IL-3, GM-CSF, TNFα, INFγ and IL-10 by blood cells of healthy donors, increases their initially low concentration, reduces initially high and does not have essential influence at an average level of their secretion. Under action of DNA IFNγ level (stimulation index=3,3 increases more significantly than IL-10 level (stimulation index =1,9. Thus, salmon soft roe DNA possesses immunomodulatory properties.

  10. The Secret Lives of Pluripotent Cells: There and Back Again

    Directory of Open Access Journals (Sweden)

    Paolo Cinelli

    2010-03-01

    Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (IPSCs hold great promise for the therapeutic treatment of human diseases, but their functional similarity, their stability and especially the mechanism underlying their derivation are not yet clearly explained. [...

  11. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Mouse mammary epithelial cells (MMEC) secrete a group of milk-specific proteins including various caseins and whey proteins. Dissociated mammary epithelial cells maintain expression of most of their differentiated functions only if cells are plated on a suitable substratum. Casein production and section, cell morphology, and production of α-lactalbumin have been used as markers to assess the degree of differentiation of mammary cells in culture. The general consensus is that cells express their differentiated properties at high levels and for longer periods of time on such substrata. In this paper, the authors demonstrate that modulation of the expression of caseins by floating collagen gels is manifested at several regulatory points

  12. Midkine secretion protects Hep3B cells from cadmium induced cellular damage

    Institute of Scientific and Technical Information of China (English)

    Nuray Yazihan; Haluk Ataoglu; Ethem Akcil; Burcu Yener; Bulent Salman; Cengiz Aydin

    2008-01-01

    AIM:To evaluate role of midkine secretion during Cadmium (Cd) exposure in the human hepatocyte cell line Hep3B cells.METHODS: Different dosages of Cd (0.5-1-5-10 μg/mL) were applied to Hep3B cells and their effects to apoptosis, lactate dehydrogenase (LDH) leakage and midkine secretion were evaluated as time dependent manner. Same experiments were repeated with exogenously applied midkine (250-5000 pg/mL) and/or 5μg/mL Cd.RESULTS: Cd exposure induced prominent apoptosis and LDH leakage beginning from lower dosages at the 48th h. Cd induced midkine secretion with higher dosages (P < 0.001), (control, Cd 0.5-1-5-10μg/mL respectively: 1123±73, 1157±63, 1242±90, 1886± 175, 1712±166 pg/mL). Exogenous 500-5000 pg/mL midkine application during 5 μg/mL Cd toxicity prevented caspase-3 activation (control, Cd toxicity, 250, 500, 1000, 2500, 5000 pg/mL midkine+ Cd toxicity, respectively:374±64, 1786±156, 1545±179, 1203±113, 974±116, 646±56, 556±63 cfu) LDH leakage and cell death in Hep3B cells (P < 0.001).CONCLUSION: Our results showed that midkine secretion from Hep3B cells during Cd exposure protects liver cells from Cd induced cellular damage. Midkine has anti-apoptotic and cytoprotective role during Cd toxicity. Further studies are needed to explain the mechanism of midkine secretion and cytoprotective role of midkine during Cd exposure. Midkine may be a promising theurapatic agent in different toxic hepatic diseases.

  13. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    Science.gov (United States)

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells. PMID:26716748

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

    Energy Technology Data Exchange (ETDEWEB)

    Boncela, Joanna; Przygodzka, Patrycja [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Wyroba, Elzbieta [Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw (Poland); Papiewska-Pajak, Izabela [Department of Molecular and Medical Biophysics, Medical University of Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Department of Molecular and Medical Biophysics, Medical University of Lodz (Poland)

    2013-05-01

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

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

    International Nuclear Information System (INIS)

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

  16. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    International Nuclear Information System (INIS)

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  17. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Robert T. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); O' Brien, Fergal J. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Hoey, David A., E-mail: david.hoey@ul.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); The Centre for Applied Biomedical Engineering Research, University of Limerick (Ireland); Materials & Surface Science Institute, University of Limerick (Ireland)

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  18. Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

    Science.gov (United States)

    Moss, Catherine E.; Glass, Leslie L.; Diakogiannaki, Eleftheria; Pais, Ramona; Lenaghan, Carol; Smith, David M.; Wedin, Marianne; Bohlooly-Y, Mohammad; Gribble, Fiona M.; Reimann, Frank

    2016-01-01

    Aims/hypothesis Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. Methods GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. Results L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. Conclusions/interpretation GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion. PMID:26144594

  19. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation.

    Science.gov (United States)

    Brady, Robert T; O'Brien, Fergal J; Hoey, David A

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24 hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. PMID:25721667

  20. Regulation of proliferation-survival decisions is controlled by FGF1 secretion in retinal pigmented epithelial cells.

    Science.gov (United States)

    Bryckaert, M; Guillonneau, X; Hecquet, C; Perani, P; Courtois, Y; Mascarelli, F

    2000-10-01

    Fibroblast growth factor 1 (FGF1) induces proliferation and differentiation in a wide variety of cells of mesodermal and neuroectodermal origin. FGF1 has no 'classical' signal sequence to direct its secretion, and there has been considerable debate concerning FGF1 secretion and its role in the biological activities of FGF1. We investigated the effects of FGF1 secretion and the signalling induced by signal peptide (SP)-containing FGFI and SP-less FGF1, on the proliferation and the apoptosis in retinal pigmented epithelial (RPE) cells. Primary RPE cell cultures were transfected with FGF1 (FGF1 cells) and SP-FGF1 (SP-FGF1 cells) cDNAs. SP-FGF1 cells secreted large amount of FGF1 and actively proliferated, whereas FGF1 and control cells did not. Secreted FGF1 induced short-term activation of both FGFR1 and ERK2, which were required for cell proliferation. In contrast, SP-FGF1 cells stopped secreting FGF1 and died rapidly, if cultured in the absence of serum. Surprisingly, FGF1 cells, but not control cells, secreted FGF1 and were resistant to apoptosis induced by serum depletion. Secreted FGF1 induced long-term activation of FGFR1 and ERK2, which was necessary to induce a constant and high level of Bcl-x production, and to induce cell survival in FGFI cells. Downregulation of ERK2 and Bcl-x increased apoptosis. Thus, the proliferation and survival activities of FGF1 depend on the secretion of FGF1 which is determined by the cell culture conditions. Cell proliferation was SP-dependent, whereas cell survival was not. The signal peptide controls the level and duration, 'whispering or shouting', of ERK2 activation cells which determines FGF1 biological function and may have important implications for anti-degenerative and anti-proliferative treatments. PMID:11039909

  1. Microparticles from kidney-derived mesenchymal stem cells act as carriers of proangiogenic signals and contribute to recovery from acute kidney injury.

    Directory of Open Access Journals (Sweden)

    Hoon Young Choi

    Full Text Available We recently demonstrated the use of in vitro expanded kidney-derived mesenchymal stem cells (KMSC protected peritubular capillary endothelial cells in acute renal ischemia-reperfusion injury. Herein, we isolated and characterized microparticles (MPs from KMSC. We investigated their in vitro biologic effects on human endothelial cells and in vivo renoprotective effects in acute ischemia-reperfusion renal injury. MPs were isolated from the supernatants of KMSC cultured in anoxic conditions in serum-deprived media for 24 hours. KMSC-derived MPs demonstrated the presence of several adhesion molecules normally expressed on KMSC membranes, such as CD29, CD44, CD73, α4, 5, and 6 integrins. Quantitative real time PCR confirmed the presence of 3 splicing variants of VEGF-A (120, 164, 188, bFGF and IGF-1 in isolated MPs. MPs labeled with PKH26 red fluorescence dye were incorporated by cultured human umbilical vein endothelial cells (HUVEC via surface molecules such as CD44, CD29, and α4, 5, and 6 integrins. MP dose dependently improved in vitro HUVEC proliferation and promoted endothelial tube formation on growth factor reduced Matrigel. Moreover, apoptosis of human microvascular endothelial cell was inhibited by MPs. Administration of KMSC-derived MPs into mice with acute renal ischemia was followed by selective engraftment in ischemic kidneys and significant improvement in renal function. This was achieved by improving proliferation, of peritubular capillary endothelial cell and amelioration of peritubular microvascular rarefaction. Our results support the hypothesis that KMSC-derived MPs may act as a source of proangiogenic signals and confer renoprotective effects in ischemic kidneys.

  2. Mechanical forces stimulate endothelial microparticle generation via caspase-dependent apoptosis-independent mechanism

    OpenAIRE

    Vion, Anne Clémence; Birukova, Anna A.; Boulanger, Chantal M; Birukov, Konstantin G.

    2013-01-01

    Microparticle release by vascular endothelium has been implicated in various cardiovascular pathologies. Ventilator-induced lung injury (VILI) is a life-threatening complication of mechanical ventilation at high tidal volumes associated with excessive mechanical stretch of pulmonary vascular endothelial cells. However, a role of VILI-relevant levels of cyclic stretch in microparticle generation by vascular endothelium remains unknown. We report microparticle formation by human pulmonary endot...

  3. Profile analysis of microparticles

    International Nuclear Information System (INIS)

    Depth resolved analyses of several types of microparticles are presented. Particles for secondary ion mass spectrometry (SIMS) depth profile analysis were collected in the working environment of glass plant, steelworks and welding station using eight-stage cascade impactor with particle size range of 0.3 μm to 15 μm. Ion beam sputtering and sample rotation technique allowed to describe morphology i.e. the elemental structure of collected sub-micrometer particles. Also model particles Iriodin 221 (Merck) were depth profiled. The core-shell structure is found for all types of investigated particles. Steelworks particles consist mainly of iron and manganese cores. At the shells of these microparticles: lead, chlorine and fluorine are found. The particles collected in the glass-works consist mainly of lead-zirconium glass cores covered by carbon and copper. Stainless-steel welding particles compose of iron, manganese and chromium cores covered by a shell rich in carbon, chlorine and fluorine. Sample rotation technique applied in SIMS appears to be an effective tool for environmental microparticle morphology studies

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

    Directory of Open Access Journals (Sweden)

    Jessica Wahlgren

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

  5. The Comparison of Adipose Stem Cell and Placental Stem Cell in Secretion Characteristics and in Facial Antiaging

    Directory of Open Access Journals (Sweden)

    Yan Xu

    2016-01-01

    Full Text Available Background. Mesenchymal stem cells are the most commonly used seed cells in biomedical research and tissue engineering. Their secretory proteins have also been proven to play an important role in tissue healing. Methods. We isolated adipose stem cells and placental stem cells and performed analysis examining characteristics. The secretory proteins were extracted from conditioned medium and analyzed by MALDI-TOF/TOF. The antiaging effect of conditioned mediums was evaluated by the results of facial skin application. Results. Adipose stem cells and placental stem cells were found to be very similar in their surface markers and multipotency. The specific proteins secreted from adipose stem cells were more adept at cell adhesion, migration, wound healing, and tissue remodeling, while the proteins secreted by placental stem cells were more adept at angiogenesis, cell proliferation, differentiation, cell survival, immunomodulation, and collagen degradation. While these two types of conditioned medium could improve the facial index, the improvement of Melanin index after injection of the adipose stem cell conditioned medium was much more significant. Conclusion. The results suggest that the secreted proteins are ideal cell-free substances for regeneration medicine, especially in the antiaging field.

  6. The Comparison of Adipose Stem Cell and Placental Stem Cell in Secretion Characteristics and in Facial Antiaging.

    Science.gov (United States)

    Xu, Yan; Guo, Shilei; Wei, Cui; Li, Honglan; Chen, Lei; Yin, Chang; Zhang, Chuansen

    2016-01-01

    Background. Mesenchymal stem cells are the most commonly used seed cells in biomedical research and tissue engineering. Their secretory proteins have also been proven to play an important role in tissue healing. Methods. We isolated adipose stem cells and placental stem cells and performed analysis examining characteristics. The secretory proteins were extracted from conditioned medium and analyzed by MALDI-TOF/TOF. The antiaging effect of conditioned mediums was evaluated by the results of facial skin application. Results. Adipose stem cells and placental stem cells were found to be very similar in their surface markers and multipotency. The specific proteins secreted from adipose stem cells were more adept at cell adhesion, migration, wound healing, and tissue remodeling, while the proteins secreted by placental stem cells were more adept at angiogenesis, cell proliferation, differentiation, cell survival, immunomodulation, and collagen degradation. While these two types of conditioned medium could improve the facial index, the improvement of Melanin index after injection of the adipose stem cell conditioned medium was much more significant. Conclusion. The results suggest that the secreted proteins are ideal cell-free substances for regeneration medicine, especially in the antiaging field. PMID:27057176

  7. Specificity of secreted proteomes from cardiac stem cells and neonatal myocytes

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Chimenti, I.; Marban, E.; Van Eyk, J.

    2009-01-01

    Roč. 276, Suppl.1 (2009), s. 346. ISSN 1742-464X. [FEBS Congress /34./. 04.07.2009-09.07.2009, Prague] Institutional research plan: CEZ:AV0Z40310501 Keywords : cardiac stem cells * secreted paracrine/autocrine factors * proteomics Subject RIV: CB - Analytical Chemistry, Separation

  8. Mucus-secreting 'signet-ring' cells in CSF revealing the site of primary cancer.

    OpenAIRE

    Agnelli, G.; Gresele, P.

    1980-01-01

    A case is reported of leptomeningeal carcinomatosis in which identification of mucus-secreting 'signet-ring' carcinoma cells in the CSF allowed diagnosis of an otherwise asymptomatic gastric cancer. When lumbar puncture is performed, careful cytological examination of the CSF should be carried out in any undiagnosed patient with neurological symptoms and signs.

  9. Intrathecal IgG synthesis and autoantibody-secreting cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn; Jensen, Claus V; Christiansen, Michael

    We studied intrathecal IgG synthesis and autoantibody-secreting cells in 148 patients with possible onset symptoms of MS (POSMS) or clinically definite MS (CDMS). In POSMS intrathecal synthesis of IgG oligoclonal bands and abnormalities on T2-weighted magnetic resonance imaging were associated bu...

  10. IGF-1 release kinetics from chitosan microparticles fabricated using environmentally benign conditions

    Energy Technology Data Exchange (ETDEWEB)

    Mantripragada, Venkata P. [Biomedical Engineering Program, The University of Toledo, Toledo, OH 43614-5807 (United States); Jayasuriya, Ambalangodage C., E-mail: a.jayasuriya@utoledo.edu [Biomedical Engineering Program, The University of Toledo, Toledo, OH 43614-5807 (United States); Department of Orthopaedic Surgery, The University of Toledo, Toledo, OH 43614-5807 (United States)

    2014-09-01

    The main objective of this study is to maximize growth factor encapsulation efficiency into microparticles. The novelty of this study is to maximize the encapsulated growth factors into microparticles by minimizing the use of organic solvents and using relatively low temperatures. The microparticles were fabricated using chitosan biopolymer as a base polymer and cross-linked with tripolyphosphate (TPP). Insulin like-growth factor-1 (IGF-1) was encapsulated into microparticles to study release kinetics and bioactivity. In order to authenticate the harms of using organic solvents like hexane and acetone during microparticle preparation, IGF-1 encapsulated microparticles prepared by the emulsification and coacervation methods were compared. The microparticles fabricated by emulsification method have shown a significant decrease (p < 0.05) in IGF-1 encapsulation efficiency, and cumulative release during the two-week period. The biocompatibility of chitosan microparticles and the bioactivity of the released IGF-1 were determined in vitro by live/dead viability assay. The mineralization data observed with von Kossa assay, was supported by mRNA expression levels of osterix and runx2, which are transcription factors necessary for osteoblasts differentiation. Real time RT-PCR data showed an increased expression of runx2 and a decreased expression of osterix over time, indicating differentiating osteoblasts. Chitosan microparticles prepared in optimum environmental conditions are a promising controlled delivery system for cells to attach, proliferate, differentiate and mineralize, thereby acting as a suitable bone repairing material. - Highlights: • Coacervation chitosan microparticles were biocompatible and biodegradable. • IGF-1 encapsulation efficiency increased with coacervation chitosan microparticles. • Coacervation chitosan microparticles support osteoblast attachment and differentiation. • Coacervation chitosan microparticles support osteoblast mineralization.

  11. IGF-1 release kinetics from chitosan microparticles fabricated using environmentally benign conditions

    International Nuclear Information System (INIS)

    The main objective of this study is to maximize growth factor encapsulation efficiency into microparticles. The novelty of this study is to maximize the encapsulated growth factors into microparticles by minimizing the use of organic solvents and using relatively low temperatures. The microparticles were fabricated using chitosan biopolymer as a base polymer and cross-linked with tripolyphosphate (TPP). Insulin like-growth factor-1 (IGF-1) was encapsulated into microparticles to study release kinetics and bioactivity. In order to authenticate the harms of using organic solvents like hexane and acetone during microparticle preparation, IGF-1 encapsulated microparticles prepared by the emulsification and coacervation methods were compared. The microparticles fabricated by emulsification method have shown a significant decrease (p < 0.05) in IGF-1 encapsulation efficiency, and cumulative release during the two-week period. The biocompatibility of chitosan microparticles and the bioactivity of the released IGF-1 were determined in vitro by live/dead viability assay. The mineralization data observed with von Kossa assay, was supported by mRNA expression levels of osterix and runx2, which are transcription factors necessary for osteoblasts differentiation. Real time RT-PCR data showed an increased expression of runx2 and a decreased expression of osterix over time, indicating differentiating osteoblasts. Chitosan microparticles prepared in optimum environmental conditions are a promising controlled delivery system for cells to attach, proliferate, differentiate and mineralize, thereby acting as a suitable bone repairing material. - Highlights: • Coacervation chitosan microparticles were biocompatible and biodegradable. • IGF-1 encapsulation efficiency increased with coacervation chitosan microparticles. • Coacervation chitosan microparticles support osteoblast attachment and differentiation. • Coacervation chitosan microparticles support osteoblast mineralization

  12. Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

    Science.gov (United States)

    White, Michael J V; Roife, David; Gomer, Richard H

    2015-08-15

    To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases. PMID:26136428

  13. Effects of radiation on IL-2 secretion and cytotoxicity of T cells

    International Nuclear Information System (INIS)

    Peripheral blood mononuclear cells (PBMC) from normal persons were irradiated with 1.6 Gy γ-rays in vitro. The method of indirect immunofluorescence was used for analysis of positive percentage of TCR, CD3 and CD25 of T cells. The method of 3H-TdR incorporation and release wa used for determination of IL-2 secretion and T cell cytotoxicity, and immunocytochemistry was used for analysis of expression of TCR and CD3. A decreasing pattern of TCR, CD3 and CD25 expression, IL-2 secretion, and T cell cytotoxicity depending on radiation dose was found. The inhibition of IL-2 secretion and cytotoxicity after irradiation of T cells was associated, in some degree, with decreasing expression and damaging activity of TCR, CD3 and CD25, whereas it was possible that the decreasing expression of TCR and CD3 at the cell surface was related to accumulation of TCR and CD3 in cytoplasm of irradiated T cells

  14. Vascular Endothelial Growth Factor A Regulates the Secretion of Different Angiogenic Factors in Lung Cancer Cells.

    Science.gov (United States)

    Frezzetti, Daniela; Gallo, Marianna; Roma, Cristin; D'Alessio, Amelia; Maiello, Monica R; Bevilacqua, Simona; Normanno, Nicola; De Luca, Antonella

    2016-07-01

    Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention. PMID:26542886

  15. Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex.

    Directory of Open Access Journals (Sweden)

    Héctor Alejandro Guidobaldi

    Full Text Available Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF, oviduct fluid, and conditioned medium from the cumulus oophorus (CU and the oocyte (O. Though several substances were confirmed as sperm chemoattractant, Progesterone (P seems to be the best chemoattractant candidate, because: 1 spermatozoa express a cell surface P receptor, 2 capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3 the CU cells produce and secrete P after ovulation; 4 a gradient of P may be kept stable along the CU; and 5 the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.

  16. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  17. Uncoupling protein 2 regulates glucagon-like peptide-1secretion in L-cells

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Zheng-Yang Li; Yan Yang; Hong-Jie Zhang

    2012-01-01

    AIM:To investigate whether uncoupling protein 2(UCP2) affects oleic acid-induced secretion of glucagonlike peptide-1 (GLP-1) in L-cells.METHODS:mRNA and protein expression of UCP2were analyzed in human NCI-H716 cells,which serve as a model for enteroendocrine L-cells,by quantitative reverse transcription-polymerase chain reaction and Western blotting before and after treatment with oleic acid.Localization of UCP2 and GLP-1 in NCI-H716 cells was assessed by immunofluorescence labeling.NCI-H716cells were transiently transfected with a small interfering RNA (siRNA) that targets UCP2 (siUCP2) or with a nonspecific siRNA using Lipofectamine 2000.The concentrations of bioactive GLP-1 in the medium were measured by enzyme linked immunosorbent assay.RESULTS:Both GLP-1 and UCP2 granules were expressed mainly in the cytoplasm of NCI-H716 cells.NCI-H716 cells that secreted GLP-1 also expressed UCP2.Time-course experiments revealed that release of GLP-1 from NCI-H716 cells into the medium reached a maximum at 120 min and remained stable until at least 180 min after treatment with oleic acid (the level of GLP-1 increased about 2.3-fold as compared with the level of GLP-1 in the control cells,P < 0.05).In an experiment to determine dose dependence,stimulation of NCI-H716 cells with ≤ 8 mmol oleic acid led to a concentration-dependent release of GLP-1 into the medium; 10 mmol oleic acid diminished the release of GLP-1.Furthermore,GLP-1 secretion induced by oleic acid from NCI-H716 cells that were transfected with siUCP2 decreased to 41.8%,as compared with NCI-H716 cells that were transfected with a non-specific siRNA (P < 0.01).CONCLUSION:UCP2 affected GLP-1 secretion induced by oleic acid.UCP2 plays an important role in L-cell secretion that is induced by free fatty acids.

  18. Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans.

    OpenAIRE

    Russell, M. W.; Czerkinsky, C; Moldoveanu, Z

    1986-01-01

    Immune responses of mice to Streptococcus mutans serotype c were analyzed by means of the enzyme-linked immunospot assay to determine the predominant specificities of the antibodies developed. In general, the numbers of splenic antibody-secreting cells correlated with serum antibody levels. A low dose (10(8) CFU) of killed whole cells injected twice intraperitoneally induced antibodies mainly against surface protein antigen I/II. A higher dose (10(9) CFU) given two to six times also resulted ...

  19. Variable stretch pattern enhances surfactant secretion in alveolar type II cells in culture

    OpenAIRE

    Arold, Stephen P.; Bartolák-Suki, Erzsébet; Suki, Béla

    2009-01-01

    Secretion of pulmonary surfactant that maintains low surface tension within the lung is primarily mediated by mechanical stretching of alveolar epithelial type II (AEII) cells. We have shown that guinea pigs ventilated with random variations in frequency and tidal volume had significantly larger pools of surfactant in the lung than animals ventilated in a monotonous manner. Here, we test the hypothesis that variable stretch patterns imparted on the AEII cells results in enhanced surfactant se...

  20. Polarization of Calcium Signaling and Fluid Secretion in Salivary Gland Cells

    OpenAIRE

    Ambudkar, I.S.

    2012-01-01

    The secretion of fluid, electrolytes, and protein by exocrine gland acinar cells is a vectorial process that requires the coordinated regulation of multiple channel and transporter proteins, signaling components, as well as mechanisms involved in vesicular fusion and water transport. Most critical in this is the regulation of cytosolic free [Ca2+] ([Ca2+]i) in response to neurotransmitter stimulation. Control of [Ca2+]i increase in specific regions of the cell is the main determinant of fluid...

  1. Sox17 regulates insulin secretion in the normal and pathologic mouse β cell.

    Directory of Open Access Journals (Sweden)

    Diva Jonatan

    Full Text Available SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature β cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.

  2. Taurine reduces the secretion of apolipoprotein B100 and lipids in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Nagao Koji

    2008-10-01

    Full Text Available Abstract Background Higher concentrations of serum lipids and apolipoprotein B100 (apoB are major individual risk factors of atherosclerosis and coronary heart disease. Therefore ameliorative effects of food components against the diseases are being paid attention in the affluent countries. The present study was undertaken to investigate the effect of taurine on apoB secretion and lipid metabolism in human liver model HepG2 cells. Results The results demonstrated that an addition of taurine to the culture media reduces triacylglycerol (TG-mass in the cells and the medium. Similarly, cellular cholesterol-mass was decreased. Taurine inhibited the incorporation of [14C] oleate into cellular and medium TG, suggesting the inhibition of TG synthesis. In addition, taurine reduced the synthesis of cellular cholesterol ester and its secretion, suggesting the inhibition of acyl-coenzyme A:cholesterol acyltransferase activity. Furthermore, taurine reduced the secretion of apoB, which is a major protein component of very low-density lipoprotein. Conclusion This is a first report to demonstrate that taurine inhibits the secretion of apoB from HepG2 cells.

  3. Secretion of interleukin-17 by CD8+ T cells expressing CD146 (MCAM).

    Science.gov (United States)

    Dagur, Pradeep K; Biancotto, Angélique; Stansky, Elena; Sen, H Nida; Nussenblatt, Robert B; McCoy, J Philip

    2014-01-01

    Interleukin-17 (IL-17) has been associated with the pathogenesis of numerous autoimmune diseases. CD4+ T cells secreting IL-17 are termed Th17 cells. CD8+ T cells, designated Tc17 cells, are also capable of secreting IL-17. Here we describe a population of Tc17 cells characterized by the expression of surface CD146, an endothelial adhesion molecule. These cells display signatures of a human Tc17 genotype and phenotype. Circulating CD8+CD146+ T cells are present in low levels in healthy adults. Elevations in CD8+CD146+ T cells are found in Behcet's disease and birdshot retinochoroidopathy, which have been reported to have HLA class I associations. Sarcoidosis does not have a class I association and displays an increase in CD4+ CD146+ T cells but not in CD8+CD146+ T cells. CD146 on these cells may facilitate their ability to bind to, and migrate through, endothelium, as has been reported for CD4+CD146+ T cells. PMID:24681356

  4. 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.

  5. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

    Science.gov (United States)

    Okuda, Satohiro; Tsutsui, Hiroki; Shiina, Keiko; Sprunck, Stefanie; Takeuchi, Hidenori; Yui, Ryoko; Kasahara, Ryushiro D; Hamamura, Yuki; Mizukami, Akane; Susaki, Daichi; Kawano, Nao; Sakakibara, Takashi; Namiki, Shoko; Itoh, Kie; Otsuka, Kurataka; Matsuzaki, Motomichi; Nozaki, Hisayoshi; Kuroiwa, Tsuneyoshi; Nakano, Akihiko; Kanaoka, Masahiro M; Dresselhaus, Thomas; Sasaki, Narie; Higashiyama, Tetsuya

    2009-03-19

    For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri. PMID:19295610

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

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

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

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

    International Nuclear Information System (INIS)

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

  8. Insulin-secreting β cells require a post-genomic concept.

    Science.gov (United States)

    Jiang, Fang-Xu; Morahan, Grant

    2016-05-25

    Pancreatic insulin-secreting β cells are essential in maintaining normal glucose homeostasis accomplished by highly specialized transcription of insulin gene, of which occupies up to 40% their transcriptome. Deficiency of these cells causes diabetes mellitus, a global public health problem. Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells (i.e., primitive cells capable of giving rise to all cell types in the body), a regenerative therapy to diabetes has not yet been established. Furthermore, the nomenclature of β cells has become inconsistent, confusing and controversial due to the lack of standardized positive controls of developmental stage-matched in vivo cells. In order to minimize this negative impact and facilitate critical research in this field, a post-genomic concept of pancreatic β cells might be helpful. In this review article, we will briefly describe how β cells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy, suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell. PMID:27226815

  9. Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion.

    Science.gov (United States)

    Liu, Shi-Hwei; Chou, Wei-I; Lin, Shu-Chuan; Sheu, Chia-Chin; Chang, Margaret Dah-Tsyr

    2005-11-01

    We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner. PMID:16176807

  10. Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

    International Nuclear Information System (INIS)

    We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4 S28N, was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4 S28N mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4 S28N gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner

  11. Androgen - secreting steroid cell tumor of the ovary

    Directory of Open Access Journals (Sweden)

    Paras Ratilal Udhreja

    2014-01-01

    Full Text Available Steroid cell tumors (SCTs, not otherwise specified of the ovary are rare subgroup of sex cord tumors, which account for less than 0.1% of all ovarian tumors and also that will present at any age. The majority of these tumors produce steroids with testosterone being the most common. A case of a 28-year-old woman who presented with symptoms of virilization is reported. Although SCTs are generally benign, there is a risk for malignant transformation. Surgery is the most important and hallmark treatment.

  12. Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells.

    OpenAIRE

    Higgins, P. J.; Darzynkiewicz, Z; Melamed, M R

    1983-01-01

    Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day ex...

  13. Combined AC electroosmosis and dielectrophoresis for controlled rotation of microparticles.

    Science.gov (United States)

    Walid Rezanoor, Md; Dutta, Prashanta

    2016-03-01

    Electrorotation is widely used for characterization of biological cells and materials using a rotating electric field. Generally, multiphase AC electric fields and quadrupolar electrode configuration are needed to create a rotating electric field for electrorotation. In this study, we demonstrate a simple method to rotate dielectrophoretically trapped microparticles using a stationary AC electric field. Coplanar interdigitated electrodes are used to create a linearly polarized nonuniform AC electric field. This nonuniform electric field is employed for dielectrophoretic trapping of microparticles as well as for generating electroosmotic flow in the vicinity of the electrodes resulting in rotation of microparticles in a microfluidic device. The rotation of barium titanate microparticles is observed in 2-propanol and methanol solvent at a frequency below 1 kHz. A particle rotation rate as high as 240 revolutions per minute is observed. It is demonstrated that precise manipulation (both rotation rate and equilibrium position) of the particles is possible by controlling the frequency of the applied electric field. At low frequency range, the equilibrium positions of the microparticles are observed between the electrode edge and electrode center. This method of particle manipulation is different from electrorotation as it uses induced AC electroosmosis instead of electric torque as in the case of electrorotation. Moreover, it has been shown that a microparticle can be rotated along its own axis without any translational motion. PMID:27014394

  14. Geniposide regulates glucose-stimulated insulin secretion possibly through controlling glucose metabolism in INS-1 cells.

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    Jianhui Liu

    Full Text Available Glucose-stimulated insulin secretion (GSIS is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM. Although the KATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.

  15. Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Michaela Norum

    Full Text Available BACKGROUND: The differentiation of an extracellular matrix (ECM at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. PRINCIPAL FINDINGS: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. CONCLUSION: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

  16. IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness

    Directory of Open Access Journals (Sweden)

    Neveu B

    2014-05-01

    Full Text Available Bertrand Neveu*, Xavier Moreel*, Marie-Pier Deschênes-Rompré, Alain Bergeron, Hélène LaRue, Cherifa Ayari, Yves Fradet, Vincent FradetDepartment of Surgery, Laval University Cancer Research Centre, CHU de Quebec Research Centre, Quebec, QC, Canada *These authors contributed equally to this workBackground: Chronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness.Materials and methods: Short-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups.Results: Baseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005.Conclusion: The baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer

  17. Evidence for regulated secretion of proteins by human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    There are two methods of protein secretion, regulated and constitutive. The purpose of this study was to determine if there is a regulated pathway of protein secretion in human umbilical vein endothelial cells (EC). EC were cultured as previously described and then incubated with 3H-leucine. Media and cell were analyzed with time (30 sec-16 hrs) for secreted TCA precipitable labelled protein which was then separated by SDS-PAGE, α-thrombin (Th) (5 x 10-12M). DFP-Th (5 x 10-12M), calcium ionophore A23187 (10-7M) but not Th stimulated rapid (125I-fibrin well assay of 1 mm gel slices of conditioned medium following SDS-PAGE. Th because of EC receptor binding, and possibly because of Ca+2 influx, stimulates a rapid secretion of EC proteins, some of which have been identified as u-PA and t-PA. This may represent a major regulatory function of ECs in maintaining normal hemostasis

  18. Tumor cell-macrophage interactions increase angiogenesis through secretion of EMMPRIN

    Directory of Open Access Journals (Sweden)

    MichalAmitRahat

    2013-07-01

    Full Text Available Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lines were co-cultured with the U937 monocytic-like cell line in the presence of TNFalpha (1 ng/ml. Membranal EMMPRIN expression was increased in the co-cultures (by 3-4 folds, p<0.01, as was the secretion of MMP-9 and VEGF (by 2-5 folds for both MMP-9 and VEGF, p<0.01, relative to the single cultures with TNFalpha. Investigating the regulatory mechanisms, we show that EMMPRIN was post-translationally regulated by miR-146a, as no change was observed in the tumoral expression of EMMPRIN mRNA during co-culture, expression of miR-146a was increased and its neutralization by its antagomir inhibited EMMPRIN expression. The secretion of EMMPRIN was also enhanced (by 2-3 folds, p<0.05, only in the A498 co-culture via shedding off of the membranal protein by a serine protease that is yet to be identified, as demonstrated by the use of wide range protease inhibitors. Finally, soluble EMMPRIN enhanced monocytic secretion of MMP-9 and VEGF, as inhibition of its expression levels by neutralizing anti-EMMPRIN or siRNA in the tumor cells lead to subsequent decreased induction of these two pro-angiogenic proteins. These results reveal a mechanism whereby tumor cell-macrophage interactions promote angiogenesis via an EMMPRIN-mediated pathway.

  19. Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae on in vitro Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Andréa Cruz e Carvalho

    2015-10-01

    Full Text Available Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage, reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

  20. Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

    OpenAIRE

    Webb Bob; Mitchell Marcus RP; Brankin Victoria; Hunter Morag G

    2003-01-01

    Abstract Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles...

  1. Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells.

    Science.gov (United States)

    Green, Alastair D; Vasu, Srividya; McClenaghan, Neville H; Flatt, Peter R

    2015-10-01

    We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity. PMID:25559846

  2. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.

    Science.gov (United States)

    Lumelsky, N; Blondel, O; Laeng, P; Velasco, I; Ravin, R; McKay, R

    2001-05-18

    Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, islet-like organization. PMID:11326082

  3. Uranium induces TNFα secretion and MAPK activation in a rat alveolar macrophage cell line

    International Nuclear Information System (INIS)

    Uranium is a toxic heavy metal found mainly in the nuclear industry, but it is also used in the manufacturing of military munitions. Inhalation studies using animal models have demonstrated that long-term exposure to uranium can lead to the development of neoplasia and fibrosis at the pulmonary level. Because it has been demonstrated that such effects are often associated with inflammation, the effect of uranium on TNFα, IL-1β, and IL-10 synthesis by macrophages was assessed in vitro using the NR8383 cell line. Our results show that a significant TNFα secretion was induced by uranium but not by other metals such as gadolinium. However, IL-1β and IL-10 secretions were unaffected by uranium treatment. TNFα secretion was detectable since 50 μM of uranium and was maximal after 24 h of exposure. Determination of the mechanisms of uranium-induced TNFα production was assessed through the evaluation of protein kinases activation. Our results showed that uranium treatment induced c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) activation. The use of pharmacological inhibitors suggested that both p38 MAPK and protein kinase C (PKC) participate in the signal transduction of uranium-induced TNFα secretion. The regulation of TNFα secretion involves TNFα mRNA accumulation at least through the stabilization of TNFα mRNA, but p38 MAPK did not appear to be involved in this stabilization. However, this observation does not exclude regulation of TNFα synthesis at the transcriptional level, which remains to be demonstrated. Taking together, these results suggest that uranium can induce TNFα secretion by macrophages, thus contributing to a better understanding of the pathological effect of uranium on the lung

  4. Glucose and acute exercise influence factors secreted by circulating angiogenic cells in vitro.

    Science.gov (United States)

    Witkowski, Sarah; Guhanarayan, Gayatri; Burgess, Rachel

    2016-02-01

    Circulating angiogenic cells (CAC) influence vascular repair through the secretion of proangiogenic factors and cytokines. While CAC are deficient in patients with diabetes and exercise has a beneficial effect on CACs, the impact of these factors on paracrine secretion from CAC is unknown. We aimed to determine whether the in vitro secretion of selected cytokines and nitric oxide (NO) from CAC is influenced by hyperglycemia and acute exercise. Colony-forming unit CAC (CFU-CAC) were cultured from young active men (n = 9, 24 ± 2 years) at rest and after exercise under normal (5 mmol/L) and elevated (15 mmol/L) glucose. Preliminary relative multiplex cytokine analysis revealed that CAC conditioned culture media contained three of six measured cytokines: transforming growth factor-beta-1 (TGFβ1), tumor necrosis factor alpha (TNFα), and monocyte chemotactic protein-1 (MCP-1). Single quantitative cytokine analysis was used to determine the concentration of each cytokine from the four conditions. NO was measured via Griess assay. There was a significant effect of CAC exposure to in vivo exercise on in vitro TGFβ1 secretion (P = 0.024) that was independent of glucose concentration. There was no effect of glucose or acute exercise on TNFα or MCP-1 concentration (both P > 0.05). The concentration of NO from CFU-CAC cultured in elevated glucose was lower following acute exercise (P = 0.002) suggesting that exercise did not maintain NO secretion under hyperglycemic conditions. Our results identify paracrine signaling factors that may be responsible for the proangiogenic function of CFU-CAC and an influence of acute exercise and elevated glucose on CFU-CAC soluble factor secretion. PMID:26847726

  5. Secretion of hyaluronic acid by mucosal and submucosal gland epithelial cell cultures derived from human trachea

    International Nuclear Information System (INIS)

    The respiratory epithelium is a target tissue for numerous airborne toxicants. We have established epithelial cell cultures form the mucosa and submucosal glands of human trachea. These epithelial cells exhibit numerous microvilli, desmosomes, and secretory granules. The objective of this study was to characterize the secretory products of these cell cultures. Neat confluent cultures at third passage were labeled with 10 μCi/ml tritiated glucosamine for 20 hours. The culture media was collected, adjusted to 5 mm DTT, and spun at 800 x g for 10 minutes to remove cellular debris. Aliquots were run on a Sepharose CL-4B column in PBS, 1 mm DTT, and 0.02% sodium azide. Void volume fractions were collected and counted by liquid scintillation. The void volume material was found to be susceptible to degradation by bovine testicular and Streptomyces Hyaluronidase. This indicates that hyaluronic acid is the major secretory product (>95%) of these cells. Submucosal gland cell secretion of hyaluronic acid was 175-200% greater than that of mucosal cells. Secretion of hyaluronic acid by the cultured cells indicates that HA may be one of the major secretory products of tracheal epithelial cells in vivo. This model provides an excellent opportunity for studying the affects of environmental agents on the target cell population

  6. Recombinant LPG3 Stimulates IFN-Γ and TNF-Α Secretion by Human NK Cells

    Directory of Open Access Journals (Sweden)

    Sanaz RASOLZADEH

    2015-12-01

    Full Text Available Background: Natural killer (NK cells play an important role in early stages of innate immune responses against viral and tumoral attacks. Activation of NK cells by leishmaniasis results in secretion of cytokines such as interferon (IFN-γ and tumor necrosis factor (TNF-α, which enhances the phagocytosis and clear­ance of parasite. Lipophosphoglycan 3 (LPG3, the Leishmania homologous with GRP94 (glucose regulated protein 94, a member of HSP90 family, contributes to LPG assembly as the most abundant macromolecule on the surface of Leishmania promastigotes.Methods: We purified NK cells from healthy individuals (n=10 using magnetic-activated cell sorting (MACS technology. Purified NK cells were co-incubated with different concentrations of recombinant LPG3 (rLPG3, and its N-terminal (NT and C-terminal (CT fragments. Finally, the production of IFN-γ and TNF-α by NK cells were measured by ELISA.Results: Recombinant LPG3 but not its fragments (CT and NT, can signifi­cantly enhance the production of TNF-α by NK cells (P<0.05. Moreover, rLPG3, CT, and NT fragments were markedly stimulated the secretion of IFN-γ by NK cells (P<0.001.Conclusion: The Leishmania LPG3 antigen can effectively activate NK cells, in vitro. Leishmania LPG3 participates in the innate immunity against leishmaniasis and thereby improves the effective parasite destruction. However, its efficiency should be tested in vivo.

  7. Artificial MicroRNAs as Novel Secreted Reporters for Cell Monitoring in Living Subjects.

    Science.gov (United States)

    Ronald, John A; D'Souza, Aloma L; Chuang, Hui-Yen; Gambhir, Sanjiv Sam

    2016-01-01

    Reporter genes are powerful technologies that can be used to directly inform on the fate of transplanted cells in living subjects. Imaging reporter genes are often employed to quantify cell number, location(s), and viability with various imaging modalities. To complement this, reporters that are secreted from cells can provide a low-cost, in vitro diagnostic test to monitor overall cell viability at relatively high frequency without knowing the locations of all cells. Whereas protein-based secretable reporters have been developed, an RNA-based reporter detectable with amplification inherent PCR-based assays has not been previously described. MicroRNAs (miRNAs) are short non-coding RNAs (18-22 nt) that regulate mRNA translation and are being explored as relatively stable blood-based disease biomarkers. We developed an artificial miRNA-based secreted reporter, called Sec-miR, utilizing a coding sequence that is not expressed endogenously and does not have any known vertebrate target. Sec-miR was detectable in both the cells and culture media of transiently transfected cells. Cells stably expressing Sec-miR also reliably secreted it into the culture media. Mice implanted with parental HeLa cells or HeLa cells expressing both Sec-miR and the bioluminescence imaging (BLI) reporter gene Firefly luciferase (FLuc) were monitored over time for tumor volume, FLuc signal via BLI, and blood levels of Sec-miR. Significantly (p<0.05) higher Sec-miR was found in the blood of mice bearing Sec-miR-expressing tumors compared to parental cell tumors at 21 and 28 days after implantation. Importantly, blood Sec-miR reporter levels after day 21 showed a trend towards correlation with tumor volume (R2 = 0.6090; p = 0.0671) and significantly correlated with FLuc signal (R2 = 0.7067; p<0.05). Finally, we could significantly (p<0.01) amplify Sec-miR secretion into the cell media by chaining together multiple Sec-miR copies (4 instead of 1 or 2) within an expression cassette. Overall, we

  8. Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Cheek, T R; Thastrup, Ole

    1989-01-01

    Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3......+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the...... sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from...

  9. Acute ablation of PERK results in ER dysfunctions followed by reduced insulin secretion and cell proliferation

    Directory of Open Access Journals (Sweden)

    McGrath Barbara C

    2009-09-01

    Full Text Available Abstract Background A deficiency in Perk (EIF2AK3 causes multiple neonatal defects in humans known as the Wolcott Rallison syndrome. Perk KO mice exhibit the same array of defects including permanent neonatal diabetes (PND. PND in mice was previously shown by us to be due to a decrease in beta cell proliferation and insulin secretion. The aim of this study was to determine if acute ablation of PERK in the 832/13 beta cells recapitulates these defects and to identify the primary molecular basis for beta cell dysfunction. Results The INS1 832/13 transformed rat beta cell line was transduced with a dominant-negative Perk transgene via an adenoviral vector. AdDNPerk-832/13 beta cells exhibited reduced expression of insulin and MafA mRNAs, reduced insulin secretion, and reduced cell proliferation. Although proinsulin content was reduced in AdDNPerk-832/13 beta cells, proinsulin was abnormally retained in the endoplasmic reticulum. A temporal study of the acute ablation of Perk revealed that the earliest defect seen was induced expression of two ER chaperone proteins, GRP78/BiP and ERp72. The oxidized states of ERp72 and ERp57 were also increased suggesting an imbalance in the redox state of the ER. Conclusion Acute ablation of Perk in INS 832/13 beta cells exhibited all of the major defects seen in Perk KO mice and revealed abnormal expression and redox state of key ER chaperone proteins. Dysregulation of ER chaperone/folding enzymes ERp72 and GRP78/BiP occurred early after ablation of PERK function suggesting that changes in ER secretory functions may give rise to the other defects including reduced insulin gene expression, secretion, and cell proliferation.

  10. Effect of house dust mite immunotherapy on interleukin-10-secreting regulatory T cells in asthmatic children

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; XIANG Li; LIU Yong-ge; WANG Yong-hong; SHEN Kun-ling

    2010-01-01

    Background Subcutaneous specific immunotherapy has been demonstrated to be capable of inducing T-cell regulatory response.Interleukin-10 (IL-10) plays a crucial role in inducing allergen-specific tolerance.However the reports of the changes of IL-10 in house dust mite (HDM)-specific immunotherapy were varied.The aim of this study was to evaluate the function of IL-10-secreting regulatory T cells in asthma children successfully treated with HDM immunotherapy.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 27 patients following 1.5--2 years of HDM-specific immunotherapy (SIT, SIT group) and from 27 matched treated asthmatic children allergic to HDM (asthmagroup).After 48 hours of in vitro stimulation with HDM extracts, IL-10-secreting regulatory T cells were measured by four colour flow cytometry.Sera were tested for allergen-specific IgG4 and IgE using the Immuno CAP 100 assay.Results PBMCs from children undergoing immunotherapy following HDM extracts stimuli produced significantly more IL-10 compared with the asthma group.The frequency of iTreg cells and aTreg cells increased in SIT group after HDM stimulation, while it was not affected in the asthma group.Among the iTreg cells and aTreg cells, the frequency of CD4+CD25-Foxp3-IL-10+ Treg cells increased the most which was 2 times higher than that in unstimulated cultures in SIT group.The levels of HDM-specific IgG4 of SIT group was significiently higher compared with asthma group, but there was no correlation of the levels of HDM-specific IgG4 and IL-10 secreting Treg cells.Conclusions HDM-specific immunotherapy can successfully upregulate the frequency of IL-10-secreting Treg cells.CD4+CD25-Foxp3-IL-10+ Treg cells may play a key role in inducing the immune tolerance in HDM-specific immunotherapy.

  11. Tumor cell-macrophage interactions increase angiogenesis through secretion of EMMPRIN

    OpenAIRE

    Amit-Cohen, Bat-Chen; Rahat, Maya M.; Rahat, Michal A.

    2013-01-01

    Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin) is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lin...

  12. Effects of opioid peptides and morphine on histamine-induced catecholamine secretion from cultured, bovine adrenal chromaffin cells.

    OpenAIRE

    Livett, B. G.; Marley, P. D.

    1986-01-01

    The effect of opioid peptides and morphine on histamine-induced catecholamine secretion has been studied in monolayer cultures of dispersed, bovine adrenal chromaffin cells. Histamine-induced a dose-dependent secretion of both adrenaline and noradrenaline with a threshold dose of approximately 5 nM, an EC50 of 150 nM and maximal secretion at 10 microM. Catecholamine secretion induced by 1 microM histamine was completely dependent on extracellular calcium, was inhibited in a dose-dependent man...

  13. Partial correction of defective Cl(-) secretion in cystic fibrosis epithelial cells by an analog of squalamine.

    Science.gov (United States)

    Jiang, C; Lee, E R; Lane, M B; Xiao, Y F; Harris, D J; Cheng, S H

    2001-11-01

    Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl(-) transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl(-) transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl(-) gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl(-) currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl(-) solution, indicating restoration of Cl(-) secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl(-) secretion. PMID:11597908

  14. The effect of protoporphyrin on histamine secretion by rat peritoneal mast cells: a dual phototoxic reaction

    International Nuclear Information System (INIS)

    Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100 ng/ml, histamine secretion was completely inhibited after 30 min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible; however, it did not develop into cell lysis. Cell lysis was evoked by increasing the light intensity between 180-950 W/m2, using a light source with emission maxima in the 350-470 nm region. Dual phototoxic effects could also be demonstrated in erythrocytes. Increased resistance to osmotic lysis was seen under moderate illumination conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60-130 W/m2) and lysis (280-950 W/m2). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350-470 nm region. (author)

  15. Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors.

    Science.gov (United States)

    Sharma, Neha; Colangelo, Nicholas W; de Toledo, Sonia M; Azzam, Edouard I

    2016-08-01

    Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or (137)Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells. PMID:27511909

  16. High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. ► Inhibition of PKC-ζ leads to significant reduction of the secreted HMGB1. ► Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. ► Activation of PKC-ζ in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

  17. Fibronectin-Alginate microcapsules improve cell viability and protein secretion of encapsulated Factor IX-engineered human mesenchymal stromal cells.

    Science.gov (United States)

    Sayyar, Bahareh; Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2015-01-01

    Continuous delivery of proteins by engineered cells encapsu-lated in biocompatible polymeric microcapsules is of considerable therapeutic potential. However, this technology has not lived up to expectations due to inadequate cell--matrix interactions and subsequent cell death. In this study we hypoth-esize that the presence of fibronectin in an alginate matrix may enhance the viability and functionality of encapsulated human cord blood-derived mesenchymal stromal cells (MSCs) expressing the human Factor IX (FIX) gene. MSCs were encapsulated in alginate-PLL microcapsules containing 10, 100, or 500 μg/ml fibronectin to ameliorate cell survival. MSCs in microcapsules with 100 and 500 μg/ml fibronectin demonstrated improved cell viability and proliferation and higher FIX secretion compared to MSCs in non-supplemented microcapsules. In contrast, 10 μg/ml fibronectin did not significantly affect the viability and protein secretion from the encapsulated cells. Differentiation studies demonstrated osteogenic (but not chondrogenic or adipogenic) differentiation capability and efficient FIX secretion of the enclosed MSCs in the fibronectin-alginate suspension culture. Thus, the use of recombinant MSCs encapsulated in fibronectin-alginate microcapsules in basal or osteogenic cultures may be of practical use in the treatment of hemophilia B. PMID:24564349

  18. Source of peritoneal proteoglycans. Human peritoneal mesothelial cells synthesize and secrete mainly small dermatan sulfate proteoglycans.

    OpenAIRE

    Yung, S; Thomas, G. J.; Stylianou, E; Williams, J D; Coles, G. A.; Davies, M.

    1995-01-01

    This study describes experiments that compare the proteoglycans (PGs) extracted from the dialysate from patients receiving continuous peritoneal ambulatory dialysis (CAPD) with those secreted by metabolically labeled human peritoneal mesothelial cells in vitro. The PGs isolated from both sources were predominantly small chondroitin sulfate/dermatan sulfate PGs. Western blot of the core proteins obtained after chondroitin ABC lyase treatment with specific antibodies identified decorin and bigl...

  19. Expression Level of IL-6 Secreted by Bone Marrow Stromal Cells in Mice with Aplastic Anemia

    OpenAIRE

    Yong Feng Chen; Zhong Min Wu; Cong Xie; Shi Bai; Li Dong Zhao

    2013-01-01

    Parasecretion of the hematopoietic cytokines is considered as one of the mechanisms account for bone marrow hematopoiesis disorder. In this study, the level of IL-6 secreted by bone marrow stromal cells from a mouse model of aplastic anemia was analyzed. The aplastic anemia mouse model was established with cyclophosphamide in combination with chloramphenicol and 60Co γ radiation. The impairment of bone marrow hematopoiesis induced by irradiation and chemotherapeutic drugs was subsequently cha...

  20. Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

    OpenAIRE

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin

    2010-01-01

    Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveol...

  1. The regulation of vasopressin secretion in a patient with oat cell carcinoma of the bronchus.

    OpenAIRE

    Spruce, B. A.; Baylis, P. H.

    1983-01-01

    We report a patient who had an oat cell bronchogenic carcinoma in association with the syndrome of inappropriate antidiuresis. There was an unusually long interval between the onset of hyponatraemia and clinically evident malignant disease. Dynamic testing of vasopressin secretion showed preservation of baroregulated, but not osmoregulated, vasopressin release. Immunoreactive vasopressin was detected in pleural fluid, which co-eluted with synthetic vasopressin on gel chromatography.

  2. Hyperglycemia affects Matrix Metalloproteinase Secretion in Human Umbilical Vein Endothelial Cells in vitro

    OpenAIRE

    2011-01-01

    Goal: The aim of the current thesis was to study the effect of hyperglycemia (HG) on MMP secretion from human primary endothelial cells. Background: Diabetes Mellitus is characterized by abnormal high levels of glucose in the blood. Endothelial dysfunction is a serious complication of sustained hyperglycemia. Diabetic complications are main reasons for mortality in diabetes. The matrix metalloproteinases (MMPs) and their corresponding inhibitors are the main regulators of the turnover of ...

  3. Mitochondrial superoxide generation is diminished during glucose-stimulated insulin secretion in INS-1E cells

    Czech Academy of Sciences Publication Activity Database

    Špaček, Tomáš; Hlavatá, Lydie; Ježek, Petr

    Elsevier. Roč. 1777, Suppl. (2008), S48-S48. ISSN 0005-2728. [European Bioenergetics Conference /15./. 19.07.2008-24.07.2008, Dublin] R&D Projects: GA AV ČR(CZ) IAA500110701; GA MZd NR9183 Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * superoxide * INS-1E cells * glucose-stimulated insulin secretion Subject RIV: ED - Physiology

  4. Superoxide generation is diminished during glucose-stimulated insulin secretion in INS-1E cells

    Czech Academy of Sciences Publication Activity Database

    Ježek, Petr; Hlavatá, Lydie; Špaček, Tomáš

    2008-01-01

    Roč. 275, Suppl.1 (2008), s. 310-310. ISSN 1742-464X. [FEBS Congress /33./ and IUBMB Conference /11./. 28.06.2008-03.07.2008, Athens] R&D Projects: GA MZd(CZ) NR7917; GA AV ČR(CZ) IAA500110701 Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * superoxide production * glucose-stimulated insulin secretion * INS-1E cells Subject RIV: ED - Physiology

  5. Inhibition of Myocardial Ischemia/Reperfusion Injury by Exosomes Secreted from Mesenchymal Stem Cells

    OpenAIRE

    Heng Zhang; Meng Xiang; Dan Meng; Ning Sun; Sifeng Chen

    2016-01-01

    Exosomes secreted by mesenchymal stem cells have shown great therapeutic potential in regenerative medicine. In this study, we performed meta-analysis to assess the clinical effectiveness of using exosomes in ischemia/reperfusion injury based on the reports published between January 2000 and September 2015 and indexed in the PUBMED and Web of Science databases. The effect of exosomes on heart function was evaluated according to the following parameters: the area at risk as a percentage of the...

  6. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

    International Nuclear Information System (INIS)

    The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain

  7. Physiological and clinical significance of enterochromaffin-like cell activation in the regulation of gastric acid secretion

    Institute of Scientific and Technical Information of China (English)

    Guanglin Cui; Helge L Waldum

    2007-01-01

    Gastric acid plays an important role in digesting food (especially protein), iron absorption, and destroying swallowed micro-organisms. H+ is secreted by the oxyntic parietal cells and its secretion is regulated by endocrine, neurocrine and paracrine mechanisms.Gastrin released from the antral G cell is the principal physiological stimulus of gastric acid secretion. Activation of the enterochromaffin-like (ECL) cell is accepted as the main source of histamine participating in the regulation of acid secretion and is functionally and trophically controlled by gastrin, which is mediated by gastrin/CCK-2 receptors expressed on the ECL cell. However, longterm hypergastrinemia will induce ECL cell hyperplasia and probably carcinoids. Clinically, potent inhibitors of acid secretion have been prescribed widely to patients with acid-related disorders. Long-term potent acid inhibition evokes a marked increase in plasma gastrin levels,leading to enlargement of oxyntic mucosa with ECL cell hyperplasia. Accordingly, the induction of ECL cell hyperplasia and carcinoids remains a topic of considerable concern, especially in long-term use. In addition, the activation of ECL cells also induces another clinical concern, i.e., rebound acid hypersecretion after acid inhibition. Recent experimental and clinical findings indicate that the activation of ECL cells plays a critical role both physiologically and clinically in the regulation of gastric acid secretion.

  8. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    International Nuclear Information System (INIS)

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8+ T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation

  9. Magnaporthe oryzae-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice.

    Science.gov (United States)

    Wang, Yiming; Wu, Jingni; Kim, Sang Gon; Tsuda, Kenichi; Gupta, Ravi; Park, Sook-Young; Kim, Sun Tae; Kang, Kyu Young

    2016-04-01

    The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity. PMID:26780420

  10. Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

    Science.gov (United States)

    Nath, Shubhankar; Christian, Laura; Tan, Sarah Youngsun; Ki, Sanghee; Ehrlich, Lauren I R; Poenie, Martin

    2016-09-15

    Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively. PMID:27534551

  11. Global impact of Salmonella type III secretion effector SteA on host cells

    International Nuclear Information System (INIS)

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration

  12. Global impact of Salmonella type III secretion effector SteA on host cells

    Energy Technology Data Exchange (ETDEWEB)

    Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es

    2014-07-11

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

  13. Human adipocytes stimulate invasion of breast cancer MCF-7 cells by secreting IGFBP-2.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available A better understanding of the effects of human adipocytes on breast cancer cells may lead to the development of new treatment strategies. We explored the effects of adipocytes on the migration and invasion of breast cancer cells both in vitro and in vivo.To study the reciprocal effects of adipocytes and cancer cells, we co-cultured human mature adipocytes and breast cancer cells in a system devoid of heterogeneous cell-cell contact. To analyze the factors that were secreted from adipocytes and that affected the invasive abilities of breast cancer cells, we detected different cytokines in various co-culture media. To study the communication of mature adipocytes and breast cancer cells in vivo, we chose 10 metastatic pathologic samples and 10 non-metastatic pathologic samples to do immunostaining.The co-culture media of human MCF-7 breast cancer cells and human mature adipocytes increased motility of MCF-7 cells. In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells. Based on our co-culture medium chip results, we chose four candidate cytokines and tested their influence on metastasis individually. We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors. In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.IGFBP-2 secreted by mature adipocytes plays a key role in promoting the metastatic ability of MCF-7 breast cancer cells.

  14. Argonaute 2 complexes selectively protect the circulating microRNAs in cell-secreted microvesicles.

    Directory of Open Access Journals (Sweden)

    Limin Li

    Full Text Available Cell-secreted miRNAs are highly stable and can serve as biomarkers for various diseases and signaling molecules in intercellular communication. The mechanism underlying the stability of circulating miRNAs, however, remains incompletely understood. Here we show that Argonaute 2 (Ago2 complexes and microvesicles (MVs provide specific and non-specific protection for miRNA in cell-secreted MVs, respectively. First, the resistance of MV-encapsulated miRNAs to RNaseA was both depended on intact vesicular structure of MVs and protease-sensitive. Second, an immunoprecipitation assay using a probe complementary to human miR-16, a miRNA primarily located in the MVs and showed a strong, protease-sensitive resistance to RNaseA, identified Ago2 as a major miR-16-associated protein. Compared with protein-free miR-16, Ago2-associated miR-16 was resistant to RNaseA in a dose- and time-dependent fashion. Third, when the miR-16/Ago2 complex was disrupted by trypaflavine, the resistance of miR-16 to RNaseA was decreased. In contrast, when the association of miR-16 with the Ago2 complexes was increased during cell apoptosis, although the total amount of miR-16 and Ago2 remained unchanged, the resistance of miR-16 to RNaseA in the MVs was enhanced. A similar correlation between the increase of miR-223/Ago2 association and the resistance of miR-223 against RNaseA was observed during all trans retinoic acid (ATRA-induced cell differentiation of promyelocytic HL60 cells. In conclusion, the association of miRNAs with Ago2 complexes, an event that is linked to cell functional status, plays a critical role in stabilizing the circulating miRNAs in cell-secreted MVs.

  15. Inhibition of voltage-gated potassium channels mediates uncarboxylated osteocalcin-regulated insulin secretion in rat pancreatic β cells.

    Science.gov (United States)

    Gao, Jingying; Zhong, Xiangqin; Ding, Yaqin; Bai, Tao; Wang, Hui; Wu, Hongbin; Liu, Yunfeng; Yang, Jing; Zhang, Yi

    2016-04-15

    Insulin secretion from pancreatic β cells is important to maintain glucose homeostasis and is regulated by electrical activities. Uncarboxylated osteocalcin, a bone-derived protein, has been reported to regulate glucose metabolism by increasing insulin secretion, stimulating β cell proliferation and improving insulin sensitivity. But the underlying mechanisms of uncarboxylated osteocalcin-modulated insulin secretion remain unclear. In the present study, we investigated the relationship of uncarboxylated osteocalcin-regulated insulin secretion and voltage-gated potassium (KV) channels, voltage-gated calcium channels in rat β cells. Insulin secretion was measured by radioimmunoassay. Channel currents and membrane action potentials were recorded using the conventional whole-cell patch-clamp technique. Calcium imaging system was used to analyze intracellular Ca(2+) concentration ([Ca(2+)]i). The data show that under 16.7mmol/l glucose conditions uncarboxylated osteocalcin alone increased insulin secretion and [Ca(2+)]i, but with no such effects on insulin secretion and [Ca(2+)]i in the presence of a KV channel blocker, tetraethylammonium chloride. In the patch-clamp experiments, uncarboxylated osteocalcin lengthened action potential duration and significantly inhibited KV currents, but had no influence on the characteristics of voltage-gated calcium channels. These results indicate that KV channels are involved in uncarboxylated osteocalcin-regulated insulin secretion in rat pancreatic β cells. By inhibiting KV channels, uncarboxylated osteocalcin prolongs action potential duration, increases intracellular Ca(2+) concentration and finally promotes insulin secretion. This finding provides new insight into the mechanisms of osteocalcin-modulated insulin secretion. PMID:26927753

  16. Porphyrin Microparticles for Biological and Biomedical Applications

    Science.gov (United States)

    Huynh, Elizabeth

    Lipids are one of the critical building blocks of life, forming the plasma membrane of cells. In addition, porphyrins also play an equally important role in life, for example, through carrying oxygen in blood. The importance of both these components is evident through the biological and biomedical applications of supramolecular structures generated from lipids and porphyrins. This thesis investigates new porphyrin microparticles based on porphyrin-lipid architecture and their potential applications in biology and medicine. In Chapter 1, a background on lipid and porphyrin-based supramolecular structures is presented and design considerations for generating multifunctional agents. Chapter 2 describes the generation of a monolayer porphyrin microparticle as a dual-modal ultrasound and photoacoustic contrast agent and subsequently, a trimodal ultrasound, photoacoustic and fluorescence contrast agent. Chapter 3 examines the optical and morphological response of these multimodality ultrasound-based contrast agents to low frequency, high duty cycle ultrasound that causes the porphyrin microparticles to convertinto nanoparticles. Chapter 4 examines the generation of bilayer micrometer-sized porphyrin vesicles and their properties. Chapter 5 presents a brief summary and potential future directions. Although these microscale structures are similar in structure, the applications of these structures greatly differ with potential applications in biology and also imaging and therapy of disease. This thesis aims to explore and demonstrate the potential of new simplified, supramolecular structures based on one main building block, porphyrin-lipid.

  17. Microparticles from apoptotic RAW 264.7 macrophage cells carry tumour necrosis factor-α functionally active on cardiomyocytes from adult mice

    Directory of Open Access Journals (Sweden)

    Edward Milbank

    2015-10-01

    Full Text Available After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs are part of the pathological process of unstable atherosclerotic plaques. The present study focused on effects of MPs, produced by apoptotic murine RAW 264.7 macrophage cell line, in adult murine cardiomyocytes. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α. Cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to these MPs. Conversely, Ca2+ transient amplitude and kinetics were not modified. The contractile effects of MPs were completely prevented after pretreatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. After 4 h of treatment with MPs, cardiomyocytes expressed increased caspase-3, caspase-8, Bax and cytochrome C. Thus, MPs from apoptotic macrophages induced a negative inotropic effect and slowing of both contraction and relaxation, similar to that observed in the presence of TNF-α. The use of specific inhibitors strongly suggests that TNF-α receptors and the guanylate cyclase/cGMP/PKG pathway were involved in the functional responses to these MPs and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. These data suggest that MPs issued from activated macrophages carrying TNF-α could contribute to propagation of inflammatory signals leading to myocardial infarction.

  18. Circulating Endothelial-Derived Activated Microparticle: A Useful Biomarker for Predicting One-Year Mortality in Patients with Advanced Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Chin-Chou Wang

    2014-01-01

    Full Text Available Background. This study tested the hypothesis that circulating microparticles (MPs are useful biomarkers for predicting one-year mortality in patients with end-stage non-small cell lung cancer (ES-NSCLC. Methods and Results. One hundred seven patients were prospectively enrolled into the study between April 2011 and February 2012, and each patient received regular follow-up after enrollment. Levels of four MPs in circulation, (1 platelet-derived activated MPs (PDAc-MPs, (2 platelet-derived apoptotic MPs (PDAp-MPs, (3 endothelial-derived activated MPs (EDAc-MPs, and (4 endothelial-derived apoptotic MPs (EDAp-MPs, were measured just after the patient was enrolled into the study using flow cytometry. Patients who survived for more than one year were categorized into group 1 (n=56 (one-year survivors and patients who survived less than one year were categorized into group 2 (n=51 (one-year nonsurvivors. Male gender, incidence of liver metastasis, progression of disease after first-line treatment, poor performance status, and the Charlson comorbidity index were significantly higher in group 2 than in group 1 (all P<0.05. Additionally, as measured by flow cytometry, only the circulating level of EDAc-MPs was found to be significantly higher in group 2 than in group 1 (P=0.006. Multivariate analysis demonstrated that circulating level of EDAc-MPs along with brain metastasis and male gender significantly and independently predictive of one-year mortality (all P<0.035. Conclusion. Circulating EDAc-MPs may be a useful biomarker predictive of one-year morality in ES-NSCLC patients.

  19. Modulation pf pulmonary surfactant secretion from alveolar type II cells by cytoplasmic free calcium ([Ca2+]/sub i/)

    International Nuclear Information System (INIS)

    Ca2+ is regulator of a variety of cellular functions including exocytosis. TPA and terbutaline have been shown to stimulate surfactant secretion from alveolar type II cells. The authors examined changes in [Ca2+]/sub i/ and surfactant secretion by secretagogues in primary culture of alveolar type II cells. Cells were isolated from adult rats and were cultured for 24 h with 3H-choline to label phosphatidylcholine. Percent secretion was determined by counting the lipids of cells and medium; cytotoxicity was excluded by measuring lactate dehydrogenase as cells and medium. [Ca2+]/sub i/ was determined by measuring quin2 fluroescence of cells cultured on a glass coverslip. Ionomycin increased secretion as well as [Ca2+] in dose dependent manner at the concentration from 25 to 400 nM. Ionomycin (50 nM) increased terbutaline-induced secretion in a synergistic manner but only increased TPA-induced secretion in an additive manner. Terbutaline mobilized [Ca2+]/sub i/ from intracellular stores and increased [Ca2+]/sub i/ by 20% from a basal level of 140 nM. TPA itself did not change [Ca2+]/sub i/ but inhibited the effect of terbutaline on [Ca2+]/sub i/. Loading of quin2 in the absence of extracellular calcium lowered [Ca2+]/sub i/ from 143 nM to 31 nM. Lowering [Ca2+]/sub i/ inhibited TPA- or terbutaline-induced secretion by 22% and 40% respectively. These results indicate that [Ca2+]/sub i/ effects cAMp-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells

  20. Modulation pf pulmonary surfactant secretion from alveolar type II cells by cytoplasmic free calcium ((Ca/sup 2 +/)/sub i/)

    Energy Technology Data Exchange (ETDEWEB)

    Sano, K.; Voelker, D.R.; Mason, R.J.

    1986-05-01

    Ca/sup 2 +/ is regulator of a variety of cellular functions including exocytosis. TPA and terbutaline have been shown to stimulate surfactant secretion from alveolar type II cells. The authors examined changes in (Ca/sup 2 +/)/sub i/ and surfactant secretion by secretagogues in primary culture of alveolar type II cells. Cells were isolated from adult rats and were cultured for 24 h with /sup 3/H-choline to label phosphatidylcholine. Percent secretion was determined by counting the lipids of cells and medium; cytotoxicity was excluded by measuring lactate dehydrogenase as cells and medium. (Ca/sup 2 +/)/sub i/ was determined by measuring quin2 fluroescence of cells cultured on a glass coverslip. Ionomycin increased secretion as well as (Ca/sup 2 +/) in dose dependent manner at the concentration from 25 to 400 nM. Ionomycin (50 nM) increased terbutaline-induced secretion in a synergistic manner but only increased TPA-induced secretion in an additive manner. Terbutaline mobilized (Ca/sup 2 +/)/sub i/ from intracellular stores and increased (Ca/sup 2 +/)/sub i/ by 20% from a basal level of 140 nM. TPA itself did not change (Ca/sup 2 +/)/sub i/ but inhibited the effect of terbutaline on (Ca/sup 2 +/)/sub i/. Loading of quin2 in the absence of extracellular calcium lowered (Ca/sup 2 +/)/sub i/ from 143 nM to 31 nM. Lowering (Ca/sup 2 +/)/sub i/ inhibited TPA- or terbutaline-induced secretion by 22% and 40% respectively. These results indicate that (Ca/sup 2 +/)/sub i/ effects cAMp-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells.

  1. L-type calcium channels in adrenal chromaffin cells: role in pace-making and secretion.

    Science.gov (United States)

    Marcantoni, A; Baldelli, P; Hernandez-Guijo, J M; Comunanza, V; Carabelli, V; Carbone, E

    2007-01-01

    Voltage-gated L-type (Cav1.2 and Cav1.3) channels are widely expressed in cardiovascular tissues and represent the critical drug-target for the treatment of several cardiovascular diseases. The two isoforms are also abundantly expressed in neuronal and neuroendocrine tissues. In the brain, Cav1.2 and Cav1.3 channels control synaptic plasticity, somatic activity, neuronal differentiation and brain aging. In neuroendocrine cells, they are involved in the genesis of action potential generation, bursting activity and hormone secretion. Recent studies have shown that Cav1.2 and Cav1.3 are also expressed in chromaffin cells but their functional role has not yet been identified despite that L-type channels possess interesting characteristics, which confer them an important role in the control of catecholamine secretion during action potentials stimulation. In intact rat adrenal glands L-type channels are responsible for adrenaline and noradrenaline release following splanchnic nerve stimulation or nicotinic receptor activation. L-type channels can be either up- or down-modulated by membrane autoreceptors following distinct second messenger pathways. L-type channels are tightly coupled to BK channels and activate at relatively low-voltages. In this way they contribute to the action potential hyperpolarization and to the pace-maker current controlling action potential firings. L-type channels are shown also to regulate the fast secretion of the immediate readily releasable pool of vesicles with the same Ca(2+)-efficiency of other voltage-gated Ca(2+) channels. In mouse adrenal slices, repeated action potential-like stimulations drive L-type channels to a state of enhanced stimulus-secretion efficiency regulated by beta-adrenergic receptors. Here we will review all these novel findings and discuss the possible implication for a specific role of L-type channels in the control of chromaffin cells activity. PMID:17561252

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

    International Nuclear Information System (INIS)

    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-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

  3. 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

  4. Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Hong, Shin-Young; Lee, Tae-Sup; Kim, Ju; Jung, Jae-Ho; Choi, Chang-Woon; Kim, Tae-Geum; Kwon, Tae-Ho; Jang, Yong-Suk; Yang, Moon-Sik

    The tumor-associated glycoprotein 72 (TAG 72) has been shown to be expressed in the majority of human adenocarcinomas. In an effort to develop a technique for the safe and inexpensive production of large quantities of anti-TAG 72 humanized antibody fragments (hzAb) as a future source of clinical......-grade proteins, we developed a transgenic rice cell suspension culture system. The in vivo assembly and secretion of hzAb were achieved in a transgenic rice cell culture under the control of the rice alpha amylase 3D (RAmy 3D) expression system, and the biological activities of plant-derived hzAb were determined...

  5. Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

    OpenAIRE

    Woo, Jung Hee; Liu, Yuan Yi; Stavrou, Scott; Neville, David M.

    2004-01-01

    The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G4S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut+) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The im...

  6. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

    OpenAIRE

    Smith, G.E.; Ju, G; Ericson, B L; Moschera, J; Lahm, H W; Chizzonite, R; Summers, M D

    1985-01-01

    A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to t...

  7. Translocation of Helicobacter pylori CagA into Gastric Epithelial Cells by Type IV Secretion

    Science.gov (United States)

    Odenbreit, Stefan; Püls, Jürgen; Sedlmaier, Bettina; Gerland, Elke; Fischer, Wolfgang; Haas, Rainer

    2000-02-01

    The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA+) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.

  8. Metabolic memory of ß-cells controls insulin secretion and is mediated by CaMKIIa

    OpenAIRE

    Santos, Gustavo Jorge dos; Ferreira, Sandra Mara; Ortis, Fernanda; Rezende, Luiz Fernando; Li, Chengyang; Naji, Ali; Carneiro, Everardo Magalhães; Kaestner, Klaus H.; Boschero, Antonio Carlos

    2014-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of “metabolic memory”, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h con...

  9. Effect of PKC on Glucose-Mediated Insulin Secretion in HIT-T15 Cells

    Directory of Open Access Journals (Sweden)

    Akiyoshi H

    2000-09-01

    Full Text Available OBJECTIVE: To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. MAIN OUTCOME MEASURES: We examined the effect of protein kinase C on the cytosolic free Ca(2+ concentration ([Ca(2+]i and the activity of Ca(2+-activated K(+ channels (K(Ca-channel in the insulinoma cell line, HIT-T15. RESULTS: Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L also attenuated the glucose-induced elevations in [Ca(2+]i. On the contrary, glibenclamide (100 nmol/L specifically blocked ATP-sensitive K(+ channels, and increased both [Ca(2+]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L, a protein kinase C activator, also decreased K(Ca channel activity. CONCLUSIONS: These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+ channel function in HIT-T15 cells.

  10. Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism

    DEFF Research Database (Denmark)

    Vandahl, Brian B S; Stensballe, Allan; Roepstorff, Peter;

    2005-01-01

    infected cells, whereas only the 65 kDa full-length Cpn0796 could be detected in purified Chlamydia. Confocal immunofluorescence microscopy demonstrated that Cpn0796 was localized in the Chlamydia membrane in young inclusions. However, at 36 h post infection and later Cpn0796 was detected in the cytoplasm...... of C. pneumoniae infected HEp-2 and BHK cells. Furthermore, Cpn0796 was detected in the cytoplasm of infected cells in the lungs of C. pneumoniae infected C57Bl mice. When cleavage was inhibited, Cpn0796 was retained in the chlamydiae. We propose that Cpn0796 is an autotransporter the N-terminal of...... which is translocated to the host cell cytoplasm. This is the first example of secretion of a Chlamydia autotransporter passenger domain into the host cell cytoplasm. Cpn0796 is specific for C. pneumoniae, where five homologous proteins are encoded by clustered genes. None of these five proteins were...

  11. Red Liriope platyphylla stimulated the insulin secretion through the regulation of calcium concentration in rat insulinoma cells and animal models

    OpenAIRE

    Lee, Hye-Ryun; Kim, Ji-Eun; Lee, Young-Ju; Kwak, Moon-Hwa; Im, Dong-Soon; Hwang, Dae-Youn

    2013-01-01

    The aim of this study was to investigate the effects of Red L. platyphylla (RLP) on calcium and glucose levels during insulin secretion. To achieve this, alteration of insulin and calcium concentrations was measured in rat insulinoma-1 (INS-1) cells and animal models in response to RLP treatment. In INS-1 cells, maximum secretion of insulin was detected upon treatment with 200 µg/mL of RLP for 20 min. Nifedipine, an L-type calcium channel blocker, effectively inhibited insulin secretion from ...

  12. The Int-2/Fgf-3 oncogene product is secreted and associates with extracellular matrix: implications for cell transformation.

    OpenAIRE

    Kiefer, P; Peters, G.; Dickson, C

    1991-01-01

    NIH3T3 cells transformed by mouse Int-2/Fgf-3 cDNA express a series of Int-2-related products representing discrete stages of processing and glycosylation. We confirm that in at least two highly transformed clonal lines, Int-2 products acquire further modifications and are efficiently secreted into the culture medium. Secreted proteins become associated with the cell surface and extracellular matrix and can be displaced by addition of soluble glycosaminoglycans, specifically heparin, heparan ...

  13. The regulatory mechanism of Hsp90α secretion from endothelial cells and its role in angiogenesis during wound healing

    International Nuclear Information System (INIS)

    Research highlights: → Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90α secretion from endothelial cells. → Secreted Hsp90α localizes on the leading edge of activated endothelial cells. → Secreted Hsp90α promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90α can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90α from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90α in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90α but not Hsp90β is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90α localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90α neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90α localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90α can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90α as a stimulator for wound repair.

  14. The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

    2010-07-16

    Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

  15. Con A-induced secretion of IL-2-like activity by mouse Thy-1+ epidermal cells

    International Nuclear Information System (INIS)

    Recently the authors reported that Con A stimulates in vitro proliferation of Thy-1+ dendritic epidermal cells (EC). They have not investigated the capacity of Con A-stimulated Thy-1+ EC to secrete IL-2-like activity (IL-2) 7-17EC is a 4 month-old line of Thy-1+ EC established from AKR/J EC by repeated stimulation with Con A and IL-2; these cells were 99% Thy-1+, but <1% L3T4+ and only 4-10% Lyt-2+. 7-17EC were harvested, washed, and plated in 96-well U-plates. Proliferation was assayed after 3H-thymidine pulsing; culture media was tested for IL-2 using the IL-2-dependent HT-2 cell line. With continuous Con A, marked IL-2 secretion (day 2 peak) was followed in parallel by proliferation (day 4 peak). In the absence of Con A, neither IL-2 nor proliferation was seen; however, both were recovered by addition of Con A to cells cultured first for 48 hr in media alone. 7-17EC proliferation was maximal at 0.5-2 μg Con A while maximal IL-2 secretion (100-200 U/ml) was seen at 4-8 μg/ml. That this amount of IL-2 was in excess of that required for their own optimal proliferation was supported by the failure of additional recombinant IL-2 (10 U/ml) to enhance Con A-stimulated proliferation of 7-17EC over a 4 day period. These findings further document the wide range of immunologic capabilities of Thy-1+ cells derived from epidermis

  16. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

    2016-08-01

    Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. PMID:27426041

  17. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

    Science.gov (United States)

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C B; Hashim, Onn H; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  18. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    Science.gov (United States)

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.

  19. Periostin secreted by mesenchymal stem cells supports tendon formation in an ectopic mouse model.

    Science.gov (United States)

    Noack, Sandra; Seiffart, Virginia; Willbold, Elmar; Laggies, Sandra; Winkel, Andreas; Shahab-Osterloh, Sandra; Flörkemeier, Thilo; Hertwig, Falk; Steinhoff, Christine; Nuber, Ulrike A; Gross, Gerhard; Hoffmann, Andrea

    2014-08-15

    True tendon regeneration in human patients remains a vision of musculoskeletal therapies. In comparison to other mesenchymal lineages the biology of tenogenic differentiation is barely understood. Specifically, easy and efficient protocols are lacking that might enable tendon cell and tissue differentiation based on adult (stem) cell sources. In the murine mesenchymal progenitor cell line C3H10T½, overexpression of the growth factor bone morphogenetic protein 2 (BMP2) and a constitutively active transcription factor, Smad8 L+MH2, mediates tendon cell differentiation in vitro and the formation of tendon-like tissue in vivo. We hypothesized that during this differentiation secreted factors involved in extracellular matrix formation exert a major impact on tendon development. Gene expression analyses revealed four genes encoding secreted factors that are notably upregulated: periostin, C-type lectin domain family 3 (member b), RNase A4, and follistatin-like 1. These factors have not previously been implicated in tendon biology. Among these, periostin showed a specific expression in tenocytes of adult mouse Achilles tendon and in chondrocytes within the nonmineralized fibrocartilage zone of the enthesis with the calcaneus. Overexpression of periostin alone or in combination with constitutively active BMP receptor type in human mesenchymal stem cells and subsequent implantation into ectopic sites in mice demonstrated a reproducible moderate tenogenic capacity that has not been described before. Therefore, periostin may belong to the factors contributing to the development of tenogenic tissue. PMID:24809660

  20. Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women

    Science.gov (United States)

    Jayachandran, Muthuvel; Litwiller, Robert D.; Owen, Whyte G.; Heit, John A.; Behrenbeck, Thomas; Mulvagh, Sharon L.; Araoz, Philip A.; Budoff, Matthew J.; Harman, S. Mitchell; Miller, Virginia M.

    2008-01-01

    While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC) was scored using 64-slice computed tomography scanners. The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score 50; range, 93–315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis. PMID:18621859

  1. Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

    Directory of Open Access Journals (Sweden)

    Webb Bob

    2003-08-01

    Full Text Available Abstract Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF; a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa, 0.01 ng/ml LH (theca or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture and with/without oocyte conditioned medium (OCM or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P

  2. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  3. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    International Nuclear Information System (INIS)

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and α-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin

  4. Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

    Directory of Open Access Journals (Sweden)

    Ruzica Livaja Koshiar

    Full Text Available Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte

  5. The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

    Science.gov (United States)

    Satchidanandam, Vijaya; Kumar, Naveen; Biswas, Sunetra; Jumani, Rajiv S; Jain, Chandni; Rani, Rajni; Aggarwal, Bharti; Singh, Jaya; Kotnur, Mohan Rao; Sridharan, Anand

    2016-04-01

    We previously reported that Rv1860 protein fromMycobacterium tuberculosisstimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulentM. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latentlyM. tuberculosis-infected individuals dominated by CD8(+)T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+)PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studiedM. tuberculosisantigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+)T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+)T-cell-stimulating antigens has the potential to prevent progression of latentM. tuberculosisinfection to TB disease. PMID:26843486

  6. Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion

    NARCIS (Netherlands)

    C. Donadee (Chenell); N.J.H. Raat (Nicolaas); T. Kanias (Tamir); J. Tejero (Jesús); J.S. Lee (Janet); E.E. Kelley (Eric); X. Zhao (Xuejun); C. Liu (Chen); H. Reynolds (Hannah); I. Azarov (Ivan); S. Frizzell (Sheila); E.M. Meyer (Michael); A.D. Donnenberg (Albert); L. Qu (Lirong); D. Triulzi (Darrel); D.B. Kim-Shapiro (Daniel); M.T. Gladwin (Mark)

    2011-01-01

    textabstractBacground-: Intravascular red cell hemolysis impairs nitric oxide (NO)-redox homeostasis, producing endothelial dysfunction, platelet activation, and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of

  7. Assessment of beta-cell function and insulin secretion in subjects that underwent renal transplantation.

    Science.gov (United States)

    Tura, Andrea; Hecking, Manfred; Wolzt, Michael; Saemann, Marcus D; Pacini, Giovanni

    2015-08-01

    In this study we aimed to assess the performance of various indices of beta-cell function derived from oral glucose tolerance test (OGTT) in subjects that underwent renal transplantation. Impaired insulin secretion seems in fact central for development of new onset diabetes after transplantation, but its assessment has not been systematically evaluated. Twenty subjects underwent a 75 g 2h-OGTT for measurement of glucose, insulin, C-peptide. OGTT indices of beta-cell function were either derived by mathematical modeling (yielding the reference index: glucose sensitivity) or were empirical: insulinogenic index (IGI), IGI derived indices, whole shape C-peptide (WHOSH_CP). Indices of beta cell function, showed significant correlation with glucose sensitivity (R(2)=0.40-0.86, all Pbody mass index, mean glycemia. In conclusion, in transplanted subjects OGTT empirical indices are typically acceptable for the estimation of beta-cell function. PMID:26736770

  8. The pre-synaptic blocker toosendanin does not inhibit secretion in exocrine cells

    Institute of Scientific and Technical Information of China (English)

    Zong-Jie Cui; Xue-Hui He

    2002-01-01

    AIM: Toosendanin is a pre-synaptic blocker at theneuromuscular junction and its inhibitory effect is dividedinto an initial facilitative/stimulatory phase followed by aprolonged inhibitory phase. The present study investigatedwhether the subsequent inhibitory phase was due toexhaustion of the secretory machinery as a result of extensivestimulation during the initial facilitative phase. Morespecifically, this paper examined whether toosendanin coulddirectly inhibit the secretory machinery in exocrine cells.METHODS: Rat pancreatic acinar cells were isolated bycollagenase digestion. Secretion was assessed by measuringthe amount of amylase released into the extracellular mediumas a percentage of the total present in the cells beforestimulation. Cholecystokinin (CCK)-induced increases inintracellular calcium in single cells were measured with fura-2 microfluorometry.RESULTS: Effects of toosendanin on CCK-induced amylasesecretion and calcium oscillations were investigated.Toosendanin of 87-870 tM had no effect on 10 pM-100 nMCCK-stimulated amylase secretion, nor did 8.7-870 μMtoosendanin inhibit 5 pM CCK-induced calcium oscillations.In contrast, 10 nM CCK1 receptor antagonist FK 480 completelyblocked 5 pM CCK-induced calcium oscillations.CONCLUSION: The pre-synaptic "blocker" toosendanin is aselective activator of the voltage-dependent calcium channels,but does not interfere with the secretory machinery itself.

  9. Natural phenylpropanoids inhibit lipoprotein-induced endothelin-1 secretion by endothelial cells.

    Science.gov (United States)

    Martin-Nizard, Françoise; Sahpaz, Sevser; Kandoussi, Abdelmejid; Carpentier, Marie; Fruchart, Jean-Charles; Duriez, Patrick; Bailleul, François

    2004-12-01

    There is increasing evidence that oxidized low-density lipoproteins (Ox-LDL) might be involved in the pathogenesis of atherosclerosis and it has been reported that polyphenols inhibit LDL peroxidation and atherosclerosis. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide isolated from endothelial cells and it induces smooth muscle cell proliferation. ET-1 secretion is increased in atheroma and induces deleterious effects such as vasospasm and atherosclerosis. The goal of this study was to test the effect of four natural phenolic compounds against copper-oxidized LDL (Cu-LDL)-induced ET-1 liberation by bovine aortic endothelial cells (BAEC). The tested compounds were phenylpropanoid glycosides previously isolated from the aerial parts of Marrubium vulgare L. (acteoside 1, forsythoside B 2, arenarioside 3 and ballotetroside 4). ET-1 secretion increased when cells were incubated with Cu-LDL but the compounds 1-4 inhibited this increase. These results were confirmed by quantitative-polymerase chain reaction (QPCR) analysis. Since ET-1 plays an important role in atherosclerosis development, our work suggests that the tested phenylpropanoids could have a beneficial effect in inhibiting atherosclerosis development. PMID:15563769

  10. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck;

    2012-01-01

    It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is...... labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited...... more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel...

  11. Towards injectable cell-based tissue-engineered bone : The effect of different calcium phosphate microparticles and pre-culturing

    NARCIS (Netherlands)

    Persson, C; Johansson, G; Dhert, WJA; Kruyt, Moyo C.; de Bruijn, Joost D.

    2006-01-01

    Bone tissue engineering by combining bone marrow stromal cells (BMSCs) with a porous scaffold is a promising technology. Current major challenges are to upscale the technique for clinical application and to improve the handling characteristics. With respect to minimal invasive surgery, moldable and/

  12. Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

    International Nuclear Information System (INIS)

    The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of [3H] thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (950C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of ≅30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth

  13. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias; Messner, Paul; Schneider, Karl; Wassermann, Klemens; Micutkova, Lucia; Fortschegger, Klaus; Maier, Andrea B; Westendorp, Rudi; Resch, Heinrich; Wolbank, Susanne; Redl, Heinz; Jansen-Dürr, Pidder; Pietschmann, Peter; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-08-01

    Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor. PMID:27146333

  14. Dielectrophoretic micropatterning with microparticle monolayers covalently linked to glass surfaces.

    Science.gov (United States)

    Suzuki, Masato; Yasukawa, Tomoyuki; Mase, Yoshiaki; Oyamatsu, Daisuke; Shiku, Hitoshi; Matsue, Tomokazu

    2004-12-01

    Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 mum) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (DSS) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity. PMID:15568852

  15. Sex Steroids Influence Brain-Derived Neurotropic Factor Secretion From Human Airway Smooth Muscle Cells.

    Science.gov (United States)

    Wang, Sheng-Yu; Freeman, Michelle R; Sathish, Venkatachalem; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S

    2016-07-01

    Brain derived neurotropic factor (BDNF) is emerging as an important player in airway inflammation, remodeling, and hyperreactivity. Separately, there is increasing evidence that sex hormones contribute to pathophysiology in the lung. BDNF and sex steroid signaling are thought to be intricately linked in the brain. There is currently little information on BDNF and sex steroid interactions in the airway but is relevant to understanding growth factor signaling in the context of asthma in men versus women. In this study, we assessed the effect of sex steroids on BDNF expression and secretion in human airway smooth muscle (ASM). Human ASM was treated with estrogen (E2 ) or testosterone (T, 10 nM each) and intracellular BDNF and secreted BDNF measured. E2 and T significantly reduced secretion of BDNF; effects prevented by estrogen and androgen receptor inhibitor, ICI 182,780 (1 μM), and flutamide (10 μM), respectively. Interestingly, no significant changes were observed in intracellular BDNF mRNA or protein expression. High affinity BDNF receptor, TrkB, was not altered by E2 or T. E2 (but not T) significantly increased intracellular cyclic AMP levels. Notably, Epac1 and Epac2 expression were significantly reduced by E2 and T. Furthermore, SNARE complex protein SNAP25 was decreased. Overall, these novel data suggest that physiologically relevant concentrations of E2 or T inhibit BDNF secretion in human ASM, suggesting a potential interaction of sex steroids with BDNF in the airway that is different from brain. The relevance of sex steroid-BDNF interactions may lie in their overall contribution to airway diseases such as asthma. J. Cell. Physiol. 231: 1586-1592, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566264

  16. Distribution of kappa and lambda light chain isotypes among human blood immunoglobulin-secreting cells after vaccination with pneumococcal polysaccharides

    DEFF Research Database (Denmark)

    Heilmann, C; Barington, T

    1989-01-01

    The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pok...

  17. [Dendritic Cells Promote the Proliferation of Peripheral Blood CRTH2 Cells (CD4(+)CD294(+)Th2) and Help B Cells to Secrete Immunoglobulin].

    Science.gov (United States)

    Tian, Fa-Qing; Li, Juan; Li, Ju-Heng; Tang, Mei-Qin; Cheng, Xiao-Hui; Huang, Ying-Cai; Li, Hui-Qing

    2016-08-01

    Objective:To investigate the promotive effect of dendritic cells(DCs) on proliferation of CRTH2 (CD4(+)CD294(+)Th2) cells and the influence of CRTH2 cells on secretion of immunoglobulin from B cells so as to provide a new approach for amplification and sorting of Th2 cells. Methods:DCs were induced from peripheral blood mononuclear cells, then the loaded-BCGV-Ag-DCs were cocultured with T cells, and the mixed lymphocyte reaction(MLR) was performed by CCK8 method. The phenotypes of DCs and CRTH2 cells were detected by flow cytometry. CRTH2 cells sorted by MACS were co-cultured with B cells for 5 days to detect the secretion of immunoglobulin. Results:The subsets and absolute number CRTH2 cells were significantly increased by loaded-BCGV-Ag-DCs. The levels of IgG, IgA and IgE were higher increased in supernatant of CRTH2 and B cell co-culture system than that in control group or that in transwell group(Pcells can be greatly promoted by loaded-BCGV-Ag-DCs, and the CRTH2 cells can help B cells to secrete IgG, IgA and IgE. PMID:27531793

  18. Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.

    Science.gov (United States)

    Pavia, J; Aguado, C; Mormeneo, S; Sentandreu, R

    2001-07-01

    The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls. PMID:11429475

  19. Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

    Directory of Open Access Journals (Sweden)

    Reiji Kojima

    Full Text Available Galectin-9 (Gal-9, a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

  20. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion

    OpenAIRE

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and tran...

  1. Influence of elevated body temperature on circulating immunoglobulin-secreting cells

    DEFF Research Database (Denmark)

    Kappel, M; Barington, T; Gyhrs, A; Pedersen, Bente Klarlund

    1995-01-01

    This work was designed to investigate the effect of in vivo hyperthermia in man on circulating immunoglobulin-secreting cells. Eight healthy male volunteers were immersed into a hot waterbath (WI) (water temperature 39.5 degrees C) for 2 h, whereby their body temperature rose to 39.5 degrees C. On...... another occasion they served as their own controls, being immersed into thermoneutral water (water temperature 34.5 degrees C) for 2 h. Blood samples were drawn before immersion, at body temperatures of 38, 39 and 39.5 degrees C, as well as 2 h after WI when their body temperatures were normalized. In the...

  2. Quantitative analysis of the secretion of the MCP family of chemokines by muscle cells

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Pedersen, Bente Klarlund; Kratchmarova, Irina

    2011-01-01

    by other tissues are still very limited. In order to comprehensively characterize the low abundant low molecular weight secreted proteins during the course of muscle differentiation we used a mass spectrometry-based proteomics strategy. The application of the triple encoding Stable Isotope Labeling...... by Amino acids in Cell culture (SILAC) method for quantitative analysis resulted in the identification and generation of quantitative profiles of 59 growth factors and cytokines, including 9 classical chemokines. The members of the CC chemokine family of proteins such as monocyte chemotactic proteins...

  3. Sox17 Regulates Insulin Secretion in the Normal and Pathologic Mouse β Cell

    OpenAIRE

    Jonatan, Diva; Spence, Jason R.; Method, Anna M.; Kofron, Matthew; Sinagoga, Katie; Haataja, Leena; Arvan, Peter; Deutsch, Gail H.; Wells, James M.

    2014-01-01

    SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin prote...

  4. Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

    OpenAIRE

    Huygen, K.; Abramowicz, D.; Vandenbussche, P; Jacobs, F.; De Bruyn, J; Kentos, A.; Drowart, A; van Vooren, J P; Goldman, M.

    1992-01-01

    Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular ...

  5. EMR2 Receptor Ligation Modulates Cytokine Secretion Profiles and Cell Survival of Lipopolysaccharide-treated Neutrophils

    Directory of Open Access Journals (Sweden)

    Tzu-Ying Chenee

    2011-10-01

    Full Text Available Background: Epidermal growth factor (EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2 is an adhesion G protein-coupled receptor previouslyshown to potentiate neutrophil responses to a number of inflammatory stimuli. EMR2 activation promotes neutrophil adhesion and migration, and augments production of reactive oxygen species and degranulation. In this study,we examined the effect of EMR2 ligation by its specific antibody on thecytokine expression profile and cell survival of lipopolysaccharide (LPS-treated neutrophils.Methods: Neutrophils were treated with LPS in the absence or presence of the antiEMR2 mAb, 2A1. Cell apoptosis was determined by flow cytometry analysisusing annexin-V and propidium iodide staining. Cell supernatants were collected for the detection of cytokine secretion by enzyme-linked immunosorbent assay.Results: We confirmed the specific priming effect of EMR2 on the response of neutrophils to formyl-Met-Leu-Phe by measuring the production of reactiveoxygen species. Furthermore, we showed that EMR2 ligation suppressesLPS-induced neutrophil survival. In addition, we demonstrated that ligationof EMR2 changes the secretion profiles of multiple cytokines, includinginterleukin (IL-6, IL-8, and monocyte chemotactic protein-1. Finally, higherlevels of EMR2 were detected on neutrophils of liver cirrhosis patients andwere correlated to a pro-apoptotic phenotype.Conclusion: Collectively, the present data indicate a functional role for EMR2 in themodulation of neutrophil activation during inflammation.

  6. Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space.

    Science.gov (United States)

    Ma, Xiao; Pietsch, Jessica; Wehland, Markus; Schulz, Herbert; Saar, Katrin; Hübner, Norbert; Bauer, Johann; Braun, Markus; Schwarzwälder, Achim; Segerer, Jürgen; Birlem, Maria; Horn, Astrid; Hemmersbach, Ruth; Waßer, Kai; Grosse, Jirka; Infanger, Manfred; Grimm, Daniela

    2014-02-01

    This study focuses on the effects of short-term [22 s, parabolic flight campaign (PFC)] and long-term (10 d, Shenzhou 8 space mission) real microgravity on changes in cytokine secretion and gene expression patterns in poorly differentiated thyroid cancer cells. FTC-133 cells were cultured in space and on a random positioning machine (RPM) for 10 d, to evaluate differences between real and simulated microgravity. Multianalyte profiling was used to evaluate 128 secreted cytokines. Microarray analysis revealed 63 significantly regulated transcripts after 22 s of microgravity during a PFC and 2881 after 10 d on the RPM or in space. Genes in several biological processes, including apoptosis (n=182), cytoskeleton (n=80), adhesion/extracellular matrix (n=98), proliferation (n=184), stress response (n=268), migration (n=63), angiogenesis (n=39), and signal transduction (n=429), were differentially expressed. Genes and proteins involved in the regulation of cancer cell proliferation and metastasis, such as IL6, IL8, IL15, OPN, VEGFA, VEGFD, FGF17, MMP2, MMP3, TIMP1, PRKAA, and PRKACA, were similarly regulated under RPM and spaceflight conditions. The resulting effect was mostly antiproliferative. Gene expression during the PFC was often regulated in the opposite direction. In summary, microgravity is an invaluable tool for exploring new targets in anticancer therapy and can be simulated in some aspects in ground-based facilities. PMID:24196587

  7. Improved endothelialization of titanium vascular implants by extracellular matrix secreted from endothelial cells.

    Science.gov (United States)

    Tu, Qiufen; Zhao, Yuancong; Xue, Xiaoqing; Wang, Jin; Huang, Nan

    2010-12-01

    A variety of metals have been widely used in construction of cardiovascular implants (CVIs), such as artificial heart valves, ventricular pumps, and vascular stents. Although great effects have been put into rigorous anticoagulation, late thrombosis still occurred due to inferior blood and cell compatibility. Natural endothelium is popularly regarded as the only substance that has long-term anticoagulant ability. So, establishment of a compact endothelial cell (EC) monolayer on CVIs surface is a guarantee for their long-term potency. In the work described here, titanium (Ti) disks were coated with extracellular matrix (ECM) directly secreted by human umbilical vein endothelial cells (HUVECs), so as to help ECs proliferate and migrate and to improve their endothelialization in vivo. Deposition of ECM on Ti disks was detected by immunofluorescence microscopy, diffuse reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The surface topography and wettability of the Ti disks significantly changed after ECM deposition. Most importantly, it was found that ECM deposition inhibited platelet adhesion, stimulated EC proliferation, increased EC migration speed in vitro, and eventually accelerated the re-cellularization speed of Ti disks in vivo. These important results render it reasonable and feasible to modify CVIs with ECM secreted from ECs for improving their long-term potency. PMID:20666613

  8. Immune-related intestinal Cl- secretion. I. Effect of histamine on the T84 cell line

    International Nuclear Information System (INIS)

    The mast cell mediator, histamine, induces a rapid and transient increase in chloride secretion across monolayers of the human colonic epithelial cell line, T84. Threshold stimulation occurred at 3 x 10-6 M histamine and a maximal effect at 10-4 M. The effect was reproduced by the H1 agonists 2-methylhistamine and 2-pyridylethylamine, but not by the H2 agonists 4-methylhistamine and dimaprit, suggesting the involvement of an H1 receptor. Additionally, histamine's action was inhibited by an H1 antagonist, diphenhydramine, but not by an H2 antagonist, cimetidine. Histamine treatment increased free cytosolic calcium levels, but not those of adenosine 3',5'-cyclic monophosphate (cAMP) or guanosine 3',5'-cyclic monophosphate (cGMP). The mechanism of chloride secretion induced by histamine resembled that of carbachol, in that both (1) were associated with an increase in free cytosolic calcium, (2) had a site of activation at a basolaterally localized K+ channel, and (3) were potentiated by both cAMP- and cGMP-mediated secretagogues. These results suggest that histamine may act as an intestinal secretagogue via direct interactions with epithelial cells

  9. Glucagon-like peptide 1 receptor playsa critical role in geniposide-regulated insulin secretion in INS-1 cells

    Institute of Scientific and Technical Information of China (English)

    Li-xia GUO; Zhi-ning XIA; Xue GAO; Fei YIN; Jian-hui LIU

    2012-01-01

    Aim:To explore the role of the glucagon-like peptide 1 receptor (GLP-1R) in geniposide regulated insulin secretion in rat INS-1 insulinoma cells.Methods:Rat INS-1 insulinoma cells were cultured.The content of insulin in the culture medium was measured with ELISA assay.GLP-1R gene in INS-1 cells was knocked down with shRNA interference.The level of GLP-1R protein in INS-1 cells was measured with Western blotting.Results:Geniposide (0.01-100 μmol/L) increased insulin secretion from INS-1 cells in a concentration-dependent manner.Geniposide (10 μmol/L) enhanced acute insulin secretion in response to both the low (5.5 mmol/L) and moderately high levels (11 mmol/L) of glucose.Blockade of GLP-1R with the GLP-1R antagonist exendin (9-39) (200 nmol/L) or knock-down of GLP-1R with shRNA interference in INS-1 cells decreased the effect of geniposide (10 μmol/L) on insulin secretion stimulated by glucose (5.5 mmol/L).Conclusion:Geniposide increases insulin secretion through glucagon-like peptide 1 receptors in rat INS-1 insulinoma cells.

  10. Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

    International Nuclear Information System (INIS)

    Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing

  11. Human Decidua-Derived Mesenchymal Stem Cells Differentiate into Functional Alveolar Type II-Like Cells that Synthesize and Secrete Pulmonary Surfactant Complexes

    OpenAIRE

    Cerrada, Alejandro; de la Torre, Paz; Grande, Jesús; Haller, Thomas; Flores, Ana I.; Pérez-Gil, Jesús

    2014-01-01

    Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alv...

  12. The alteration in intestinal secretory immunoglobulin A and its secreting cells during ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Li-qun SUN

    2012-04-01

    Full Text Available Objective To investigate the change in intestinal secretion immunoglobulin A (sIgA level and IgA-secreting cells during ischemia/reperfusion (I/R injury. Methods Forty-eight BALB/c mice were randomly divided into 6 experimental groups in accordance with different reperfusion times (R2h, R6h, R12h, R24h, and R72h group, and one sham group (n=8. Bacterial translocation to distant organs (lung, spleen, and mesenteric lymph nodes was observed. The sIgA level of the intestinal tract was measured by enzyme-linked immunosorbent assay (ELISA. The B cell subgroup in the lymphocytes related to the intestinal tract was measured by flow cytometry. Results The bacterial translocation occurred during I/R injury, and the intestinal sIgA level decreased, and they showed an obvious negative correlation (r2=0.729. With the increase in intestinal I/R injury, the ratio of IgM+B220+ cells in the gut-associated lymphoid tissue increased, whereas the proportion of IgA+B220+ cells decreased. The most significant change was found in R12h group (P < 0.01. Conclusions The proportion of IgM+ B cells in the gut-associated lymphoid tissue increased, whereas that of IgA+ B cells reduced during I/R injury. These phenomena may cause sIgA level to reduce and bacterial translocation of the distant organs to occur.

  13. Combined MSC-Secreted Factors and Neural Stem Cell Transplantation Promote Functional Recovery of PD Rats.

    Science.gov (United States)

    Yao, Yuan; Huang, Chen; Gu, Ping; Wen, Tieqiao

    2016-01-01

    Stem cell transplantation has enormous potential for the treatment of neurodegenerative disorders like Parkinson's disease (PD). Mesenchymal stem cells (MSCs) have attracted much attention because they can secrete a wide variety of cellular factors that promote cell growth. In this study, we prepared a conditioned medium (CM) using lyophilized MSC culture medium that contained the secretome of MSCs and applied this CM to the culture of neural stem cells (CM-NSCs) for the transplantation of PD model rats. Quantitative real-time PCR, Western blot, and immunocytochemistry were used to identify cell differentiation and expression of dopaminergic neuron-specific genes in vitro. Behavioral tests including rotational behavior and MWM training tests were also performed to assess the recovery. Our results indicated that combined treatment of CM and neural stem cell transplantation can significantly reduce apomorphine-induced rotational asymmetry and improve spatial learning ability. The CM-NSCs were able to differentiate into dopaminergic neurons in the ventral tegmental area (VTA) and medial forebrain bundle (MFB), and migrated around the lesion site. They showed a higher activity than untreated NSCs in cell survival, migration, and behavior improvement in the dopa-deficit rat model. These findings suggest that the neural stem cells treated with conditioned medium possess a great potential as a graft candidate for the treatment of Parkinson's disease. PMID:26607204

  14. Visualization of the gas flow in fuel cell bipolar plates using molecular flow seeding and micro-particle image velocimetry

    Energy Technology Data Exchange (ETDEWEB)

    Hecht, Christian; Wlokas, Irenaeus; Schulz, Christof [University of Duisburg-Essen, IVG and CeNIDE, Duisburg (Germany); Schoot, Nadine van der; Lindken, Ralph [Center for Fuel Cell Technology ZBT GmbH, Duisburg (Germany); Kronemayer, Helmut [University of Duisburg-Essen, IVG and CeNIDE, Duisburg (Germany); BASF SE, Ludwigshafen (Germany)

    2012-03-15

    Main components of proton exchange membrane fuel cells are bipolar plates that electrically connect the electrodes and provide a gas flow to the membrane. We investigate the flow in the channel structures of bipolar plates. Flow seeding is used to visualize the propagating and mixing gas stream. It is shown that a part of the gas is transported perpendicularly to the channel structure. An analysis of the diffusion compared with the convection shows different transport behavior for both flow directions. Additionally, the convective flow field is investigated in detail near the channel wall using Micro-PIV in a Reynolds-number-scaled liquid fluid system. For a more exact comparison of the experimental setups, flow seeding in both gas and liquid systems is performed. (orig.)

  15. Insulin stimulates endothelin-1 secretion from human endothelial cells and modulates its circulating levels in vivo.

    Science.gov (United States)

    Ferri, C; Pittoni, V; Piccoli, A; Laurenti, O; Cassone, M R; Bellini, C; Properzi, G; Valesini, G; De Mattia, G; Santucci, A

    1995-03-01

    Endothelin-1 (ET-1) is a potent vasoactive and mitogenic peptide produced by the vascular endothelium. In this study, we evaluated whether insulin stimulates ET-1 secretion by human endothelial cells derived from umbilical cord veins and by human permanent endothelial hybrid cells Ea.hy 926. Moreover, to provide evidence that insulin may stimulate ET-1 secretion in vivo, plasma ET-1 levels were evaluated in 7 type II diabetic normotensive males (mean age, 54.3 +/- 4.0 yr) during 2-h hyperinsulinemic euglycemic clamps (287 pmol insulin/m2.min-1) as well as in 12 obese hypertensive males (mean age, 44.2 +/- 4.6 yr) before and after a 12-week period of caloric restriction. Our results showed that insulin stimulated ET-1 release from cultured endothelial cells in a dose-dependent fashion. ET-1 release persisted for 24 h and was also observed at physiological insulin concentrations (10(-9) mol/L). The insulin-induced ET-1 secretion was inhibited by genistein, a tyrosine kinase inhibitor, and by cycloheximide, a protein synthesis inhibitor, suggesting that it requires de novo protein synthesis rather than ET-1 release from intracellular stores. In the in vivo experiments, plasma ET-1 levels rapidly increased during euglycemic hyperinsulinemic clamps (from 0.76 +/- 0.18 pg/mL at time zero to 1.65 +/- 0.21 pg/mL at 60 min; P < 0.05) and persisted elevated until the end of insulin infusion (1.37 +/- 0.37 pg/mL at 120 min; P < 0.05 vs. time zero). In obese hypertensives, plasma ET-1 levels significantly decreased after 12 weeks of caloric restriction (from 0.85 +/- 0.51 to 0.48 +/- 0.28 pg/mL; P < 0.04). The decrease in body weight induced by caloric restriction was accompanied by a significant reduction in fasting insulin levels (from 167.2 +/- 94.0 to 98.9 +/- 44.9 pmol/L; P < 0.05) which correlated with the reduction in plasma ET-1 levels (r = 0.78; P < 0.003). In conclusion, our data show that insulin stimulates both in vitro and in vivo ET-1 secretion. Such interaction

  16. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Cobos Everardo

    2005-01-01

    Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

  17. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

    OpenAIRE

    1982-01-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secr...

  18. Pattern secretion of matrix Metalloproteinases and their biological tissue inhibitors by human glomerular mesangial cells in culture

    OpenAIRE

    "Hosseini R; Hampel G; Jung K

    2001-01-01

    The glomerular mesangial cells (GMC) play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between of a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC, and any disturbance in the balance may result in appearance of various pathological states such as glomerulosclerosis. We therefore studied pattern secretion of matrix metalloproteinases (MMP...

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

    KAUST Repository

    Abdallah, Abdallah

    2011-09-28

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

  20. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

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

    Science.gov (United States)

    Abdallah, Abdallah M; Bestebroer, Jovanka; Savage, Nigel D L; de Punder, Karin; van Zon, Maaike; Wilson, Louis; Korbee, Cees J; van der Sar, Astrid M; Ottenhoff, Tom H M; van der Wel, Nicole N; Bitter, Wilbert; Peters, Peter J

    2011-11-01

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. PMID:21957139

  2. Peptide production and secretion in GLUTag, NCI-H716 and STC-1 cells: a comparison to native L-cells

    DEFF Research Database (Denmark)

    Kuhre, Rune Ehrenreich; Albrechtsen, Nicolai Jacob Wewer; Deacon, Carolyn F.;

    2016-01-01

    samples; n=9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n=6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2......GLUTag, NCI-H716 and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis......, glicentin and oxyntomodulin in a pattern (prohormone convertase (PC)1/3 dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and -2 which increased after stimulation. In contrast to freshly isolated murine L-cells, all lines also expressed PC2 and secreted...

  3. Electrophysiological identification and characterization of ion currents in mast cells and their involvement in the stimulus secretion coupling process

    International Nuclear Information System (INIS)

    The process, known as stimulus-secretion coupling, is the aim of this study with particular emphasis on the involvement of ion currents. Mucosal-type mast cells (RBL-2H3 line) were used as a well-established model for study of molecular processes involved in stimulus-secretion coupling. Immunological stimulation was performed with IgE saturated RBL-cells using the corresponding antigen. The patch-clamp technique was employed to measure ion currents. Complementary, secretion of allergy-causing mediators was monitored by the release of radioactive serotonin. The initial characterization of ion currents of unstimulated RBL cells revealed only one major current carried by K+ ions. Upon immunological stimulation of RBL cells activation of Cl- channels occurred, which were further characterized with respect to their activation behaviour, channel conductance, open probability and permeability to other ions. The Cl-channels were blocked by the well-known Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). In order to examine an involvement of this Cl- channel activity in the stimulus-secretion coupling process, serotonin release from immunologically stimulated RBL cells was determined in the presence of the Cl- channel blocker NPPB. Secretion was clearly inhibited by this compound, whereby inhibition of secretion occurred in a similar concentration range as inhibition of Cl- channel activity. This correlation emphasized an involvement of Cl- channel activity in the stimulus-secretion coupling and confirmed a current hypothesis on the function of Cl- current in mast cells. Further the widely used antiallergicum sodiumchromoglycat was identified as a potent blocker of the immunologically activated Cl- channel. Experiments with further five antiallergical compounds revealed a common potency to block Cl- channel activity. One of these compounds showed even an affinity in the micromolar range, which is in comparison to other well-known Cl- channel blockers

  4. Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria.

    LENUS (Irish Health Repository)

    Sibartie, Shomik

    2009-01-01

    BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

  5. Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix.

    Science.gov (United States)

    Luecke, Nina; Templin, Christian; Muetzelburg, Marika Victoria; Neumann, Detlef; Just, Ingo; Pich, Andreas

    2010-03-15

    Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration. PMID:20127908

  6. Modulation of human B cell immunoglobulin secretion by the C3b component of complement.

    Science.gov (United States)

    Tsokos, G C; Berger, M; Balow, J E

    1984-02-01

    The human C3b component of complement was found to inhibit the differentiation of human B lymphocytes into immunoglobulin-secreting cells in vitro. Pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses were inhibited by C3-coated zymosan particles and by purified human C3b. C3b inhibited the PWM-driven responses in a dose-dependent fashion, and it was necessary for C3b to be present in the early phases of the cultures. C3b acted directly on B cells rather than on helper T cells because it inhibited the PFC responses of MNC depleted of T cells and subsequently stimulated with a T cell-independent Epstein Barr virus mitogen. Furthermore, C3b failed to stimulate the generation of suppressor lymphocytes and/or monocytes that might have been responsible for the inhibition of B cell responses. Our results indicate that C3b or its fragments exert negative modulatory effects on human B lymphocyte responses. PMID:6228593

  7. Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

    Science.gov (United States)

    Parise, Carolina Bellini; Sá-Rocha, Vanessa Moura; Moraes, Jane Zveiter

    2015-12-25

    Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity. PMID:26482336

  8. Factors secreted from dental pulp stem cells show multifaceted benefits for treating experimental rheumatoid arthritis.

    Science.gov (United States)

    Ishikawa, Jun; Takahashi, Nobunori; Matsumoto, Takuya; Yoshioka, Yutaka; Yamamoto, Noriyuki; Nishikawa, Masaya; Hibi, Hideharu; Ishigro, Naoki; Ueda, Minoru; Furukawa, Koichi; Yamamoto, Akihito

    2016-02-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and chronic inflammation, which lead to the progressive destruction of cartilage and bone in the joints. Numerous studies have reported that administrations of various types of MSCs improve arthritis symptoms in animal models, by paracrine mechanisms. However, the therapeutic effects of the secreted factors alone, without the cell graft, have been uncertain. Here, we show that a single intravenous administration of serum-free conditioned medium (CM) from human deciduous dental pulp stem cells (SHED-CM) into anti-collagen type II antibody-induced arthritis (CAIA), a mouse model of rheumatoid arthritis (RA), markedly improved the arthritis symptoms and joint destruction. The therapeutic efficacy of SHED-CM was associated with an induction of anti-inflammatory M2 macrophages in the CAIA joints and the abrogation of RANKL expression. SHED-CM specifically depleted of an M2 macrophage inducer, the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9), exhibited a reduced ability to induce M2-related gene expression and attenuate CAIA. SHED-CM also inhibited the RANKL-induced osteoclastogenesis in vitro. Collectively, our findings suggest that SHED-CM provides multifaceted therapeutic effects for treating CAIA, including the ED-Siglec-9-dependent induction of M2 macrophage polarization and inhibition of osteoclastogenesis. Thus, SHED-CM may represent a novel anti-inflammatory and reparative therapy for RA. PMID:26603475

  9. Diverse signaling systems activated by the sweet taste receptor in human GLP-1-secreting cells.

    Science.gov (United States)

    Ohtsu, Yoshiaki; Nakagawa, Yuko; Nagasawa, Masahiro; Takeda, Shigeki; Arakawa, Hirokazu; Kojima, Itaru

    2014-08-25

    Sweet taste receptor regulates GLP-1 secretion in enteroendocrine L-cells. We investigated the signaling system activated by this receptor using Hutu-80 cells. We stimulated them with sucralose, saccharin, acesulfame K and glycyrrhizin. These sweeteners stimulated GLP-1 secretion, which was attenuated by lactisole. All these sweeteners elevated cytoplasmic cyclic AMP ([cAMP]c) whereas only sucralose and saccharin induced a monophasic increase in cytoplasmic Ca(2+) ([Ca(2+)]c). Removal of extracellular calcium or sodium and addition of a Gq/11 inhibitor greatly reduced the [Ca(2+)]c responses to two sweeteners. In contrast, acesulfame K induced rapid and sustained reduction of [Ca(2+)]c. In addition, glycyrrhizin first reduced [Ca(2+)]c which was followed by an elevation of [Ca(2+)]c. Reductions of [Ca(2+)]c induced by acesulfame K and glycyrrhizin were attenuated by a calmodulin inhibitor or by knockdown of the plasma membrane calcium pump. These results indicate that various sweet molecules act as biased agonists and evoke strikingly different patterns of intracellular signals. PMID:25017733

  10. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (∼7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. PMID:26627201

  11. In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

    Directory of Open Access Journals (Sweden)

    Andreas Kupz

    Full Text Available Natural killer (NK cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18, independently of Toll-like receptor (TLR-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

  12. Cafestol, a Bioactive Substance in Coffee, Stimulates Insulin Secretion and Increases Glucose Uptake in Muscle Cells: Studies in Vitro.

    Science.gov (United States)

    Mellbye, Fredrik Brustad; Jeppesen, Per Bendix; Hermansen, Kjeld; Gregersen, Søren

    2015-10-23

    Diet and exercise intervention can delay or prevent development of type-2-diabetes (T2D), and high habitual coffee consumption is associated with reduced risk of developing T2D. This study aimed to test whether selected bioactive substances in coffee acutely and/or chronically increase insulin secretion from β-cells and improve insulin sensitivity in skeletal muscle cells. Insulin secretion from INS-1E rat insulinoma cells was measured after acute (1-h) and long-term (72-h) incubation with bioactive substances from coffee. Additionally, we measured uptake of radioactive glucose in human skeletal muscle cells (SkMC) after incubation with cafestol. Cafestol at 10(-8) and 10(-6) M acutely increased insulin secretion by 12% (p coffee drinkers and be of therapeutic interest. PMID:26465380

  13. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.

    OpenAIRE

    M. L. Li; Aggeler, J; Farson, D A; Hatier, C; Hassell, J; Bissell, M.J.

    1987-01-01

    When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of beta-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows greater than 90% of cells to produce hi...

  14. Optical and non-optical methods for detection and characterization of microparticles and exosomes

    NARCIS (Netherlands)

    E. van der Pol; A.G. Hoekstra; A. Sturk; C. Otto; T.G. van Leeuwen; R. Nieuwland

    2010-01-01

    Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available fo

  15. Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

    Directory of Open Access Journals (Sweden)

    Lotta E Andersson

    Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

  16. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

    International Nuclear Information System (INIS)

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4+ IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4+ IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4+ IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4+ IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4+ LPLs and primed splenic CD4+ T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4+ IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

  17. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  18. Mechanisms of fat-induced gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide secretion from K cells.

    Science.gov (United States)

    Yamane, Shunsuke; Harada, Norio; Inagaki, Nobuya

    2016-04-01

    Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, which are gastrointestinal hormones released in response to nutrient ingestion and potentiate glucose-stimulated insulin secretion. Single fat ingestion stimulates GIP secretion from enteroendocrine K cells; chronic high-fat diet (HFD) loading enhances GIP secretion and induces obesity in mice in a GIP-dependent manner. However, the mechanisms of GIP secretion from K cells in response to fat ingestion and GIP hypersecretion in HFD-induced obesity are not well understood. We generated GIP-green fluorescent protein knock-in (GIP (gfp/+)) mice, in which K cells are labeled by enhanced GIP-green fluorescent protein. Microarray analysis of isolated K cells from GIP (gfp/+) mice showed that both fatty acid-binding protein 5 and G protein-coupled receptor 120 are highly expressed in K cells. Single oral administration of fat resulted in significant reduction of GIP secretion in both fatty acid-binding protein 5- and G protein-coupled receptor 120-deficient mice, showing that fatty acid-binding protein 5 and G protein-coupled receptor 120 are involved in acute fat-induced GIP secretion. Furthermore, the transcriptional factor, regulatory factor X6 (Rfx6), is highly expressed in K cells. In vitro experiments using the mouse enteroendocrine cell line, STC-1, showed that GIP messenger ribonucleic acid levels are upregulated by Rfx6. Expression levels of Rfx6 messenger ribonucleic acid as well as that of GIP messenger ribonucleic acid were augmented in the K cells of HFD-induced obese mice, in which GIP content in the small intestine is increased compared with that in lean mice fed a control diet. These results suggest that Rfx6 is involved in hypersecretion of GIP in HFD-induced obese conditions by increasing GIP gene expression. PMID:27186351

  19. Relationships among CFTR expression, HCO3- secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies.

    Science.gov (United States)

    Shah, Viral S; Ernst, Sarah; Tang, Xiao Xiao; Karp, Philip H; Parker, Connor P; Ostedgaard, Lynda S; Welsh, Michael J

    2016-05-10

    Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl(-) secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3 (-) secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3 (-) at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR(+/-) or CFTR(+/∆F508)) expressed CFTR and secreted HCO3 (-) at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3 (-) secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl(-) secretion, the amount of CFTR is rate-limiting for HCO3 (-) secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers. PMID:27114540

  20. Inhalable Antitubercular Therapy Mediated by Locust Bean Gum Microparticles.

    Science.gov (United States)

    Alves, Ana D; Cavaco, Joana S; Guerreiro, Filipa; Lourenço, João P; Rosa da Costa, Ana M; Grenha, Ana

    2016-01-01

    Tuberculosis remains a major global health problem and alternative therapeutic approaches are needed. Considering the high prevalence of lung tuberculosis (80% of cases), the pulmonary delivery of antitubercular drugs in a carrier system capable of reaching the alveoli, being recognised and phagocytosed by alveolar macrophages (mycobacterium hosts), would be a significant improvement to current oral drug regimens. Locust bean gum (LBG) is a polysaccharide composed of galactose and mannose residues, which may favour specific recognition by macrophages and potentiate phagocytosis. LBG microparticles produced by spray-drying are reported herein for the first time, incorporating either isoniazid or rifabutin, first-line antitubercular drugs (association efficiencies >82%). Microparticles have adequate theoretical properties for deep lung delivery (aerodynamic diameters between 1.15 and 1.67 μm). The cytotoxic evaluation in lung epithelial cells (A549 cells) and macrophages (THP-1 cells) revealed a toxic effect from rifabutin-loaded microparticles at the highest concentrations, but we may consider that these were very high comparing with in vivo conditions. LBG microparticles further evidenced strong ability to be captured by macrophages (percentage of phagocytosis >94%). Overall, the obtained data indicated the potential of the proposed system for tuberculosis therapy. PMID:27240337

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

    International Nuclear Information System (INIS)

    Highlights: ► G protein coupled receptor TGR5 is expressed in mouse and human islets. ► TGR5 is coupled to activation of Gs and Ca2+ release via cAMP/Epac/PLC-ε pathway. ► Activation of TGR5 by bile salts and selective ligands causes insulin secretion. ► TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gαs and caused an increase in intracellular cAMP and Ca2+. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gαs inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on Gs/cAMP/Ca2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  2. [Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells].

    Science.gov (United States)

    Feng, Ying; Lu, Shi-Hong; Wang, Xin; Cui, Jun-Jie; Li, Xue; DU, Wen-Jing; Wang, Ying; Li, Juan-Juan; Song, Bao-Quan; Chen, Fang; Ma, Feng-Xia; Chi, Ying; Yang, Shao-Guang; Han, Zhong-Chao

    2014-06-01

    This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization. PMID:24989260

  3. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    Directory of Open Access Journals (Sweden)

    Goldbach Rob W

    2011-07-01

    Full Text Available Abstract Background Chikungunya virus (CHIKV is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

  4. A SELF-PRIMING EFFECT OF LHRH ON LH SECRETION IN DISPERSED ANTERIOR PITUITARY CELLS OF ADULT MALE RAT

    Institute of Scientific and Technical Information of China (English)

    QUZhi-Chao; GUOJing; GUOJian

    1989-01-01

    LHRH self-priming effect is simply defmed as an enhancement of LH response to LHRH, i. e., a second challenge with LHRH elicits more LH secretion as compared to the first challenge. The present study is to observe whether this phenomenon exists in perfused anterior pituitary (AP) cells of adult male rat. Dispersed AP cells of adult SD

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-06-01

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

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

    International Nuclear Information System (INIS)

    The influence of in-vitro conditions on the production of inhibin by Sertoli cells from 21-day-old normal and prenatally irradiated rat testes was studied by measuring inhibin activity in culture media, using the suppression of the release of FSH from cultured rat pituitary cells. Sertoli cells secreted inhibin-like activity during at least 21 days of culture, and cells cultured at 370C produced significantly more inhibin than those cultured at 320C. The presence of fetal calf serum had no significant effect on inhibin production at 320C, while at 370C the production was decreased. The presence of ovine FSH stimulated inhibin secretion, while inhibin concentrations in Sertoli cell culture media were decreased after the addition of testosterone. Testosterone, added together with ovine FSH, suppressed inhibin secretion when compared with the levels found in the presence of FSH alone. The presence of spermatogenic cells decreased the release of inhibin. From these results it was concluded that both Sertoli cells isolated from normal immature rat testes and those from testes without spermatogenic cells can secrete inhibin-like activity in culture. A number of discrepancies with in-vivo observations was observed. (author)

  7. 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. PMID:19160674

  8. Microparticles as mediators of cellular cross-talk in inflammatory disease.

    Science.gov (United States)

    Distler, Jörg H W; Huber, Lars C; Gay, Steffen; Distler, Oliver; Pisetsky, David S

    2006-12-01

    Microparticles are a heterogeneous population of membrane-coated vesicles which can be released from virtually all cell types during activation or apoptosis. Release occurs from the cell surface in an exogenous budding process involving local rearrangement of the cytoskeleton. Given their origin, these particles can be identified by staining for cell surface markers and annexin V. As shown in in vitro studies, microparticles may represent a novel subcellular element for intercellular communication in inflammation. Thus, microparticles can transfer chemokine receptors and arachidonic acid between cells, activate complement, promote leukocyte rolling and stimulate the release of pro-inflammatory mediators. Under certain conditions, however, microparticles may also exert anti-inflammatory properties by inducing immune cell apoptosis and the production of anti-inflammatory mediators. Microparticles may play an important role in the pathogenesis of rheumatologic diseases as evidenced by their elevation in diseases such as systemic sclerosis (SSc), systemic vasculitis and antiphospholipid antibody syndrome and correlation with clinical events. A role in inflammatory arthritis is suggested by the finding that leukocyte-derived microparticles induce the production of matrix metalloproteinases and cytokines by synovial fibroblasts. Together, these findings point to novel signaling pathways of cellular cross-talk that may operate along the spectrum of soluble cytokines and mediators of direct cell-cell contact. PMID:17178565

  9. Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

    Science.gov (United States)

    Ao, Mei; Domingue, Jada C; Khan, Nabihah; Javed, Fatima; Osmani, Kashif; Sarathy, Jayashree; Rao, Mrinalini C

    2016-06-01

    Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of

  10. Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

    OpenAIRE

    Hellerman, J G; Cone, R C; Potts, J T; Rich, A; Mulligan, R C; Kronenberg, H M

    1984-01-01

    In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recomb...

  11. Differential inhibitory effects of opioids on cigarette smoke, capsaicin and electrically-induced goblet cell secretion in guinea-pig trachea.

    OpenAIRE

    Kuo, H. P.; Rohde, J. A.; Barnes, P. J.; Rogers, D. F.

    1992-01-01

    1. Goblet cell secretion in guinea-pig airways is under neural control. Opioids have previously been shown to inhibit neurogenic plasma exudation and bronchoconstriction in guinea-pig airways. We have now examined the effects of morphine and opioid peptides on tracheal goblet cell secretion induced by either electrical stimulation of the cervical vagus nerves, exogenous capsaicin, or acute inhalation of cigarette smoke. The degree of goblet cell secretion was determined by a morphometric meth...

  12. Inhibition of acid secretion from parietal cells by non-human-infecting Helicobacter species: a factor in colonization of gastric mucosa?

    OpenAIRE

    Vargas, M; Lee, A; Fox, J.G.; Cave, D. R.

    1991-01-01

    Helicobacter pylori has been shown to produce a protein that inhibits acid secretion from parietal cells. We have examined other non-human-infecting Helicobacter species for this property by measuring the uptake of [14C]aminopyrine into rabbit parietal cells as an indirect assessment of acid secretion. Helicobacter felis and an isolate from a rhesus monkey were shown to inhibit acid secretion. Isolates of Helicobacter mustelae gave variable responses. Whole bacteria and cell-free sonicates im...

  13. Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

    OpenAIRE

    Vasilopoulou, E.; Loubière, L.S.; Lash, G.E.; Ohizua, O.; McCabe, C.J.; Franklyn, J A; Kilby, M. D.; Chan, S Y

    2014-01-01

    STUDY QUESTION Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY Maternal thyroid dysfunction...

  14. Differential Cl-and HCO3-mediated anion secretion by different colonic cell types in response to tetromethylpyrazine

    Institute of Scientific and Technical Information of China (English)

    Jin-Xia Zhu; Ning Yang; Qiong He; Lai-Ling Tsang; Wen-Chao Zhao; Yiu-Wa Chung; Hsiao-Chang Chan

    2004-01-01

    AIM: Colonic epithelium is known to secrete both Cl- and HCO3-, but the secretory mechanisms of different colonic cell types are not fully understood. The present study aimed to investigate the differential activation of Cl-and HCO3-secretion by tetramethylpyrazine (TMP) in human crypt-like cell line, T84, and villus-like cell line, Caco-2, in comparison to the TMP-induced secretory response in freshly isolated rat colonic mucosa.METHODS: Colonic epithelial anion secretion was studied by using the short circuit current (Isc) technique. RT-PCR was used to examine the expression of Na+-HCO3--cotranspoter in different epithelial cell types.RESULTS: TMP produced a concentration-dependent Isc which was increase in both T84 and Caco-2 cells. When extracellular Cl- was removed, TMP-induced Isc was abolished by 76.6% in T84 cells, but not in Caco-2 cells. However,after both Cl- and HCO3- were removed, TMlP-induced ISC in Caco-2 cells was reduced to 10%. Bumetanide, an inhibitor of Na+-K+-Cl--cotranspoter, inhibited the TMP-induced ISC by 96.7% in T84 cells, but only 47.9% in Caco-2 cells. In the presence of bumetanide and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Na+-HCO3- cotransporter,inhibited the TMP-induced current in Caco-2 cells by 93.3%.In freshly isolated rat colonic mucosa, TMP stimulated distinct ISC responses similar to that observed in T84 and Caco-2cells depending on the concentration used. RT-PCR revealed that the expression of Na+-HCO3- cotransporter in Caco-2cells was 4-fold more greater than that in T84 cells.CONCLUSION: TMP exerts concentration-dependent differential effects on different colonic cell types with stimulation of predominant Cl- secretion by crypt cells at a lower concentration, but predominant HCO3- secretion by villus cells at a higher concentration, suggesting different roles of these cells in colonic Cl- and HCO3- secretion.

  15. Control of Secretion by Encodes of Action Potentials in Neuronal Cells

    Institute of Scientific and Technical Information of China (English)

    Kailai Duan; Zhuan Zhou

    2003-01-01

    @@ Action potentials (APs) are principle physiological stimuli of neurotransmitter secretion or synaptic transmis sion. Most studies on stimulus-secretion-coupling have been performed under voltage clamp using artificial electric stimulations.

  16. Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells.

    Science.gov (United States)

    Kageyama, Y; Kawakami, S; Fujii, Y; Kihara, K; Oshima, H

    1997-03-01

    Intravesical administration of bacillus Calmette-Guérin (BCG) is an effective and widely accepted treatment for superficial bladder cancer. Rapid progression of the disease after BCG therapy, however, has been reported in some cases refractory to the treatment. We examined whether BCG treatment and coexistence of peripheral blood mononuclear cells (PBMCs) alter the invasive potential of bladder cancer cells. Production and secretion of two type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP 9, by PBMCs from five healthy donors or bladder cancer cells (T24, JTC 30, and JTC 32) were evaluated by gelatin zymography, western blot analysis, and northern blot analysis. Invasion of bladder cancer cells was also examined using reconstituted basement membrane (Matrigel). BCG (5, 50, and 500 micrograms/ml) had no effect on secretion of MMP 2 and MMP 9 by bladder cancer cells, but increased the production and secretion of MMP 9 by PBMCs in a dose-dependent manner. The coexistence of PBMCs increased invasion of T24 cells and BCG further enhanced the invasion. Thus, BCG promotes invasion of bladder cancer cells under certain conditions. An increase in the secretion of MMP 9 by PBMCs may account in part for the effect. PMID:9140113

  17. VAMP7 Regulates Autophagy to Maintain Mitochondrial Homeostasis and to Control Insulin Secretion in Pancreatic β-Cells.

    Science.gov (United States)

    Aoyagi, Kyota; Ohara-Imaizumi, Mica; Itakura, Makoto; Torii, Seiji; Akimoto, Yoshihiro; Nishiwaki, Chiyono; Nakamichi, Yoko; Kishimoto, Takuma; Kawakami, Hayato; Harada, Akihiro; Takahashi, Masami; Nagamatsu, Shinya

    2016-06-01

    VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic β-cells is largely unknown. To elucidate the physiological role of VAMP7 in β-cells, we generated pancreatic β-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient β-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient β-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre β-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in β-cells. PMID:26953164

  18. RFX6 Regulates Insulin Secretion by Modulating Ca2+ Homeostasis in Human β Cells

    Directory of Open Access Journals (Sweden)

    Vikash Chandra

    2014-12-01

    Full Text Available Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca2+-channel genes resulting in the reduction in L-type Ca2+-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca2+-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

  19. 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. PMID:27283618

  20. Effect of addition of human follicular fluid on progesterone secretion by cultured sheep granulosa cells.

    Science.gov (United States)

    Kumari, G L; Vohra, S; Raghavan, V

    1982-10-01

    The effect of addition of human follicular fluid to cultures of granulosa cells of large sheep follicles (4-6 mm in diameter) on basal and LH-stimulated progesterone secretion was investigated. Both luteinization and progesterone secretion were inhibited by addition of 10% (w/v) charcoal-treated follicular fluid from medium (2-6 mm) and large (7-16 mm) follicles which had low concentrations of estradiol-17 beta, progesterone and LH. In comparison, the fluid from large follicles, having high levels of the same hormones, stimulated both the parameters, and addition of LH along with the fluid had no further effect. Fluid collected from cystic follicles appeared to be stimulatory which also had elevated levels of estradiol-17 beta and progesterone. These findings indicate the presence of both the inhibitors and stimulators of luteinization in human follicular fluid. The effectiveness of any of them either to inhibit or stimulate luteinization probably will depend upon the composition of the follicular fluid and the stage of maturation of the follicles from which it was collected. PMID:6218983

  1. Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion.

    Directory of Open Access Journals (Sweden)

    Meghali P Nighot

    Full Text Available The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05 and 53% (P<0.001 respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025 lower by 9.4 fold (89% in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84-95%, P<0.005 in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.

  2. Clonal expansions of CD8+ T cells with IL-10 secreting capacity occur during chronic Mycobacterium tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Joshua C Cyktor

    Full Text Available The exact role of CD8(+ T cells during Mycobacterium tuberculosis (Mtb infection has been heavily debated, yet it is generally accepted that CD8(+ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8(+ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1(+, Tim-3(+, CD122(+. CD8(+ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10, although in vivo CD8(+ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8(+ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR Vβ chain 8 (8.2, 8.3 or Vβ 14. Although Vβ8(+ CD8(+ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8(+ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8(+ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.

  3. Rising Intracellular Zinc by Membrane Depolarization and Glucose in Insulin-Secreting Clonal HIT-T15 Beta Cells

    OpenAIRE

    Slepchenko, Kira G.; Li, Yang V

    2012-01-01

    Zinc (Zn2+) appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30–60 mM) was able to induce a rapid increase in...

  4. Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells

    Energy Technology Data Exchange (ETDEWEB)

    McNutt, Markey C.; Kwon, Hyock Joo; Chen, Chiyuan; Chen, Justin R.; Horton, Jay D.; Lagace, Thomas A.; (USMC); (UTSMC)

    2009-07-10

    PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

  5. Measurements of prolactin and growth hormone synthesis and secretion by rat pituitary cells in culture.

    Science.gov (United States)

    Gautvik, K M; Kriz, M

    1976-02-01

    A specific and sensitive immunoprecipitation method for measurements of biosynthesized radioactive prolactin and growth hormone is described. Antisera to rat prolactin and growth hormone were developed in the rabbit and monkey, respectively. The specificity of the immune sera was assessed by polyacylamide gel electrophoresis of the dissolved immunoprecipitates. The two antisera showed cross-reactions with the nonhomologous hormone of less than 1%. Separation of tritium-labelled prolactin and growth hormone by immunoprecipitation, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was shown to be 95-57% complete. When both hormones were measured in the same microsample by sequential immunoprecipitation, the reaction was 97% complete for determination of intra- and extracellular prolactin and extracellular growth hormone, but 85% complete for determination of intracellular growth hormone. This method has been used to characterize the basal synthesis and secretion of prolactin and growth hormone in three different but related, pituitary cell strains. Radioactive prolactin and growth hormone was obtained from monolayer cultures when the cells were grown in the presence of [3H]L-leucine. The rate of prolactin synthesis and extracellular accumulation was higher than that of growth hormone in a cell strain which produced both hormones. In these cells prolactin synthesis represents 1-5%, and growth hormone 0.1-0.6% of total protein synthesis. PMID:942913

  6. Stimulatory effect of trans-cinnamaldehyde on norepinephrine secretion in cultured pheochromocytoma (PC-12) cells

    Institute of Scientific and Technical Information of China (English)

    TSAI Chin-Chuan; L1U I-Min; CHENG Juei-Tang

    2000-01-01

    AIM: The effect of trans-cinnamaldehyde (CNMA) on the release of norepinephrine (NE) from nerve terminal was investigated using rat pheochromocytoma 12 (PC-12) cells. METHODS: The amount of NA released from PC-12 cells incubated with CNMA or related substances was quantified by high performance fiquid chromatography (HPLC)-electrochemical detection. The lipophilic anion bisoxonol was used to monitor the effect of CNMA on the membrane potential. RESULTS: CNMA stimulated the secretion of NE in a concentration-dependent manner from 5μmol/L to 50 μmol/L, while the value of lactate dehydrogenase in the incubated medium was not influenced by CNMA. However, acetaldehyde, cinnamic acid, cinnamoyl chloride and cinnamamide failed to produce similar effect. The action of CNMA can thus be considered specific. The depolarizing effect of CNMA on the membrane potential was also illustrated by a concentration-dependent increase in the fluorescence of bisoxonol, a potential-sensitive dye. Saxitoxin attenuated the depolarizing action of CNMA at concentrations sufficient to block sodium channels. Besides, the effect of CNMA to depolarize the membrane potential in PC-12 cells is greater than that of 4-aminopyridine (4-AP). The action of CNMA on NE releasing depends on extmcellular Ca2 + and is attenuated by 8-bromo-cAMP at concentrations sufficient to inhibit the action of cyclic AMP. CONCLUSION: These findings suggest that CNMA can depolarize the membrane to result in a Cas+-dependent and cyclic AMP-related release of NE from PC-12 cells.

  7. Inflammatory Cytokine Secretion Status of Bone Marrow Cells and Clinical Significance in Immune-related Hematocytopenia

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective To observe the expression of inlfammatory molecules in bone marrow immune cells of patients with immune-related hematocytopenia (IRH), and to investigate the immune mechanism and clinical signiifcance of the disease. Methods Total of 36 IRH patients were selected as observation group and 30 healthy people were taken as control group. Serum cytokines levels, activity of immunocytes and expression of HLA-DR were detected. Immune lfuorescence was applied to observe the expression state of immunologic molecules and cytokines in IRH patients. Results Serum cytokines were elevated in various degrees in observation group. Compared with the control group, the cytokines levels were significantly higher (P Conclusions It is demonstrated that antibodies or self-reactive lymphocytes were produced in IRH marrow, which would cause lesions of hemocytes, and lead to pathological process ifnally. Structure of hematopoietic cells mutated and these cells might be acted as target cells of immunocytes in the pathological process. Immunocytes could secrete inlfammatory factors and lead to immunologic injury of hemocyte.

  8. Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion.

    Science.gov (United States)

    Ilegems, E; van Krieken, P P; Edlund, P K; Dicker, A; Alanentalo, T; Eriksson, M; Mandic, S; Ahlgren, U; Berggren, P-O

    2015-01-01

    The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status. PMID:26030284

  9. Effects of follistatin on testosterone secretion of rat Leydig cell in vitro

    Institute of Scientific and Technical Information of China (English)

    李江源; 邵迎红; 窦京涛; 李明

    2003-01-01

    Objective: To investigate the effects of follistatin(rhFS-288) on biosynthesis andsecretion of testosterone in rat Leydig cell in vitro.Methods: Leydig cells were isolated from Wistar rat testes by a discontinuous Per-coll gradient procedure. Purified cells were incubated in 24-well plate(105 cell/ml/well)and maintained for 24 h in a CO2 incubator. rhFS-288 and Ca2+ were added to the wellsindependently or jointly in both baseline (without hCG) and stimulation condition (1.0IU/ml of hCG) to observe the change of testosterone concentration in the media.Results: rhFS-288 showed a dose-dependent inhibiting effect on testosterone releasein baseline and stimulating condition. Ca2+ presented inhibitory effect either. Whereas,escape phenomenon emerged while Ca2+ concentration reached to 100 mmol/L. A com-bination of rhFS-288 with Ca2+ displayed a dose-dependent inhibition on testosterone se-cretion.Conclusion: rhFS-288 inhibits testosterone secretion in a dose-dependent manner.Calcium is thought to be the second messenger of FS action. The mechanism of escapephenomenon during high dose of Ca2+ along is unknown.

  10. Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Bruce A Stanton

    Full Text Available P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF. Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770.F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR.The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials.

  11. Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

    Science.gov (United States)

    Martin, David; Allagnat, Florent; Gesina, Emilie; Caille, Dorothee; Gjinovci, Asllan; Waeber, Gerard; Meda, Paolo; Haefliger, Jacques-Antoine

    2012-01-01

    The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival. PMID:23029270

  12. Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

    Directory of Open Access Journals (Sweden)

    David Martin

    Full Text Available The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice, and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

  13. The role of membrane lipids in the induction of macrophage apoptosis by microparticles.

    Science.gov (United States)

    Huber, Lars C; Jüngel, Astrid; Distler, Jörg H W; Moritz, Falk; Gay, Renate E; Michel, Beat A; Pisetsky, David S; Gay, Steffen; Distler, Oliver

    2007-02-01

    Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the

  14. Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.

    Science.gov (United States)

    Worrell, Roger T; Best, Alison; Crawford, Oscar R; Xu, Jie; Soleimani, Manoocher; Matthews, Jeffrey B

    2005-10-01

    Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is

  15. Sildenafil Effect on Nitric Oxide Secretion by Normal Human Endometrial Epithelial Cells Cultured In vitro

    Directory of Open Access Journals (Sweden)

    Farzaneh Chobsaz

    2011-01-01

    Full Text Available Background: Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specificphosphodiesterase type 5. It increases intracellular nitric oxide (NO production in some cells.There are reports on its positive effect on uterine circulation, endometrial thickness, and infertilityimprovement. Endometrial epithelial cells (EEC play an important role in embryo attachment andimplantation. The present work investigates the effect of sildenafil on human EEC and their NOsecretion in vitro.Materials and Methods: In this experimental in vitro study, endometrial biopsies (n=10 werewashed in a phosphate buffered solution (PBS and digested with collagenase I (2 mg/ml in DMEM/F12 medium at 37°C for 90 minutes. Epithelial glands were collected by sequential filtrationthrough nylon meshes (70 and 40 μm pores, respectively. Epithelial glands were then treated withtrypsin to obtain individual cells. The cells were counted and divided into four groups: control and1, 10, and 20 μM sildenafil concentrations. Cells were cultured for 15 days at 37ºC and 5% CO2; themedia were changed every 3 days, and their supernatants were collected for the NO assay. NO wasmeasured by standard Greiss methods. Data were analyzed by one way ANOVA.Results: There was no significant difference between groups in cell count and NO secretion, but thelevel of NO increased slightly in the experimental groups. The 10 μM dose showed the highest cellcount. EEC morphology changed into long spindle cells in the case groups.Conclusion: Sildenafil (1, 10, and 20 μM showed a mild proliferative effect on human EECnumbers, but no significant change was seen in NO production.

  16. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

    OpenAIRE

    Maddi, Abhiram; Bowman, Shaun M.; Free, Stephen J.

    2009-01-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and 6 secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and 9 secreted proteins. Most ...

  17. Thyroid Hormone Promotes Postnatal Rat Pancreatic β-Cell Development and Glucose-Responsive Insulin Secretion Through MAFA

    OpenAIRE

    Aguayo-Mazzucato, Cristina; Zavacki, Ann Marie; Marinelarena, Alejandra; Hollister-Lock, Jennifer; El Khattabi, Ilham; Marsili, Alessandro; Weir, Gordon C.; Sharma, Arun; Larsen, P. Reed; Bonner-Weir, Susan

    2013-01-01

    Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectivel...

  18. In vitro effects of mesenchymal stem cells on secreting function of T lymphocytes and CD4~+CD25~+T cells from patients with immune thrombo-cytopenia

    Institute of Scientific and Technical Information of China (English)

    赵霞

    2014-01-01

    Objective To analyze in vitro the effect of mesenchymal stem cells(MSCs)on secreting cytokines by T lymphocytes and ratio of CD4+CD25+T cells from patients with immune thrombocytopenia(ITP).Methods Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells.Allogeneic T lymphocytes

  19. Review: the Multiple Roles of Monocytic Microparticles.

    Science.gov (United States)

    Halim, Ahmad Tarmizi Abdul; Ariffin, Nur Azrah Fazera Mohd; Azlan, Maryam

    2016-08-01

    Monocytic microparticles (mMP) are microparticles derived from human monocytes either under in vivo or in vitro conditions. The size of mMP is between 0.1 and 1.0 μm. Apart from the size range, mMPs are also identified based on phosphatidylserine and CD14 expression on their surface, though this is not always the case. Monocytic MP are critical players in inflammation, endothelial cell function, and blood coagulation. They exhibit dual function by either helping the progression of such conditions or limiting it, depending on certain factors. Furthermore, the numbers of mMP are elevated in some autoimmune diseases, infectious diseases, and metabolic disorders. However, it is unknown whether mMP play an active role in these diseases or are simply biomarkers. The mechanism of mMP modulation is yet to be identified. In this review, we highlight the mechanism of mMP formation and the roles that they play in inflammation, blood coagulation, and different disease settings. PMID:27216803

  20. Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

    OpenAIRE

    Ellen Melaleuca Menkhorst; Natalie Lane; Amy Louise Winship; Priscilla Li; Joanne Yap; Katie Meehan; Adam Rainczuk; Andrew Stephens; Evdokia Dimitriadis

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by alteri...

  1. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

    Directory of Open Access Journals (Sweden)

    Yanan Hou

    Full Text Available The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1. Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

  2. Glycated albumin suppresses glucose-induced insulin secretion by impairing glucose metabolism in rat pancreatic β-cells

    Directory of Open Access Journals (Sweden)

    Muto Takashi

    2011-04-01

    Full Text Available Abstract Background Glycated albumin (GA is an Amadori product used as a marker of hyperglycemia. In this study, we investigated the effect of GA on insulin secretion from pancreatic β cells. Methods Islets were collected from male Wistar rats by collagenase digestion. Insulin secretion in the presence of non-glycated human albumin (HA and GA was measured under three different glucose concentrations, 3 mM (G3, 7 mM (G7, and 15 mM (G15, with various stimulators. Insulin secretion was measured with antagonists of inducible nitric oxide synthetase (iNOS, and the expression of iNOS-mRNA was investigated by real-time PCR. Results Insulin secretion in the presence of HA and GA was 20.9 ± 3.9 and 21.6 ± 5.5 μU/3 islets/h for G3 (P = 0.920, and 154 ± 9.3 and 126.1 ± 7.3 μU/3 islets/h (P = 0.046, for G15, respectively. High extracellular potassium and 10 mM tolbutamide abrogated the inhibition of insulin secretion by GA. Glyceraldehyde, dihydroxyacetone, methylpyruvate, GLP-1, and forskolin, an activator of adenylate cyclase, did not abrogate the inhibition. Real-time PCR showed that GA did not induce iNOS-mRNA expression. Furthermore, an inhibitor of nitric oxide synthetase, aminoguanidine, and NG-nitro-L-arginine methyl ester did not abrogate the inhibition of insulin secretion. Conclusion GA suppresses glucose-induced insulin secretion from rat pancreatic β-cells through impairment of intracellular glucose metabolism.

  3. Cortactin and Exo70 mediated invasion of hepatoma carcinoma cells by MMP-9 secretion.

    Science.gov (United States)

    Zhao, Gang; Zhang, Hongyi; Huang, Ziming; Lv, Liping; Yan, Fan

    2016-05-01

    This study was aimed to evaluate the regulation mechanism of cortactin (CTTN) on matrix metalloproteinases 9 (MMP-9) and its relations with Exo70 in invasion of hepatoma carcinoma (HCC) cells. The expression levels of CTTN, Exo70 and MMP-9 were detected in normal hepatocytes and various HCC cells by real-time PCR. Then the migration and invasion ability of these cells was revealed by scratch and invasion assay. The effects of CTTN on MMP-9 and the ability of migration and invasion were evaluated by silence and overexpress CTTN. During this process, the expression of CTTN was detected by Western blot, the activity and concentration of MMP-9 in supernatant of culture medium was detected by zymography and ELISA assay. Besides, Exo70 was also inhibited to reveal its effects on MMP-9 and the migration and invasion ability of LM3. Increased expression of CTTN, MMP-9, Exo70, reduced scratch area and increased puncture cell numbers were found in HCC cells (p < 0.05). The expression of CTTN was significantly correlated with Exo70 and the migration and invasion ability of HCC (p < 0.05). In addition, the activity and concentration of MMP-9 were significantly affected by the expression level of CTTN, while the expression of MMP-9 was not influenced. Besides, Exo70-si also exhibited significantly inhibition effects on the activity and concentration of MMP-9 and puncture cell numbers (p < 0.05). A synergistic reaction may exhibited on CTTN and Exo70, which could mediate the secretion of MMPs thereby regulate tumor invasion. PMID:27025610

  4. Mechanically robust microfluidics and bulk wave acoustics to sort microparticles

    Science.gov (United States)

    Dauson, Erin R.; Gregory, Kelvin B.; Greve, David W.; Healy, Gregory P.; Oppenheim, Irving J.

    2016-04-01

    Sorting microparticles (or cells, or bacteria) is significant for scientific, medical and industrial purposes. Research groups have used lithium niobate SAW devices to produce standing waves, and then to align microparticles at the node lines in polydimethylsiloxane (PDMS, silicone) microfluidic channels. The "tilted angle" (skewed) configuration is a recent breakthrough producing particle trajectories that cross multiple node lines, making it practical to sort particles. However, lithium niobate wafers and PDMS microfluidic channels are not mechanically robust. We demonstrate "tilted angle" microparticle sorting in novel devices that are robust, rapidly prototyped, and manufacturable. We form our microfluidic system in a rigid polymethyl methacrylate (PMMA, acrylic) prism, sandwiched by lead-zirconium-titanate (PZT) wafers, operating in through-thickness mode with inertial backing, that produce standing bulk waves. The overall configuration is compact and mechanically robust, and actuating PZT wafers in through-thickness mode is highly efficient. Moving to this novel configuration introduced new acoustics questions involving internal reflections, but we show experimental images confirming the intended nodal geometry. Microparticles in "tilted angle" devices display undulating trajectories, where deviation from the straight path increases with particle diameter and with excitation voltage to create the mechanism by which particles are sorted. We show a simplified analytical model by which a "phase space" is constructed to characterize effective particle sorting, and we compare our experimental data to the predictions from that simplified model; precise correlation is not expected and is not observed, but the important physical trends from the model are paralleled in the measured particle trajectories.

  5. Intracellular kinases mediate increased translation and secretion of netrin-1 from renal tubular epithelial cells.

    Directory of Open Access Journals (Sweden)

    Calpurnia Jayakumar

    Full Text Available BACKGROUND: Netrin-1 is a laminin-related secreted protein, is highly induced after tissue injury, and may serve as a marker of injury. However, the regulation of netrin-1 production is not unknown. Current study was carried out in mouse and mouse kidney cell line (TKPTS to determine the signaling pathways that regulate netrin-1 production in response to injury. METHODS AND PRINCIPAL FINDINGS: Ischemia reperfusion injury of the kidney was induced in mice by clamping renal pedicle for 30 minutes. Cellular stress was induced in mouse proximal tubular epithelial cell line by treating with pervanadate, cisplatin, lipopolysaccharide, glucose or hypoxia followed by reoxygenation. Netrin-1 expression was quantified by real time RT-PCR and protein production was quantified using an ELISA kit. Cellular stress induced a large increase in netrin-1 production without increase in transcription of netrin-1 gene. Mitogen activated protein kinase, ERK mediates the drug induced netrin-1 mRNA translation increase without altering mRNA stability. CONCLUSION: Our results suggest that netrin-1 expression is suppressed at the translational level and MAPK activation leads to rapid translation of netrin-1 mRNA in the kidney tubular epithelial cells.

  6. Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells.

    Science.gov (United States)

    Burrows, G G; Chou, Y K; Wang, C; Chang, J W; Finn, T P; Culbertson, N E; Kim, J; Bourdette, D N; Lewinsohn, D A; Lewinsohn, D M; Ikeda, M; Yoshioka, T; Allen, C N; Offner, H; Vandenbark, A A

    2001-10-15

    Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases. PMID:11591763

  7. Microparticles in Stored RBC as Potential Mediators of Transfusion Complications

    OpenAIRE

    Jy, Wenche; Ricci, Marco; Shariatmadar, Sherry; Gomez-Marin, Orlando; Horstman, Lawrence H; Ahn, Yeon S

    2011-01-01

    This article reviews evidence for the involvement of cell-derived microparticles (MP) in transfusion-related adverse events. The controversy concerning possible added risk of older vs. fresher stored blood is also reviewed, and is consistent with the hypothesis that MP are involved with adverse events. Although all types of circulating MP are discussed, the emphasis is on red cell-derived MP (RMP). The evidence is particularly strong for involvement of RMP in transfusion-related acute lung in...

  8. Vectorial secretion of CTGF as a cell-type specific response to LPA and TGF-β in human tubular epithelial cells

    Directory of Open Access Journals (Sweden)

    Zuehlke Jonathan

    2012-09-01

    Full Text Available Abstract Background Increased expression of the pro-fibrotic protein connective tissue growth factor (CTGF has been detected in injured kidneys and elevated urinary levels of CTGF are discussed as prognostic marker of chronic kidney disease. There is evidence that epithelial cells lining the renal tubular system contribute to uptake and secretion of CTGF. However, the role of different types of tubular epithelial cells in these processes so far has not been addressed in primary cultures of human cells. Results Tubular epithelial cells of proximal and distal origin were isolated from human kidneys and cultured as polarized cells in insert wells. The pro-fibrotic stimuli lysophosphatidic acid (LPA and transforming growth factor β (TGF-β were used to induce CTGF secretion. LPA activated CTGF secretion in proximal tubular cells when applied from either the apical or the basolateral side as shown by immunocytochemistry. CTGF was secreted exclusively to the apical side. Signaling pathways activated by LPA included MAP kinase and Rho kinase signaling. TGF-β applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release. Interestingly, TGF-β activation induced different signaling pathways depending on the side of TGF-β application. Smad signaling was almost exclusively activated from the basolateral side most prominently in cells of distal origin. Only part of these cells also synthesized CTGF indicating that Smad activation alone was not sufficient for CTGF induction. MAP kinases were involved in apical TGF-β-mediated activation of CTGF synthesis in proximal cells and a subset of epithelial cells of distal origin. This subpopulation of distal tubular cells was also able to internalize recombinant apical CTGF, in addition to proximal cells which were the main cells to take up exogenous CTGF. Conclusions Analysis of polarized human primary renal epithelial cells in a transwell system shows that

  9. Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

    OpenAIRE

    Coconnier, M H; Klaenhammer, T R; Kernéis, S; Bernet, M F; Servin, A L

    1992-01-01

    The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and i...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-26

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

  11. Supracellular microtubule alignments in cell layers associated with the secretion of certain fish scales.

    Science.gov (United States)

    Dane, P J; Tucker, J B

    1986-01-01

    Intercellularly aligned microtubule arrays are present in cell layers associated with the growth and secretion of scales in the zebra fish Brachydanio rerio and the neon tetra fish Hyphessobrycon innesi. The layers in question are: the osteoblast layer that covers the ossified outer surface of a scale, and the layer of fibroblasts that is situated immediately underneath the inner collagenous surface of a scale's fibrillary plate. In certain osteoblasts, the proximal portions of microtubules (with respect to centrosomes) run closely alongside the anterior margin of each cell where it flanks one of a scale's ridge-shaped circuli. These osteoblasts and microtubule portions are arranged in aligned rows that are parallel to circuli. However, the distal portions of the microtubules curve into an orientation that is approximately at right angles to circuli and they are aligned with each other and similar microtubule portions in adjacent osteoblasts. Such microtubule alignments only occur in osteoblasts that are associated with circuli. In Hyphessobrycon osteoblasts situated elsewhere on a scale's surface, microtubules radiate from cell centres but their distal portions curve into alignment with each other and are oriented alongside cell margins. The proximal portions of fibroblast microtubules radiate from centrally positioned centrosomes but the distal portions curve into alignment with each other and distal microtubule portions in neighbouring fibroblasts. The overall pattern of microtubule alignment is similar to that of collagen fibres, which these fibroblasts are secreting onto the fibrillary plate. The immunofluorescence protocol that was used to demonstrate the microtubule alignments described above did not reveal such alignments in the osteoblast and fibroblast layers associated with scales of the brown trout Salmo trutta fario. These findings are assessed in terms of intra-and inter-cellular control of microtubule alignment, and decentralized reorientation of

  12. Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women

    OpenAIRE

    Jayachandran, Muthuvel; Litwiller, Robert D.; Owen, Whyte G.; Heit, John A.; Behrenbeck, Thomas; Mulvagh, Sharon L.; Araoz, Philip A; Budoff, Matthew J.; Harman, S. Mitchell; Virginia M Miller

    2008-01-01

    While the risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened ...

  13. Leptin promotes breast cancer cell migration and invasion via IL-18 expression and secretion.

    Science.gov (United States)

    Li, Kuangfa; Wei, Lan; Huang, Yunxiu; Wu, Yang; Su, Min; Pang, Xueli; Wang, Nian; Ji, Feihu; Zhong, Changli; Chen, Tingmei

    2016-06-01

    In recent years, crosstalk between tumor microenvironment and cancer cells have received increasing attention. Accumulating research data suggests that leptin, a key adipokine secreted from adipocytes, plays important roles in breast cancer development. In our study, the effects of leptin on polarization of tumor-associated macrophages (TAMs) and promotion of the invasiveness of tumor cells were investigated. THP1 cells were used to differentiate M2 polarization macrophages. After stimulated by leptin, we established a co-culture system of tumor cells and macrophages to evaluate the function of leptin-induced macrophages in the migration and invasion of breast cancer cells. The gene and protein expressions were analyzed and the underlying mechanisms were evaluated. Moreover, pathological human specimens, and xenografts in nude mice, were detected to strengthen the in vitro results. Leptin elevated the expression of an array of cytokines in TAMs, IL-18 was the most increased, with an activation of the NF-κB/NF-κB1 signalling pathway. Additionally, after treated with leptin, TAMs significantly promoted the migration and invasion of breast cancer cells. However, these effects of leptin were abolished by the co-incubation of Bay11‑7082, a pharmacological NF-κB inhibitor. Leptin also directly stimulated IL-18 expression in breast cancer cells, which, differently, was via the PI3K/AKT-ATF-2 signaling pathway. In vivo studies showed that malignant breast carcinoma exhibited strong higher expression of Leptin, IL-8, and TAMs markers. Xenograft tumor-bearing mouse models showed that leptin significantly increased tumor volume, enhanced lung metastases, and increased expression of IL-8 and TAM markers, which were abolished by depletion of macrophages by clophosome-clodronate liposomes (CCL). Leptin could induce IL-18 expression both in TAMs and breast cancer cells. Leptin-induced IL-18 expression was regulated via NF-κB/NF-κB1 signaling in TAMs, while via PI3K

  14. Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells

    Directory of Open Access Journals (Sweden)

    Michael Kittl

    2016-06-01

    which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. Conclusion: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

  15. Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells: dependence on extracellular sodium

    DEFF Research Database (Denmark)

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

    1992-01-01

    Purified populations of rat peritoneal mast cells were used to study the effect of ouabain on compound 48/80-induced histamine secretion and on 86Rb+ uptake. 86Rb+ was used as a tracer for extracellular K+. The calculated value of the ouabain-sensitive uptake of K+ and 86Rb+ was considered a meas...

  16. Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

    International Nuclear Information System (INIS)

    A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S

  17. Chloride-dependent secretion of alveolar wall liquid determined by optical-sectioning microscopy.

    Science.gov (United States)

    Lindert, Jens; Perlman, Carrie E; Parthasarathi, Kaushik; Bhattacharya, Jahar

    2007-06-01

    The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr(-/-) mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr(-/-) mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease. PMID:17290033

  18. Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells.

    Science.gov (United States)

    Atienzar-Aroca, Sandra; Flores-Bellver, Miguel; Serrano-Heras, Gemma; Martinez-Gil, Natalia; Barcia, Jorge M; Aparicio, Silvia; Perez-Cremades, Daniel; Garcia-Verdugo, Jose M; Diaz-Llopis, Manuel; Romero, Francisco J; Sancho-Pelluz, Javier

    2016-08-01

    The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell-to-cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT-PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes. PMID:26999719

  19. Biofouling control using microparticles carrying a biocide

    OpenAIRE

    Ferreira, C.; Rosmaninho, R; Simoes, M.; Pereira, MC; Bastos, MMSM; Nunes, OC; Coelho, M; Melo, LF

    2010-01-01

    This study presents a new technological approach to minimize the use of antimicrobial (AMB) agents and their deleterious effects, based on the principle of drug-delivery systems whereby the AMB chemicals are transported on microparticles. The efficacy of microparticles carrying the quaternary ammonium compound (QAC), benzyldimethyldodecyl ammonium chloride (BDMDAC), was assessed against Pseudomonas fluorescens in both the planktonic and the biofilm state. The microparticles were prepared usin...

  20. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells.

    Science.gov (United States)

    Hayashi, M; Inagaki, A; Novak, I; Matsuda, H

    2016-07-01

    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl(-) channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (V te) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl(-) currents in Capan-1 single cells. The effects of adenosine on V te, an equivalent short-circuit current (I sc), and whole-cell Cl(-) currents were inhibited by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitor. The adenosine A2B receptor agonist, BAY 60-6583, increased I sc and whole-cell Cl(-) currents through CFTR Cl(-) channels, whereas the A2A receptor agonist, CGS 21680, had negligible effects. The A2B receptor antagonist, PSB 603, inhibited the response of I sc to adenosine. Immunohistochemical analysis showed that the A2A and A2B receptors colocalized with Ezrin in the luminal membranes of Capan-1 monolayers and in rat pancreatic ducts. Adenosine elicited the whole-cell Cl(-) currents in guinea pig duct cells. These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl(-) channels via adenosine A2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion. PMID:26965147

  1. Intrinsic optical signal imaging of glucose-stimulated physiological responses in the insulin secreting INS-1 β-cell line

    Science.gov (United States)

    Li, Yi-Chao; Cui, Wan-Xing; Wang, Xu-Jing; Amthor, Franklin; Yao, Xin-Cheng

    2011-03-01

    Intrinsic optical signal (IOS) imaging has been established for noninvasive monitoring of stimulus-evoked physiological responses in the retina and other neural tissues. Recently, we extended the IOS imaging technology for functional evaluation of insulin secreting INS-1 cells. INS-1 cells provide a popular model for investigating β-cell dysfunction and diabetes. Our experiments indicate that IOS imaging allows simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid image sequences reveal transient optical responses that have time courses comparable to glucose-evoked β-cell electrical activities.

  2. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

    OpenAIRE

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Jing WANG; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L.; Bottino, Rita; Kim, Seung K.

    2013-01-01

    eLife digest Diabetes mellitus is a disease that can lead to dangerously high blood sugar levels, causing numerous complications such as heart disease, glaucoma, skin disorders, kidney disease, and nerve damage. In healthy individuals, beta cells in the pancreas produce a hormone called insulin, which stimulates cells in the liver, muscles and fat to take up glucose from the blood. However, this process is disrupted in people with diabetes, who either have too few pancreatic beta cells (type ...

  3. Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation

    OpenAIRE

    Wafa Markikou-Ouni; Sima Drini; Narges Bahi-Jaber; Mehdi Chenik; Amel Meddeb-Garnaoui

    2015-01-01

    Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vit...

  4. The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa.

    Science.gov (United States)

    Gouran, Hossein; Gillespie, Hyrum; Nascimento, Rafael; Chakraborty, Sandeep; Zaini, Paulo A; Jacobson, Aaron; Phinney, Brett S; Dolan, David; Durbin-Johnson, Blythe P; Antonova, Elena S; Lindow, Steven E; Mellema, Matthew S; Goulart, Luiz R; Dandekar, Abhaya M

    2016-01-01

    Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa. PMID:27492542

  5. Activation of Chloride Secretion by Isoflavone Genistein in Endometrial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Chatsri Deachapunya

    2013-11-01

    Full Text Available Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (Isc which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl- current and barium-sensitive basolateral K+ current while inhibiting the apical K+ current. Genistein failed to increase the Isc in the presence of forskolin or IBMX, but did increase the Isc in UTP. Pretreatment with genistein also abolished the increase in the Isc when induced by forskolin, IBMX or UTP. However, Ca2+-chelating BAPTA-AM did not affect the genistein-induced increase in the Isc. The genistein-stimulated Isc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia.

  6. Sertoli cell synthesizes and secretes a protease inhibitor, α2-macroglobulin

    International Nuclear Information System (INIS)

    The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial components of the seminiferous epithelium, release a protease inhibitor, testicular α2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular α2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that α2-macroglubulin rather than α1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of α2-macroglobulin to inactivate proteases and growth factors such as TGF-β by a common mechanism suggests that this protein may have a dual function in the testis

  7. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    Directory of Open Access Journals (Sweden)

    Morteza Abouzaripour

    2016-02-01

    Full Text Available Objective: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone marrow have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1 positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS followed by characterization with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR, immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4 detected by immunocytochemistry and C-X-C chemokine receptor type 4 (CXCR4 and stem cell antigen-1 (SCA-1 detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors, Ngn3 (endocrine progenitor marker, Insulin1 and Insulin2 (pancreaticβ-cell markers. Additionally, our results demonstrate expression of PDX1 and GLUT2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion: Our study clearly demonstrates the potential of SSEA-1

  8. Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion.

    Science.gov (United States)

    Rubí, Blanca; Antinozzi, Peter A; Herrero, Laura; Ishihara, Hisamitsu; Asins, Guillermina; Serra, Dolors; Wollheim, Claes B; Maechler, Pierre; Hegardt, Fausto G

    2002-01-01

    Lipid metabolism in the beta-cell is critical for the regulation of insulin secretion. Pancreatic beta-cells chronically exposed to fatty acids show higher carnitine palmitoyltransferase I (CPT I) protein levels, higher palmitate oxidation rates and an altered insulin response to glucose. We examined the effect of increasing CPT I levels on insulin secretion in cultured beta-cells. We prepared a recombinant adenovirus containing the cDNA for the rat liver isoform of CPT I. The overexpression of CPT I in INS1E cells caused a more than a 5-fold increase in the levels of CPT I protein (detected by Western blotting), a 6-fold increase in the CPT activity, and an increase in fatty acid oxidation at 2.5 mM glucose (1.7-fold) and 15 mM glucose (3.1-fold). Insulin secretion was stimulated in control cells by 15 mM glucose or 30 mM KCl. INS1E cells overexpressing CPT I showed lower insulin secretion on stimulation with 15 mM glucose (-40%; P<0.05). This decrease depended on CPT I activity, since the presence of etomoxir, a specific inhibitor of CPT I, in the preincubation medium normalized the CPT I activity, the fatty-acid oxidation rate and the insulin secretion in response to glucose. Exogenous palmitate (0.25 mM) rescued glucose-stimulated insulin secretion (GSIS) in CPT I-overexpressing cells, indicating that the mechanism of impaired GSIS was through the depletion of a critical lipid. Depolarizing the cells with KCl or intermediary glucose concentrations (7.5 mM) elicited similar insulin secretion in control cells and cells overexpressing CPT I. Glucose-induced ATP increase, glucose metabolism and the triacylglycerol content remained unchanged. These results provide further evidence that CPT I activity regulates insulin secretion in the beta-cell. They also indicate that up-regulation of CPT I contributes to the loss of response to high glucose in beta-cells exposed to fatty acids. PMID:11988095

  9. Naproxen sodium decreases prostaglandins secretion from cultured human endometrial stromal cells modulating metabolizing enzymes mRNA expression.

    Science.gov (United States)

    Carrarelli, Patrizia; Funghi, Lucia; Bruni, Simone; Luisi, Stefano; Arcuri, Felice; Petraglia, Felice

    2016-04-01

    Dysmenorrhea, defined as painful cramps occurring immediately before or during the menstrual period, is a common symptom of different gynecological diseases. An acute uterine inflammatory response driven by prostaglandins (PGs) is responsible for painful symptoms. Progesterone withdrawal is responsible for activation of cyclooxygenase (COX-2) enzyme and decrease of hydroxyprostaglandin dehydrogenase (HPDG) with consequent increased secretion of PGs secretion, inducing uterine contractility and pain. The most widely used drugs for the treatment of pelvic pain associated with menstrual cycle are non steroidal anti-inflammatory drugs (NSAIDs). The uterine site of action of these drugs is still not defined and the present study evaluated the effect of naproxen sodium in cultured human endometrial stromal cells (HESC) collected from healthy women. PGE2 release was measured by ELISA; COX-2 and HPDG mRNA expression were assessed by qRT-PCR. Naproxen sodium did not affect HESC vitality. Naproxen sodium significantly decreased PGE2 secretion (p dysmenorrhea. PMID:26634864

  10. Effects of saxagliptin on β-cell stimulation and insulin secretion in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Henry, R R; Smith, Susanne; Schwartz, Saul; Mudaliar, S R; Deacon, C F; Holst, Jens Juul; Duan, R Y; Chen, R S; List, J F

    2011-01-01

    Aim: To study the effect of dipeptidyl peptidase-4 (DPP-4) inhibition with saxagliptin on ß-cell function as reflected by the stimulated insulin secretion rate after an enteral glucose load in patients with type 2 diabetes. Methods: Patients in this randomized, parallel-group, double-blind, placebo......-controlled study were drug-naÏve, aged 43–69 years, with baseline haemoglobin A1c (HbA1c) 5.9–8.1%. Twenty patients received saxagliptin 5 mg once daily; 16 received placebo. Patients were assessed at baseline and week 12 by intravenous hyperglycaemic clamp (0–180 min, fasting state), and intravenous......-oral hyperglycaemic clamp (180–480 min, postprandial state) following oral ingestion of 75 g glucose. Primary and secondary endpoints were percent changes from baseline in insulin secretion during postprandial and fasting states, respectively. Insulin secretion was calculated by C-peptide deconvolution. Results...

  11. Bile Acids Acutely Stimulate Insulin Secretion of Mouse β-Cells via Farnesoid X Receptor Activation and KATP Channel Inhibition

    OpenAIRE

    Düfer, Martina; Hörth, Katrin; Wagner, Rebecca; Schittenhelm, Björn; Prowald, Susanne; Wagner, Thomas F. J.; Oberwinkler, Johannes; Lukowski, Robert; Gonzalez, Frank J.; Krippeit-Drews, Peter; Drews, Gisela

    2012-01-01

    Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by...

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

    OpenAIRE

    Claes, Ingmar; Schoofs, Geert; K. Regulski; Courtin, P.; Chapot-Chartier, M.-P.; Rolain, T.; Hols, P; von Ossowski, I; Reunanen, J.; DE VOS W.M.; Palva, A.; Vanderleyden, Jos; De Keersmaecker, Sigrid; Lebeer, Sarah

    2012-01-01

    Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mu...

  13. Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

    OpenAIRE

    1987-01-01

    Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an underst...

  14. Pharmaceutical microparticle engineering with electrospraying

    DEFF Research Database (Denmark)

    Bohr, Adam; Wan, Feng; Kristensen, Jakob;

    2015-01-01

    Microparticles of Celecoxib, dispersed in a matrix of poly(lactic-co-glycolic acid) (PLGA), were prepared by electrospraying using different solvent mixtures to investigate the influence upon particle formation and the resulting particle characteristics. Mixtures consisting of a good solvent, ace...... demonstrated by the increasingly higher drug release rates. The results demonstrate the importance of solvent composition in particle preparation and indicate potential for exploiting this dependence to improve pharmaceutical particle design and performance....

  15. 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. PMID:26348137

  16. Hyperpolarization of the Membrane Potential Caused by Somatostatin in Dissociated Human Pituitary Adenoma Cells that Secrete Growth Hormone

    Science.gov (United States)

    Yamashita, Naohide; Shibuya, Naohiko; Ogata, Etsuro

    1986-08-01

    Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 ± 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 ± 4.3 mV in 6 mM K+ medium and -97.2 ± 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 ± 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.

  17. 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.

  18. αB-crystallin stimulates VEGF secretion and tumor cell migration and correlates with enhanced distant metastasis in head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    αB-crystallin is able to modulate vascular endothelial growth factor (VEGF) secretion. In many solid tumors VEGF is associated with angiogenesis, metastasis formation and poor prognosis. We set out to assess whether αB-crystallin expression is correlated with worse prognosis and whether this is related to VEGF secretion and cell motility in head and neck squamous cell carcinoma (HNSCC). αB-crystallin expression was determined immunohistochemically in tumor biopsies of 38 HNSCC patients. Locoregional control (LRC) and metastasis-free survival (MFS) of the patients were analyzed in relation to αB-crystallin expression. Additionally, the effects of αB-crystallin knockdown on VEGF secretion and cell motility were studied in vitro. Patients with higher staining fractions of αB-crystallin exhibited a significantly shorter MFS (Log-Rank test, p < 0.005). Under normoxic conditions αB-crystallin knockdown with two different siRNAs in a HNSCC cell line reduced VEGF secretion 1.9-fold and 2.1-fold, respectively. Under hypoxic conditions, a similar reduction of VEGF secretion was observed, 1.9-fold and 2.2-fold, respectively. The effect on cell motility was assessed by a gap closure assay, which showed that αB-crystallin knockdown decreased the rate by which HNSCC cells were able to close a gap by 1.5- to 2.0-fold. Our data suggest that αB-crystallin expression is associated with distant metastases formation in HNSCC patients. This association might relate to the chaperone function of αB-crystallin in mediating folding and secretion of VEGF and stimulating cell migration

  19. Production of microparticles of molinate degrading biocatalysts using the spray drying technique.

    Science.gov (United States)

    Lopes, Ana R; Sousa, Vera M; Estevinho, Berta N; Leite, José P; Moreira, Nuno F F; Gales, Luís; Rocha, Fernando; Nunes, Olga C

    2016-10-01

    Previous studies demonstrated the capability of mixed culture DC1 to mineralize the thiocarbamate herbicide molinate through the activity of molinate hydrolase (MolA). Because liquid suspensions are not compatible with long-term storage and are not easy to handle when bioremediation strategies are envisaged, in this study spray drying was evaluated as a cost-effective method to store and transport these molinate biocatalysts. Microparticles of mixed culture DC1 (DC1) and of cell free crude extracts containing MolA (MA) were obtained without any carrier polymer, and with calcium alginate (CA) or modified chitosan (MCt) as immobilizing agents. All the DC1 microparticles showed high molinate degrading activity upon storage for 6 months, or after 9 additions of ∼0.4 mM molinate over 1 month. The DC1-MCt microparticles were those with the highest survival rate and lowest heterogeneity. For MA microparticles, only MA-MCt degraded molinate. However, its Vmax was only 1.4% of that of the fresh cell free extract (non spray dried). The feasibility of using the DC1-MCt and MA-MCt microparticles in bioaugmentation processes was assessed in river water microcosms, using mass (g):volume (L) ratios of 1:13 and 1:0.25, respectively. Both type of microparticles removed ∼65-75% of the initial 1.5 mg L(-1) molinate, after 7 days of incubation. However, only DC1-MCt microparticles were able to degrade this environmental concentration of molinate without disturbing the native bacterial community. These results suggest that spray drying can be successfully used to produce DC1-MCt microparticles to remediate molinate polluted sites through a bioaugmentation strategy. PMID:27421102

  20. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    Directory of Open Access Journals (Sweden)

    Ida M Smith

    Full Text Available Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications

  1. Secreted trophic factors of mesenchymal stem cells support neurovascular and musculoskeletal therapies.

    Science.gov (United States)

    Hofer, Heidi R; Tuan, Rocky S

    2016-01-01

    Adult mesenchymal stem cells (MSCs) represent a subject of intense experimental and biomedical interest. Recently, trophic activities of MSCs have become the topic of a number of revealing studies that span both basic and clinical fields. In this review, we focus on recent investigations that have elucidated trophic mechanisms and shed light on MSC clinical efficacy relevant to musculoskeletal applications. Innate differences due to MSC sourcing may play a role in the clinical utility of isolated MSCs. Pain management, osteochondral, nerve, or blood vessel support by MSCs derived from both autologous and allogeneic sources have been examined. Recent mechanistic insights into the trophic activities of these cells point to ultimate regulation by nitric oxide, nuclear factor-kB, and indoleamine, among other signaling pathways. Classic growth factors and cytokines-such as VEGF, CNTF, GDNF, TGF-β, interleukins (IL-1β, IL-6, and IL-8), and C-C ligands (CCL-2, CCL-5, and CCL-23)-serve as paracrine control molecules secreted or packaged into extracellular vesicles, or exosomes, by MSCs. Recent studies have also implicated signaling by microRNAs contained in MSC-derived exosomes. The response of target cells is further regulated by their microenvironment, involving the extracellular matrix, which may be modified by MSC-produced matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs. Trophic activities of MSCs, either resident or introduced exogenously, are thus intricately controlled, and may be further fine-tuned via implant material modifications. MSCs are actively being investigated for the repair and regeneration of both osteochondral and other musculoskeletal tissues, such as tendon/ligament and meniscus. Future rational and effective MSC-based musculoskeletal therapies will benefit from better mechanistic understanding of MSC trophic activities, for example using analytical "-omics" profiling approaches. PMID:27612948

  2. Barbiturates inhibit ATP-K+ channels and voltage-activated currents in CRI-G1 insulin-secreting cells.

    OpenAIRE

    Kozlowski, R. Z.; Ashford, M. L.

    1991-01-01

    1. Patch-clamp recording techniques were used to examine the effects of barbiturates upon the ATP-K+ channel, and voltage-activated channels present in the plasma membrane of CRI-G1 insulin-secreting cells. 2. Thiopentone inhibited ATP-K+ channel activity when applied to cell-attached patches or the intracellular or extracellular surface of cell-free patches. Secobarbitone and pentobarbitone were also effective inhibitors of ATP-K+ channels in cell-free patches, whereas phenobarbitone was ine...

  3. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

    DEFF Research Database (Denmark)

    Kalis, Martins; Bolmeson, Caroline; Esguerra, Jonathan L.S.;

    2011-01-01

    -blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses...... revealed altered islet morphology, marked decreased ß-cell mass, reduced numbers of granules within the ß-cells and reduced granule docking in adult RIP-Cre Dicer1(¿/¿) mice. ß-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal ß-cell development as 2-week old RIP-Cre Dicer1...... and diabetes development....

  4. Tumor-targeted paclitaxel-loaded folate conjugated poly(ethylene glycol)-poly(L-lactide) microparticles produced by supercritical fluid technology.

    Science.gov (United States)

    Huang, Xiaobei; Zhang, Yanzhi; Yin, Guangfu; Pu, Ximing; Liao, Xiaoming; Huang, Zhongbing; Chen, Xianchun; Yao, Yadong

    2015-02-01

    The new biodegradable diblock copolymers poly(ethylene glycol)-poly(L-lactide) (PEG-PLLA) were synthesized and were chemically conjugated with folate (FA) in the PEG terminal ends to form FA-PEG-PLLA. Then the hydrophobic drug paclitaxel (PTX) loaded microparticles (PTX/FA-PEG-PLLA) were produced via solution enhanced dispersion by supercritical fluids (SEDS). These microparticles exhibited sphere-like shape by scanning electron microscopy observation and showed narrow hydrodynamic size distributions by dynamic light scattering measurement. Drug loading of PTX loaded microparticles was about 7-9% and the encapsulation efficiency of PTX loaded microparticles was about 18-23%. Flow cytometry and confocal laser scanning microscope analyses revealed that fluorescein isothiocyanate labeled FA conjugated microparticles presented significantly higher cellular uptake than FA-free group due to the FA-receptor-mediated endocytosis. In vitro cytotoxicity evaluation indicated that FA-PEG-PLLA expressed negligible cytotoxicity to mouse fibroblasts L929 cells. Moreover, PTX/FA-PEG-PLLA microparticles exhibited much higher anti-cancer efficacy than PTX/PEG-PLLA microparticles against human ovarian cancer SKOV3 cells. Nude mice xenografted with SKOV3 cells were used in biodistribution studies, the results indicated that an increased amount of PTX was accumulated in the tumor tissue deal with PTX/FA-PEG-PLLA microparticles. These results collectively suggested that PTX/FA-PEG-PLLA microparticles prepared by SEDS would have potential in anti-tumor applications as a tumor-targeted drug delivery formulation. PMID:25649516

  5. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte;

    2010-01-01

    directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high...... hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the...... in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are...

  6. Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH3 pituitary cells

    International Nuclear Information System (INIS)

    The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH3 cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated 45calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockage of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH3 cells. These observations may have implications for RHC 80267 action in other cell types

  7. Glucagon-like peptide-1 receptor is present in pancreatic acinar cells and regulates amylase secretion through cAMP.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Heidenreich, Kaeli; Williams, John A

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic β-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway. PMID:26542397

  8. Analysis of the Proteins Secreted from the Oryza meyeriana Suspension-Cultured Cells Induced by Xanthomonas oryzae pv. oryzae

    Science.gov (United States)

    Chen, Xian; Dong, Yan; Yu, Chulang; Fang, XianPing; Deng, Zhiping; Yan, Chengqi; Chen, Jianping

    2016-01-01

    Oryza meyeriana, a wild species of rice from China, shows high resistance to Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial blight, one of the most serious rice pathogens. To better understand the resistance mechanism, a proteomic study was conducted to identify changes in the proteins secreted in embryo cell suspension cultures in response to Xoo. After two-dimensional difference gel electrophoresis (2D-DIGE), 72 differentially expressed protein spots corresponding to 34 proteins were identified by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. Of the 34 proteins, 10 were up regulated and 24 down regulated. The secreted proteins identified were predicted to be involved in various biological processes, including signal transduction, defense, ROS and cell wall modification. 77% of the 34 proteins were predicted to have a signal peptide by Signal P. Quantitative Real-Time PCR showed that transcript levels of 14 secreted proteins were not well correlated with secreted protein levels. Peroxidase activity was up regulated in both O. meyriana and susceptible rice but was about three times higher in O. meyeriana. This suggests that peroxidases may play an important role in the early response to Xoo in O. meyeriana. These results not only provide a better understanding of the resistance mechanism of O. meyeriana, but have implications for studies of the interactions between other plants and their pathogens. PMID:27196123

  9. Analysis of the Proteins Secreted from the Oryza meyeriana Suspension-Cultured Cells Induced by Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Chen, Xian; Dong, Yan; Yu, Chulang; Fang, XianPing; Deng, Zhiping; Yan, Chengqi; Chen, Jianping

    2016-01-01

    Oryza meyeriana, a wild species of rice from China, shows high resistance to Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial blight, one of the most serious rice pathogens. To better understand the resistance mechanism, a proteomic study was conducted to identify changes in the proteins secreted in embryo cell suspension cultures in response to Xoo. After two-dimensional difference gel electrophoresis (2D-DIGE), 72 differentially expressed protein spots corresponding to 34 proteins were identified by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. Of the 34 proteins, 10 were up regulated and 24 down regulated. The secreted proteins identified were predicted to be involved in various biological processes, including signal transduction, defense, ROS and cell wall modification. 77% of the 34 proteins were predicted to have a signal peptide by Signal P. Quantitative Real-Time PCR showed that transcript levels of 14 secreted proteins were not well correlated with secreted protein levels. Peroxidase activity was up regulated in both O. meyriana and susceptible rice but was about three times higher in O. meyeriana. This suggests that peroxidases may play an important role in the early response to Xoo in O. meyeriana. These results not only provide a better understanding of the resistance mechanism of O. meyeriana, but have implications for studies of the interactions between other plants and their pathogens. PMID:27196123

  10. Pattern secretion of matrix Metalloproteinases and their biological tissue inhibitors by human glomerular mesangial cells in culture

    Directory of Open Access Journals (Sweden)

    "Hosseini R

    2001-08-01

    Full Text Available The glomerular mesangial cells (GMC play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between of a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC, and any disturbance in the balance may result in appearance of various pathological states such as glomerulosclerosis. We therefore studied pattern secretion of matrix metalloproteinases (MMPs, MMP-1, MMP-2, MMP-3, MMP-9 and their biological tissue inhibitor of matrix metalloproteinases (TIMPs, TIMP-1 and TIMP-2 by cultured human GMC. We also measured MMP-1/TIMP-1 complex level in the cell culture supernatants. For this purpose, the GMC were incubated under serum-free conditions with medium (RPMI-1640 alone or in combination with TNF-α (30 ng/ml or phorbol myristate acetate (PMA (50 ng/ml for exactly 24, 48 and 72 hours. The above parameters were assayed by established ELISA techniques. Our results showed that the lowest and largest secretions were related to MMP-9 and MMP-2, respectively. The results indicated that the MMPs and TIMPs secretion were increased by TNF-α (MMP-1, MMP-2, TIMP-1 and TIMP-2 and PMA (MMP-2, TIMP-1 and TIMP-2, significantly (P<0.05. These results suggest that the GMC can synthesis and release various MMPs and their inhibitors (TIMPs that, in part, control turnover of extracellular matrix proteins.

  11. Red Liriope platyphylla stimulated the insulin secretion through the regulation of calcium concentration in rat insulinoma cells and animal models.

    Science.gov (United States)

    Lee, Hye-Ryun; Kim, Ji-Eun; Lee, Young-Ju; Kwak, Moon-Hwa; Im, Dong-Soon; Hwang, Dae-Youn

    2013-06-01

    The aim of this study was to investigate the effects of Red L. platyphylla (RLP) on calcium and glucose levels during insulin secretion. To achieve this, alteration of insulin and calcium concentrations was measured in rat insulinoma-1 (INS-1) cells and animal models in response to RLP treatment. In INS-1 cells, maximum secretion of insulin was detected upon treatment with 200 µg/mL of RLP for 20 min. Nifedipine, an L-type calcium channel blocker, effectively inhibited insulin secretion from INS-1 cells. Regarding calcium levels, the maximum concentration of intracellular calcium in INS-1 cells was obtained by treatment with 100 µg/mL of RLP, whereas this level was reduced under conditions of 200 µg/mL of RLP. Further, RLP-treated INS-1 cells showed a higher level of intracellular calcium than that of L. platyphylla (LP), Korea White Ginseng (KWG), or Korea Red Ginseng (KRG)-treated cells. This RLP-induced increase in intracellular calcium was abrogated but not completely abolished upon treatment with 40 µM nifedipine in a dose-dependent manner. Furthermore, the insulin level was dramatically elevated upon co-treatment with high concentrations of glucose and RLP, whereas it was maintained at a low level in response to glucose and RLP co-treatment at low concentrations. In an animal experiment, the serum concentration of calcium increased or decreased upon RLP treatment according to glucose level compared to vehicle treatment. Therefore, these results suggest that insulin secretion induced by RLP treatment may be tightly correlated with calcium regulation, which suggests RLP is an excellent candidate for diabetes treatment. PMID:23825481

  12. Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Nahuel A Garcia

    Full Text Available Cardiomyocytes (CMs and endothelial cells (ECs have an intimate anatomical relationship that is essential for maintaining normal development and function in the heart. Little is known about the mechanisms that regulate cardiac and endothelial crosstalk, particularly in situations of acute stress when local active processes are required to regulate endothelial function. We examined whether CM-derived exosomes could modulate endothelial function. Under conditions of glucose deprivation, immortalized H9C2 cardiomyocytes increase their secretion of exosomes. CM-derived exosomes are loaded with a broad repertoire of miRNA and proteins in a glucose availability-dependent manner. Gene Ontology (GO analysis of exosome cargo molecules identified an enrichment of biological process that could alter EC activity. We observed that addition of CM-derived exosomes to ECs induced changes in transcriptional activity of pro-angiogenic genes. Finally, we demonstrated that incubation of H9C2-derived exosomes with ECs induced proliferation and angiogenesis in the latter. Thus, exosome-mediated communication between CM and EC establishes a functional relationship that could have potential implications for the induction of local neovascularization during acute situations such as cardiac injury.

  13. Tolvaptan Treatment in Syndrome of Inappropriate ADH Secretion due to Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Mucahit Gur

    2014-06-01

    Full Text Available Experience of ADH receptor antagonist (-vaptanes treatment in hyponatremia in malign patient is very limited. 68 years old male patient admitted to our department with a complain of nause, vomitting and epigastric pain. He has advanced stage of small cell lung cancer. He had treated with cisplatin and etoposide regimen 10 days ago as a first cure. We diagnosed inapropriate secretion of antidiuretic hormone syndrome (SIADH with low sodium level (118 meq/dl. Although the treatment with water restriction and 3% NaCl infusion, sodium level was not in normal. So we ordered 30 mg tolvaptan tablet. And then sodium levels were reached normal. After one month of discharge from hospital, he has hospitilized with same symptom and diagnosis. And again we ordered same treatment procedure and tolvaptane treatment. He had normal sodium (136 mEq/dl level during his follow up. This case demostrate that tolvaptane treatment is suitable aproaches in hyponatremia due to SIADH in oncologic patient.

  14. Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yue Tang; Yongchun Cui; Fuliang Luo; Xiaopeng Liu; Xiaojuan Wang; Aili Wu; Junwei Zhao; Zhong Tian; Like Wu

    2012-01-01

    In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

  15. In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans.

    Science.gov (United States)

    Liew, Kah Leong; Jee, Jap Meng; Yap, Ivan; Yong, Phelim Voon Chen

    2016-01-01

    Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection. PMID:27054608

  16. Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells.

    Science.gov (United States)

    Clapham, D E; Neher, E

    1984-08-01

    Using patch-clamp techniques, excitation and secretion in chromaffin cells were studied by measurement of unitary inward currents and of stimulus-evoked increments in membrane capacitance. The effect of the calmodulin inhibitor trifluoperazine (TFP) on Na, Ca and acetylcholine-induced (ACh) currents as well as on capacitance increments was investigated. TFP in concentrations up to 10 microM had no effect on Na channel currents. TFP was a potent anticholinergic agent. TFP in concentrations of 100 nM-1 microM decreased net ACh-induced currents by a slow block or allosteric modification of the channel. The effect was only partially reversible. Recovery from desensitization was retarded in direct relation to [TFP]. At the single channel level, TFP was found to slightly shorten open times in 0.5 and 20 microM-ACh. As reported previously, desensitization can be modelled by at least two desensitized states, as reflected by the bursting and clustering behaviour of single channels. TFP shortened clusters mainly by reducing the number of bursts per cluster. Whole-cell Ca currents (ICa) were reduced in 10 microM-TFP from an average of 29 microA cm-2-13 microA cm-2. Changes in capacitance of 1-200 fF were elicited in controls by maximal activation of the Ca current. We interpreted these steps to be the summed result of many exocytotic vesicular fusion events. Capacitance steps depended on ICa and were absent when extracellular Ca was removed. Application of 10 microM-TFP inhibited capacitance steps. The block of capacitance steps by TFP was shown to be independent of the reduction of ACh and Ca inward ionic currents. We conclude that the prevention of exocytosis by TFP is not completely described by its inhibition of electrical excitability but also results from intracellular actions. PMID:6090644

  17. In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans

    Science.gov (United States)

    2016-01-01

    Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection. PMID:27054608

  18. Effect of Src Tyrosine Kinase Inhibition on Secretion of MMP-2 and MMP-9 by Non-small Cell Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Rui ZHENG

    2011-01-01

    Full Text Available Background and objective Src tyrosine kinase and matrix metalloproteinase play the pivotal roles in lung cancer invasion and metastasis. The aim of this study is to evaluate the effect of Src tyrosine kinase inhibition on secretion of matrix metalloproteinase 2 (MMP-2 and matrix metalloproteinase 9 (MMP-9 by non-small cell lung cancer (NSCLC cells. Methods ELISA was used to examine the activity of MMP-2 and MMP-9 produced by NSCLC cells (PC14PE6, H226, PC-9, A549 as well as the effect of Src tyrosine kinase inhibition on secretion of MMP-2 and MMP-9 by NSCLC cells. Boyden chamber assay was used to assess the effect of Src tyrosine kinase inhibition on invasion of NSCLC cells in vitro. Results The levels of MMP-2 and MMP-9 in PC14PE6 and H226 cells were high, whereas the level of MMP-9 in A549 cell was low. MMP-2 and MMP-9 levels in PC-9 cell could not be detected. Src tyrosine kinase inhibitor obviously decreased the secretion of MMP-9 by PC14PE6, H226 and A549 cells, as well as MMP-2 by PC14PE6 cells in a dose-dependent manner. 10 μM Src tyrosine kinase inhibitor suppressed the secretion of MMP-9 by H226 and A549 cells, as wells as MMP-2 by PC14PE6 cells by more than 50%, while the same concentration of Src tyrosine kinase inhibitor almost had no effect on the level of MMP-2 in H226 cell. Invasiveness of NSCLC cells was suppressed by Src tyrosine kinase inhibitor in a dose-dependent manner, though there was minor difference in degree of the inhibition among four cell lines. 3 μM Src tyrosine kinase inhibitor suppressed the cell invasiveness of PC14PE6, H226, A549 and PC-9 cells by 79.1%, 68.09%, 90.96% and 96.98%, respectively (P < 0.001. Conclusion Inhibition of Src tyrosine kinase could suppress the invasion of NSCLC cells as well as the secretion of MMP-2 and MMP-9 by NSCLC cells in vitro. MMP-2 and MMP-9 were involved in regulating cell migration and invasion.

  19. Secreted recombinant human IL-24 protein inhibits the proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro and in vivo.

    Science.gov (United States)

    Ma, Qunfeng; Jin, Bangming; Zhang, Yao; Shi, Yinan; Zhang, Chi; Luo, Dan; Wang, Pengkun; Duan, Cuimi; Song, Heyu; Li, Xue; Deng, Xuefeng; Chen, Zhinan; Wang, Ziling; Jiang, Hong; Liu, Yan

    2016-05-01

    Interleukin-24 (IL-24) displays cancer-specific apoptosis-inducing properties in a broad spectrum of human tumors without harmful effects on normal cells. The human IL-24 protein is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and a potent antiangiogenic molecule. However, the function of secreted recombinant human IL-24 (srhIL-24) protein in esophageal squamous cell carcinoma (ESCC) cells has not been studied. In the present study, we prepared a stable site-specific-integrated cell line, Flp-InTMCHO/IL-24 (FCHO/IL-24), which secreted rhIL-24 at a higher level than three random-integrated cell lines. In vitro, we identified that the purified srhIL-24 inhibited proliferation and induced the apoptosis of ESCC Eca-109 cells and activated STAT3, which was related with the IL-20 receptors. In vivo, the tumorigenicity of Eca-109 cells was significantly inhibited by s.c. injection of FCHO/IL-24 cells. Decreased tumor microvessel density and an increased number of TUNEL-positive tumor cells were associated with tumor growth inhibition, indicating the presence of antiangiogenic activity and induction of apoptotic activity. In summary, the present study demonstrated that srhIL-24 induced growth inhibition and apoptosis in ESCC Eca-109 cells in vitro and in vivo, which may be mediated by the receptor pathway. PMID:26935394

  20. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Directory of Open Access Journals (Sweden)

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  1. Modulation of α-enolase post-translational modifications by dengue virus: increased secretion of the basic isoforms in infected hepatic cells.

    Directory of Open Access Journals (Sweden)

    Luiza M Higa

    Full Text Available Hepatic cells are major sites of dengue virus (DENV replication and liver injury constitutes a characteristic of severe forms of dengue. The role of hepatic cells in dengue pathogenesis is not well established, but since hepatocytes are the major source of plasma proteins, changes in protein secretion by these cells during infection might contribute to disease progression. Previously, we showed that DENV infection alters the secretion pattern of hepatic HepG2 cells, with α-enolase appearing as one of the major proteins secreted in higher levels by infected cells. ELISA analysis demonstrated that DENV infection modulates α-enolase secretion in HepG2 cells in a dose-dependent manner, but has no effect on its gene expression and on the intracellular content of the protein as assessed by PCR and western blot analyses, respectively. Two-dimensional western blots showed that both intracellular and secreted forms of α-enolase appear as five spots, revealing α-enolase isoforms with similar molecular weights but distinct isoeletric points. Remarkably, quantification of each spot content revealed that DENV infection shifts the isoform distribution pattern of secreted α-enolase towards the basic isoforms, whereas the intracellular protein remains unaltered, suggesting that post-translational modifications might be involved in α-enolase secretion by infected cells. These findings provide new insights into the mechanisms underlying α-enolase secretion by hepatic cells and its relationship with the role of liver in dengue pathogenesis. In addition, preliminary results obtained with plasma samples from DENV-infected patients suggest an association between plasma levels of α-enolase and disease severity. Since α-enolase binds plasminogen and modulates its activation, it is plausible to speculate the association of the increase in α-enolase secretion by infected hepatic cells with the haemostatic dysfunction observed in dengue patients including the

  2. Circulating microparticles and plasma levels of soluble E- and P-selectins in patients with systemic sclerosis

    DEFF Research Database (Denmark)

    Iversen, Lars; Østergaard, O; Ullman, S; Nielsen, C T; Halberg, P; Karlsmark, T; Heegaard, Niels Henrik Helweg; Jacobsen, Søren

    2013-01-01

    Microparticles (MPs) may be involved in the pathogenesis of systemic sclerosis (SSc), which includes vasculopathy, endothelial cell activation, and coagulation activation. Circulating MPs from SSc patients were characterized and their relationship with soluble markers of vascular activation inves...

  3. Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

    OpenAIRE

    Torisu, Takehiro; Torisu, Kumiko; Lee, In Hye; Liu, Jie; Malide, Daniela; Combs, Christian A.; Wu, Xufeng S.; Rovira, Ilsa I.; Fergusson, Maria M; Weigert, Roberto; Connelly, Patricia S.; Daniels, Mathew P.; Komatsu, Masaaki; Cao, Liu; Finkel, Toren

    2013-01-01

    Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here, we demonstrate that WPBs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy, or knockdown of the essential autophagy genes Atg5 or Atg7, inhibits the in vitro secretion of VWF. Furthermore, while mice...

  4. Pleiotropic effects of cancer cells' secreted factors on human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Almusa, Abdulaziz; Almajed, Mohammed;

    2013-01-01

    from MCF7 and HT-29, developed an elongated, spindle-shaped morphology with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK...... MSCs exposed to tumor CM, which was found to be positively regulated by FAK and MAPK signaling and negatively regulated by TGFβ signaling. Thus, our data support a model where MSCs could promote cancer progression through becoming pro-inflammatory cells within the cancer stroma....

  5. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on release type I collagen gels show greatly enhanced mRNA levels and secretion rates of β-casein and of some other milk proteins. The authors show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows > 90% of cells to produce high levels of β-casein. By comparison, 30-40% of cells on released type 1 gels and only 2-10% of cells on plastic express β-casein after 6 days in culture. Because only 40% of cells from late pregnant gland produced β-casein before culture, the EHS matrix can both induce and maintain an increased level of casein gene expression. Individual basal lamina components were also evaluated. Type IV collagen and fibronectin had little effect on morphology and β-casein mRNA levels. In contrast, both laminin and heparan sulfate proteoglycan increased β-casein mRNA levels. Profound morphological differences were evident between cells cultured on plastic and on EHS matrix, the latter cells forming ducts, ductules, and lumina and resembling secretory alveoli. These results emphasize the vital role of the extracellular matrix in receiving and integrating structural and functional signals that can direct specific gene expression in differentiated tissues

  6. Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

    Directory of Open Access Journals (Sweden)

    Didsbury Alicia

    2011-12-01

    Full Text Available Abstract Background Nonstructural glycoprotein 4 (NSP4 encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types. Methods NSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay. Results Secreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane. Conclusion Our study is the first to analyze an authentic (i.e. non-recombinant form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain

  7. Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE).

    Science.gov (United States)

    Chandramouleeswaran, Prasanna M; Shen, Dawen; Lee, Anna J; Benitez, Alain; Dods, Kara; Gambanga, Fiona; Wilkins, Benjamin J; Merves, Jamie; Noah, Yuliana; Toltzis, Sarit; Yearley, Jennifer H; Spergel, Jonathan M; Nakagawa, Hiroshi; Malefyt, Rene deWaal; Muir, Amanda B; Wang, Mei-Lun

    2016-01-01

    Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface

  8. Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE.

    Directory of Open Access Journals (Sweden)

    Prasanna M Chandramouleeswaran

    Full Text Available Eosinophilic esophagitis (EoE is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP, an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC, we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf, inactive EoE (<15 eos/hpf and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C. To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA, wheat, and milk proteins beta-lactoglobulin (blg and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the

  9. Effects of mitochondrial ATP-sensitive potassium channels on the proliferation and secretion of human airway smooth muscle cells.

    OpenAIRE

    2014-01-01

    Bronchial asthma is the common chronic inflammatory disease and is characterized by chronic airway inflammation, airway remodeling, and airway hyperreactivity (AHR). Aim of this study was to investigate the effects of mitochondrial ATP-sensitive potassium channels (MitoKATP) on the proliferation and secretion of human airway smooth muscle cells (HASMCs). HASMCs were treated with the serum from asthmatic patients to establish HASMCs asthma model of passive sensitization. Rhodamine 123 (R-123) ...

  10. Small cell gall bladder carcinoma complicated by syndrome of inappropriate secretion of antidiuretic hormone (SIADH) treated with mozavaptan

    OpenAIRE

    TAMURA, Tetsuo; Takeuchi, Kazuo

    2013-01-01

    Small cell gall bladder carcinoma (Scc-GB) is a very rare entity. Although some cases present with endocrine manifestations, paraneoplastic hyponatraemia has been reported in only one previous case. Recently, the antidiuretic hormone (ADH) receptor antagonist mozavaptan has become available. Herein we report a case with Scc-GB complicated with syndrome of inappropriate secretion of antidiuretic hormone (SIADH) treated with mozavaptan. A 47-year-old woman was referred to our hospital for hypon...

  11. A Granulin-Like Growth Factor Secreted by the Carcinogenic Liver Fluke, Opisthorchis viverrini, Promotes Proliferation of Host Cells

    OpenAIRE

    Smout, Michael J.; Laha, Thewarach; Mulvenna, Jason; Sripa, Banchob; Suttiprapa, Sutas; Jones, Alun; Brindley, Paul J.; Loukas, Alex

    2009-01-01

    The human liver fluke, Opisthorchis viverrini, infects millions of people throughout south-east Asia and is a major cause of cholangiocarcinoma, or cancer of the bile ducts. The mechanisms by which chronic infection with O. viverrini results in cholangiocarcinogenesis are multi-factorial, but one such mechanism is the secretion of parasite proteins with mitogenic properties into the bile ducts, driving cell proliferation and creating a tumorigenic environment. Using a proteomic approach, we i...

  12. Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles

    Directory of Open Access Journals (Sweden)

    Fabiana Fernandes Bressan

    2015-10-01

    Full Text Available This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

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

    Czech Academy of Sciences Publication Activity Database

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

    2011-01-01

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

  14. Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.

    Science.gov (United States)

    Amao, Michiko; Kitahara, Yoshiro; Tokunaga, Ayaka; Shimbo, Kazutaka; Eto, Yuzuru; Yamada, Naoyuki

    2015-03-01

    Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells. PMID:25461479

  15. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

    Science.gov (United States)

    Suchard, S J; Lattanzio, F A; Rubin, R W; Pressman, B C

    1982-09-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation. PMID:7130269

  16. Secretion of beta-human chorionic gonadotropin by non-small cell lung cancer: a case report

    OpenAIRE

    Varma Seema; Vivekanandarajah Abhirami; Khattri Saakshi; Kong Frank

    2011-01-01

    Abstract Introduction We describe a case of non-small cell lung cancer that was found to stain positive for beta-human chorionic gonadotropin on immunohistochemistry. Only a few case reports have described lung cancers that secrete beta-human chorionic gonadotropin. Case presentation A 68-year-old Caucasian man presented with symptoms of weakness, fatigue and weight loss for the past two months. On examination, he was found to have generalized lymphadenopathy, and radiologic workup revealed n...

  17. Observations on the discharge of the secretion from ependymal cells of the subcommissural organ (SCO) of some South American primates.

    Science.gov (United States)

    Hofer, H O

    1986-01-01

    The secretion of the subcommissural organ (SCO) is synthesized in the intrinsic cells of that structure, a neuroendocrine gland. The organelles involved in the synthesis of this secretion are rough-surfaced endoplasmic reticulum (RER) and the Golgi apparatus. It is still uncertain whether and to what extent the latter participates in the synthesis. In highly active secretory ependymal cells of the SCO, the Golgi apparatus exhibits distinct signs of intense activity. This suggests that the Golgi apparatus is involved in the preparation of the secretion, even if this is difficult to prove electron microscopically. The secretion is discharged from the optical part of the SCO-cells into the cerebrospinal fluid (CSF) of the IIIrd ventricle, where Reissner's fibre is formed. Different phases of release of the content of the secretory granula into the IIIrd ventricle are described; our findings are in agreement with previously published observations. In 2 cases delicately granulated and moderately electron-dense material was found in circumscriptly dilated vaults of the intercellular spaces. Release of secretory material into intercellular spaces appears to be possible, but is evidently infrequent. Regular occurrence of a basal (peripheral) discharge into the hypendymal capillaries is not unanimously agreed upon as yet. In this paper, a passage of granula is described from the cytoplasm of the end feet of SCO cells through the walls of the capillaries into the systemic circulation. This provides structural evidence that secretory material synthesized in the SCO is released into the capillaries. These observations must ultimately be confirmed with the aid of ultracytochemical methods, particularly using labelled material. PMID:3086178

  18. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

    Directory of Open Access Journals (Sweden)

    Sebastian Gehmert

    2014-01-01

    Full Text Available Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs, these cells (ASCs provide a therapeutic option for Duchenne Muscular Dystrophy (DMD. But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

  19. Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

    Directory of Open Access Journals (Sweden)

    Henriques-Normark Birgitta

    2009-04-01

    Full Text Available Abstract Background Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness. Results We demonstrate that INPs affect Chlamydia growth in a dose dependent manner after bacterial invasion. The efficiency of C. trachomatis L2 and C. caviae GPIC entry into host cells was not altered in the presence of INPs. In C. caviae, entry appears to proceed normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the site of bacterial entry. Conclusion INPs have a strong inhibitory effect on Chlamydia growth. However, bacterial invasion is not altered in the presence of these drugs. In the light of these results, we discuss several hypotheses regarding the mode of action of INPs on type III secretion during the Chlamydia infectious cycle.

  20. Drug carrier systems based on collagen-alginate composite structures for improving the performance of GDNF-secreting HEK293 cells.

    Science.gov (United States)

    Lee, M; Lo, A C; Cheung, P T; Wong, D; Chan, B P

    2009-02-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor. Development of drug delivery technologies facilitating controlled release of GDNF is critical to applying GDNF in treating neurodegenerative diseases. We previously developed 3D collagen microspheres and demonstrated enhanced GDNF secretion after encapsulation of HEK293 cells, which were transduced to overexpress GDNF in these microspheres. However, the entrapped HEK293 cells were able to migrate out of the collagen microspheres, making it undesirable for clinical applications. In this report, we investigate two new carrier designs, namely collagen-alginate composite gel and collagen microspheres embedded in alginate gel in preventing cell leakage, maintaining cell growth and controlling GDNF secretion in the HEK293 cells. We demonstrated that inclusion of alginate gel in both designs is efficient in preventing cell leakage to the surrounding yet permitting the GDNF secretion, although the cellular growth rate is reduced in an alginate concentration dependent manner. Differential patterns of GDNF secretion in the two designs were demonstrated. The collagen-alginate composite gel maintains a more or less constant GDNF secretion over time while the collagen microspheres embedded in alginate gel continue to increase the secretion level of GDNF over time. This study contributes towards the development of cell-based GDNF delivery devices for the future therapeutics of neurodegenerative diseases. PMID:19059641

  1. Fabrication and characterization of a novel microparticle with gyrus-patterned surface and growth factor delivery for cartilage tissue engineering

    International Nuclear Information System (INIS)

    Microparticles can serve as substrates for cell amplification and deliver the expanded cells to the site of the defect. It was hypothesized that a novel microparticle combined of sustained and localized delivery of proliferative growth factors and gyrus-patterned surface would influence the cell behaviours of adherence and expansion on the microparticle in the present study. To test the hypothesis, gelatin particles with diameter ranging from 280 to 350 μm were fabricated and were modified by cryogenic freeze-drying treatment and basic fibroblast growth factor (bFGF) incorporation. The results of in vitro chondrocyte culture illustrated that cells could proliferate more obviously on the microparticles with bFGF addition, but no correlation between attachment rate and bFGF was observed. On the other hand, microparticles with gyrus-patterned surface demonstrated the highest cell attachment rate and higher rate of cell growth, in particular on bFGF combined ones. It seems to be a promising candidate as a chondrocyte microparticle and could be the potential application in cartilage tissue engineering.

  2. Fabrication and characterization of a novel microparticle with gyrus-patterned surface and growth factor delivery for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Huang Sha [Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi' an 710032 (China); Wang Yijuan [Key Laboratory for Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi' an 710062 (China); Liang Tang [Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi' an 710032 (China); Jin Fang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Liu Shouxin [Key Laboratory for Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi' an 710062 (China); Jin Yan, E-mail: yanjin@fmmu.edu.cn [Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi' an 710032 (China)

    2009-05-05

    Microparticles can serve as substrates for cell amplification and deliver the expanded cells to the site of the defect. It was hypothesized that a novel microparticle combined of sustained and localized delivery of proliferative growth factors and gyrus-patterned surface would influence the cell behaviours of adherence and expansion on the microparticle in the present study. To test the hypothesis, gelatin particles with diameter ranging from 280 to 350 {mu}m were fabricated and were modified by cryogenic freeze-drying treatment and basic fibroblast growth factor (bFGF) incorporation. The results of in vitro chondrocyte culture illustrated that cells could proliferate more obviously on the microparticles with bFGF addition, but no correlation between attachment rate and bFGF was observed. On the other hand, microparticles with gyrus-patterned surface demonstrated the highest cell attachment rate and higher rate of cell growth, in particular on bFGF combined ones. It seems to be a promising candidate as a chondrocyte microparticle and could be the potential application in cartilage tissue engineering.

  3. Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Dagbjort H Petursdottir

    Full Text Available Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC can influence the functionality of resident pulmonary macrophages (PuM. We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS1; c AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS, secretion of nitric oxide (NO, or IL-12, d in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

  4. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    Science.gov (United States)

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The mat