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Sample records for cell barrier alterations

  1. Loss of caveolin-1 causes blood-retinal barrier breakdown, venous enlargement, and mural cell alteration.

    Science.gov (United States)

    Gu, Xiaowu; Fliesler, Steven J; Zhao, You-Yang; Stallcup, William B; Cohen, Alex W; Elliott, Michael H

    2014-02-01

    Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

  2. Loss of EP2 Receptor Subtype in Colonic Cells Compromise Epithelial Barrier Integrity by Altering Claudin-4

    OpenAIRE

    Manigandan Lejeune; France Moreau; Kris Chadee

    2014-01-01

    Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high...

  3. Peanut Allergens Alter Intestinal Barrier Permeability and Tight Junction Localisation in Caco-2 Cell Cultures1

    Directory of Open Access Journals (Sweden)

    Dwan B. Price

    2014-05-01

    Full Text Available Background/Aims: Allergen absorption by epithelia may play an important role in downstream immune responses. Transport mechanisms that can bypass Peyer's patches include transcellular and paracellular transport. The capacity of an allergen to cross via these means can modulate downstream processing of the allergen by the immune system. The aim of this study was to investigate allergen-epithelial interactions of peanut allergens with the human intestinal epithelium. Methods: We achieved this using the human Caco-2 cell culture model, exposed to crude peanut extract. Western and immunofluorescence analysis were used to identify the cellular and molecular changes of peanut extract on the intestinal epithelium. Results: Following exposure of Caco-2 cells to peanut extract, binding of the peanut allergens Ara h 1 and Ara h 2 to the apical cellular membrane and transcytosis across the monolayers were observed. Additionally, the co-localisation of the transmembrane tight junction proteins occludin, JAM-A and claudin-1, with the intracellular adhesion protein ZO-1 was modified. Conclusion: Disruption of Caco-2 barrier integrity through tight junction disruption may enable movement of peanut proteins across the intestinal epithelium. This accounts for peanut's increased allergenicity, compared to other food allergens, and provides an explanation for the potency of peanut allergens in immune response elicitation.

  4. Hemorheological alterations of red blood cells induced by non-thermal dielectric barrier discharge plasma

    Science.gov (United States)

    Kim, Jeongho; Kim, Jae Hyung; Chang, Boksoon; Choi, Eun Ha; Park, Hun-Kuk

    2016-11-01

    Atmospheric pressure non-thermal plasma has been introduced in various applications such as wound healing, sterilization of infected tissues, blood coagulation, delicate surgeries, and so on. The non-thermal plasma generates reactive oxygen species (ROS), including ozone. Various groups have reported that the produced ROS influence proliferation and differentiation of cells, as well as apoptosis and growth arrest of tumor cells. In this study, we investigated the effects of non-thermal plasma on rheological characteristics of red blood cells (RBC). We experimentally measured the extent of hemolysis, deformability, and aggregation of red blood cells (RBC) with respect to exposure times of non-thermal plasma. RBC morphology was also examined using field-emission scanning electron microscopy. The absorbance of hemoglobin released from the RBCs increased with increasing exposure time of the non-thermal plasma. Values of the elongation index and aggregation index were shown to decrease significantly with increasing plasma exposure times. Therefore, hemorheological properties of RBCs could be utilized to assess the performance of various non-thermal plasmas.

  5. Altered permeability barrier structure in cholesteatoma matrix

    DEFF Research Database (Denmark)

    Svane-Knudsen, Viggo; Halkier-Sørensen, Lars; Rasmussen, Gurli

    2002-01-01

    The stratum corneum of the cholesteatoma epithelium comprises the greater part of the cholesteatoma matrix. The permeability barrier that militates against diffusion and penetration of infectious and toxic agents into and through the epithelium is situated here. The multiple long sheets of lamellar...... lipid structures filling the intercellular spaces mainly control the barrier function. The barrier in cholesteatoma epithelium is several times thicker than in unaffected skin but presents distinctive features of a defective barrier as seen in other scaling skin diseases. The intercellular spaces appear...

  6. Alteration of blood-brain barrier integrity by retroviral infection.

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    Philippe V Afonso

    2008-11-01

    Full Text Available The blood-brain barrier (BBB, which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans, both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

  7. Alterations of blood brain barrier function in hyperammonemia: an overview.

    Science.gov (United States)

    Skowrońska, Marta; Albrecht, Jan

    2012-02-01

    Ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia, including hepatic encephalopathy, a condition associated with acute--(ALF) or chronic liver failure. This article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells, ammonia influences the passage of different molecules across the blood brain barrier (BBB). A brief description is provided of the tight junctions, which couple adjacent cerebral capillary endothelial cells to each other to form the barrier. Ammonia modulates the transcellular passage of low-to medium-size molecules, by affecting their carriers located at the BBB. Ammonia induces interrelated aberrations of the transport of the large neutral amino acids and aromatic amino acids (AAA), whose influx is augmented by exchange with glutamine produced in the course of ammonia detoxification, and maybe also modulated by the extracellularly acting gamma-glutamyl moiety transferring enzyme, gamma-glutamyl-transpeptidase. Impaired AAA transport affects neurotransmission by altering intracerebral synthesis of catecholamines (serotonin and dopamine), and producing "false neurotransmitters" (octopamine and phenylethylamine). Ammonia also modulates BBB transport of the cationic amino acids: the nitric oxide precursor, arginine, and ornithine, which is an ammonia trap, and affects the transport of energy metabolites glucose and creatine. Moreover, ammonia acting either directly or in synergy with liver injury-derived inflammatory cytokines also evokes subtle increases of the transcellular passage of molecules of different size (BBB "leakage"), which appears to be responsible for the vasogenic component of cerebral edema associated with ALF.

  8. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  9. Plastic Schottky barrier solar cells

    Science.gov (United States)

    Waldrop, James R.; Cohen, Marshall J.

    1984-01-24

    A photovoltaic cell structure is fabricated from an active medium including an undoped, intrinsically p-type organic semiconductor comprising polyacetylene. When a film of such material is in rectifying contact with a magnesium electrode, a Schottky-barrier junction is obtained within the body of the cell structure. Also, a gold overlayer passivates the magnesium layer on the undoped polyacetylene film.

  10. Opiates Upregulate Adhesion Molecule Expression in Brain MicroVascular Endothelial Cells (BMVEC: Implications for Altered Blood Brain Barrier (BBB Permeability

    Directory of Open Access Journals (Sweden)

    Madhavan P.N. Nair

    2006-01-01

    Full Text Available The blood-brain barrier (BBB is an intricate cellular system composed of vascular endothelial cells and perivascular astrocytes that restrict the passage of immunocompetent cells into the central nervous system (CNS. Expression of the adhesion molecules, intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 on brain microvascular endothelial cells (BMVEC and their interaction with human immunodeficiency virus (HIV-1 viral proteins may help enhance viral adhesion and virus-cell fusion resulting in increased infectivity. Additionally, transmigration through the BBB is facilitated by both endothelial and monocyte/macrophage-derived nitric oxide (NO. Dysregulated production of NO by BMVEC due to opiates and HIV-1 viral protein interactions play a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB. Opioids act as co-factors in the neuropathogenesis of HIV-1 by facilitating BBB dysfunction however, no studies have been done to investigate the role of opiates alone or in combination with HIV-1 viral proteins on adhesion molecule expression in BMVEC. We hypothesize that opiates such as heroin and morphine in conjunction with the HIV-1 viral protein gp120 increase the expression of adhesion molecules ICAM-1 and VCAM-1 and these effects are mediated via the modulation of NO. Results show that opiates alone and in synergy with gp120 increase both the genotypic and phenotypic expression of ICAM-1 and VCAM-1 by BMVEC, additionally, these opiate induced effects may be the result of increased NO production. These studies will provide a better understanding of how opiate abuse in conjunction with HIV-1 infection facilitates the breakdown of the BBB and exacerbates the neuropathogenesis of HIV-1. Elucidation of the mechanisms of BBB modulation will provide new therapeutic approaches to maintain BBB integrity

  11. Alterations of intestinal barrier and microbiota in chronic kidney disease.

    Science.gov (United States)

    Sabatino, Alice; Regolisti, Giuseppe; Brusasco, Irene; Cabassi, Aderville; Morabito, Santo; Fiaccadori, Enrico

    2015-06-01

    Recent studies have highlighted the close relationship between the kidney and the gastrointestinal (GI) tract--frequently referred to as the kidney--gut axis--in patients with chronic kidney disease (CKD). In this regard, two important pathophysiological concepts have evolved: (i) production and accumulation of toxic end-products derived from increased bacterial fermentation of protein and other nitrogen-containing substances in the GI tract, (ii) translocation of endotoxins and live bacteria from gut lumen into the bloodstream, due to damage of the intestinal epithelial barrier and quantitative/qualitative alterations of the intestinal microbiota associated with the uraemic milieu. In both cases, these gut-centred alterations may have relevant systemic consequences in CKD patients, since they are able to trigger chronic inflammation, increase cardiovascular risk and worsen uraemic toxicity. The present review is thus focused on the kidney-gut axis in CKD, with special attention to the alterations of the intestinal barrier and the local microbiota (i.e. the collection of microorganisms living in a symbiotic coexistence with their host in the intestinal lumen) and their relationships to inflammation and uraemic toxicity in CKD. Moreover, we will summarize the most important clinical data suggesting the potential for nutritional modulation of gut-related inflammation and intestinal production of noxious by-products contributing to uraemic toxicity in CKD patients.

  12. Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

    Science.gov (United States)

    Kim, Jin Hyoung; Hossain, Ferdaus Mohd Altaf; Patil, Ajit Mahadev; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Park, Sang-Youel; Lee, John-Hwa; Kim, Bumseok; Kim, Koanhoi; Eo, Seong Kug

    2016-10-01

    Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules.

  13. Alterations of Blood Brain Barrier Function in Hyperammonemia: An Overview

    OpenAIRE

    2011-01-01

    Ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia, including hepatic encephalopathy, a condition associated with acute—(ALF) or chronic liver failure. This article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells, ammonia influences the passage of different molecules across the blood brain barrier (BBB). A brief description is provided of the tight junctions, which c...

  14. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

    Science.gov (United States)

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M O; Rajan, Binoy; Tinsley, John W; Bickerdike, Ralph; Martin, Samuel A M; Bowman, Alan S

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  15. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein

    Science.gov (United States)

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M. O.; Rajan, Binoy; Tinsley, John W.; Bickerdike, Ralph

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts. PMID:28046109

  16. Plastic Schottky-barrier solar cells

    Science.gov (United States)

    Waldrop, J.R.; Cohen, M.J.

    1981-12-30

    A photovoltaic cell structure is fabricated from an active medium including an undoped polyacetylene, organic semiconductor. When a film of such material is in rectifying contact with a metallic area electrode, a Schottky-barrier junction is obtained within the body of the cell structure. Also, a gold overlayer passivates a magnesium layer on the undoped polyacetylene film. With the proper selection and location of elements a photovoltaic cell structure and solar cell are obtained.

  17. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

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    Ferranda Puig

    Full Text Available Acute lung injury (ALI is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC on mechanical tension and barrier integrity in human alveolar epithelial cells (A549 exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml or vehicle (control. Subsequently, thrombin (50 nM or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  18. Alterations of intestinal mucosa structure and barrier function following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    Chun-Hua Hang; Ji-Xin Shi; Jie-Shou Li; Wei Wu; Hong-Xia Yin

    2003-01-01

    AIM: Gastrointestinal dysfunction is a common complication in patients with traumatic brain injury (TBI). However, the effect of traumatic brain injury on intestinal mucosa has not been studied previously. The aim of the current study was to explore the alterations of intestinal mucosa morphology and barrier function, and to determine how rapidly the impairment of gut barrier function occurs and how long it persists following traumatic brain injury.METHODS: Male Wistar rats were randomly divided into six groups (6 rats each group) including controls without brain injury and traumatic brain injury groups at hours 3,12, 24, and 72, and on day 7. The intestinal mucosa structure was detected by histopathological examination and electron microscopy. Gut barrier dysfunction was evaluated by detecting serum endotoxin and intestinal permeability. The level of serum endotoxin and intestinal permeability was measured by using chromogenic limulus amebocyte lysate and lactulose/mannitol (L/M) ratio, respectively.RESULTS: After traumatic brain injury, the histopathological alterations of gut mucosa occurred rapidly as early as 3 hours and progressed to a serious state, including shedding of epithelial cells, fracture of villi, focal ulcer, fusion of adjacent villi, dilation of central chyle duct, mucosal atrophy,and vascular dilation, congestion and edema in the villous interstitium and lamina propria. Apoptosis of epithelial cells,fracture and sparseness of microvilli, loss of tight junction between enterocytes, damage of mitochondria and endoplasm, were found by electron microscopy. The villous height, crypt depth and surface area in jejunum decreased progressively with the time of brain injury. As compared with that of control group (183.7±41.8 EU/L), serum endotoxin level was signnificantly increased at 3, 12, and 24 hours following TBI (434.8±54.9 EU/L, 324.2±61.7 EU/L and 303.3±60.2 EU/L, respectively), and peaked at 72 hours (560.5±76.2 EU/L), then declined on day 7

  19. Entamoeba histolytica contains an occludin-like protein that can alter colonic epithelial barrier function.

    Science.gov (United States)

    Goplen, Michael; Lejeune, Manigandan; Cornick, Steve; Moreau, France; Chadee, Kris

    2013-01-01

    The exact mechanism by which Entamoeba histolytica disrupts the human colonic epithelium and invades the mucosa has yet to be clearly elucidated. E. histolytica produces a diverse array of putative virulent factors such as glycosidase, cysteine proteinases and amebapore that can modulate and/or disrupt epithelial barrier functions. However, it is currently thought that E. histolytica produces numerous other molecules and strategies to disrupt colonic mucosal defenses. In this study, we document a putative mechanism whereby the parasite alters the integrity of human epithelium by expressing a cognate tight junction protein of the host. We detected this protein as "occludin-like" as revealed by immunoblotting and immunoprecipitation studies and visualization by confocal microscopy using antibodies highly specific for human occludin. We propose that E. histolytica occludin-like protein might displace mucosal epithelial occludin-occludin tight junction interactions resulting in epithelial disruption analogous to sub mucosal human dendritic cells sampling luminal contents. These results indicate that E. histolytica occludin is a putative virulent component that can play a role in the pathogenesis of intestinal amebiasis.

  20. Blood-Brain Barrier Alterations Provide Evidence of Subacute Diaschisis in an Ischemic Stroke Rat Model

    Science.gov (United States)

    Garbuzova-Davis, Svitlana; Rodrigues, Maria C. O.; Hernandez-Ontiveros, Diana G.; Tajiri, Naoki; Frisina-Deyo, Aric; Boffeli, Sean M.; Abraham, Jerry V.; Pabon, Mibel; Wagner, Andrew; Ishikawa, Hiroto; Shinozuka, Kazutaka; Haller, Edward; Sanberg, Paul R.; Kaneko, Yuji; Borlongan, Cesario V.

    2013-01-01

    Background Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB) competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas. Methodology/Principal Findings In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO), significant BBB alterations characterized by large Evans Blue (EB) parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices. Conclusions/Significance These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke. PMID:23675488

  1. Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model.

    Directory of Open Access Journals (Sweden)

    Svitlana Garbuzova-Davis

    Full Text Available BACKGROUND: Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas. METHODOLOGY/PRINCIPAL FINDINGS: In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO, significant BBB alterations characterized by large Evans Blue (EB parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices. CONCLUSIONS/SIGNIFICANCE: These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke.

  2. Graphene applications in Schottky barrier solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Lancellotti, L., E-mail: laura.lancellotti@enea.it [ENEA Research Centre Portici, Piazzale E. Fermi 1, 80055 Portici (Napoli) (Italy); Polichetti, T.; Ricciardella, F. [ENEA Research Centre Portici, Piazzale E. Fermi 1, 80055 Portici (Napoli) (Italy); Tari, O., E-mail: orlando.tari@unina.it [University of Naples ' Federico II' , Dept. of Electronic Engineering, Via Claudio 21, 80125 Napoli (Italy); Gnanapragasam, S. [ENEA Research Centre Portici, Piazzale E. Fermi 1, 80055 Portici (Napoli) (Italy); Daliento, S. [University of Naples ' Federico II' , Dept. of Electronic Engineering, Via Claudio 21, 80125 Napoli (Italy); Di Francia, G. [ENEA Research Centre Portici, Piazzale E. Fermi 1, 80055 Portici (Napoli) (Italy)

    2012-11-01

    We report a theoretical study about the performances of graphene on semiconductor Schottky barrier solar cells with the aim to show the potentiality of this kind of device. The simulations are carried by a generalized equivalent circuit model, where the circuital parameters are strictly dependent on the physical properties of the graphene and semiconductor which form the Schottky junction. We have realized graphene samples and characterized them by optical and atomic force microscopy, and Raman spectroscopy. Capacitance-voltage measurements have been made on some 'ad hoc' graphene based devices in order to obtain graphene workfunction, a very essential physical parameter. The estimated value is compatible with four layer graphene. This result is in agreement with the morphological characterizations of our material. - Highlights: Black-Right-Pointing-Pointer An equivalent circuit model simulates graphene based Schottky barrier solar cells. Black-Right-Pointing-Pointer Graphene flakes are identified through Raman spectroscopy and Atomic Force Microscopy. Black-Right-Pointing-Pointer Workfunction estimation by Capacitance-Voltage (C-V) on graphene based devices Black-Right-Pointing-Pointer A multilayered structure is evidenced by morphological and C-V characterization.

  3. Enteric Pathogens and Their Toxin-Induced Disruption of the Intestinal Barrier through Alteration of Tight Junctions in Chickens

    Directory of Open Access Journals (Sweden)

    Wageha A. Awad

    2017-02-01

    Full Text Available Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing bird’s health. The intestinal epithelial barrier serves as the first line of defense between the host and the luminal environment. It consists of a continuous monolayer of intestinal epithelial cells connected by intercellular junctional complexes which shrink the space between adjacent cells. Consequently, free passing of solutes and water via the paracellular pathway is prevented. Tight junctions (TJs are multi-protein complexes which are crucial for the integrity and function of the epithelial barrier as they not only link cells but also form channels allowing permeation between cells, resulting in epithelial surfaces of different tightness. Tight junction’s molecular composition, ultrastructure, and function are regulated differently with regard to physiological and pathological stimuli. Both in vivo and in vitro studies suggest that reduced tight junction integrity greatly results in a condition commonly known as “leaky gut”. A loss of barrier integrity allows the translocation of luminal antigens (microbes, toxins via the mucosa to access the whole body which are normally excluded and subsequently destroys the gut mucosal homeostasis, coinciding with an increased susceptibility to systemic infection, chronic inflammation and malabsorption. There is considerable evidence that the intestinal barrier dysfunction is an important factor contributing to the pathogenicity of some enteric bacteria. It has been shown that some enteric pathogens can induce permeability defects in gut epithelia by altering tight junction proteins, mediated by their toxins. Resolving the strategies that microorganisms use to hijack the functions of tight junctions is important for our understanding of microbial pathogenesis

  4. Enteric Pathogens and Their Toxin-Induced Disruption of the Intestinal Barrier through Alteration of Tight Junctions in Chickens

    Science.gov (United States)

    Awad, Wageha A.; Hess, Claudia; Hess, Michael

    2017-01-01

    Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing bird’s health. The intestinal epithelial barrier serves as the first line of defense between the host and the luminal environment. It consists of a continuous monolayer of intestinal epithelial cells connected by intercellular junctional complexes which shrink the space between adjacent cells. Consequently, free passing of solutes and water via the paracellular pathway is prevented. Tight junctions (TJs) are multi-protein complexes which are crucial for the integrity and function of the epithelial barrier as they not only link cells but also form channels allowing permeation between cells, resulting in epithelial surfaces of different tightness. Tight junction’s molecular composition, ultrastructure, and function are regulated differently with regard to physiological and pathological stimuli. Both in vivo and in vitro studies suggest that reduced tight junction integrity greatly results in a condition commonly known as “leaky gut”. A loss of barrier integrity allows the translocation of luminal antigens (microbes, toxins) via the mucosa to access the whole body which are normally excluded and subsequently destroys the gut mucosal homeostasis, coinciding with an increased susceptibility to systemic infection, chronic inflammation and malabsorption. There is considerable evidence that the intestinal barrier dysfunction is an important factor contributing to the pathogenicity of some enteric bacteria. It has been shown that some enteric pathogens can induce permeability defects in gut epithelia by altering tight junction proteins, mediated by their toxins. Resolving the strategies that microorganisms use to hijack the functions of tight junctions is important for our understanding of microbial pathogenesis, because some pathogens

  5. Hematotesticular barrier is altered from early stages of liver cirrhosis:Effect of insulin-like growth factor 1

    Institute of Scientific and Technical Information of China (English)

    Inma Castilla-Cortázar; Isabel Varela-Nieto; Jesús Prieto; Salvador González-Barón; Nieves Diez; María García-Fernández; Juan Enrique Puche; Fernando Diez-Caballero; Jorge Quiroga; Matías Díaz-Sánchez; Alberto Castilla; Amelia Díaz Casares

    2004-01-01

    AIM: The pathogenesis of hypogonadism in liver cirrhosis is not well understood. Previous results from our laboratory showed that IGF-1 deficiency might play a pathogenetic role in hypogonadism of cirrhosis. The administration of IGF-1 for a short period of time reverted the testicular atrophy associated with advanced experimental cirrhosis.The aim of this study was to establish the historical progression of the described alterations in the testes,explore testicular morphology, histopathology, cellular proliferation, integrity of testicular barrier and hypophysogonadal axis in rats with no ascitic cirrhosis.METHODS: Male Wistar rats with histologically-proven cirrhosis induced with carbon tetrachloride (CCl4) for 11 wk,were allocated into two groups (n = 12, each) to receive vehicle. Healthy rats receiving vehicle were used as control group (n = 12).RESULTS: Compared to controls, rats with compensated cirrhosis showed a normal testicular size and weight and very few histopathological testicular abnormalities.However, these animals showed a significant diminution of cellular proliferation and a reduction of testicular transferrin expression. In addition, pituitary-gonadal axis was altered, with significant higher levels of FSH (P<0.001vs controls) and increased levels of LH in untreated cirrhotic animals. Interestingly, IGF-1 treatment normalized testicular transferrin expression and cellular proliferation and reduced serum levels of LH (P = ns vs controls, and P<0.01 vs untreated cirrhotic group).CONCLUSION: The testicular barrier is altered from an early stage of cirrhosis, shown by a reduction of transferrin expression in Sertoli cells, a diminished cellular proliferation and an altered gonadal axis. The treatment with IGF-1 could be also useful in this initial stage of testicular disorder associated with compensated cirrhosis.

  6. Plant cells in vitro under altered gravity.

    Science.gov (United States)

    Klymchuk, D O

    1998-07-01

    Establishing the role of gravity in plant requires information about how gravity regulates the metabolism of individual cells. Plant cells and tissues in vitro are valuable models for such purpose. Disrupted intercellular relations in such models have allowed to elucidate both the gravity role in non-specialised to gravity plant cells and the correlative relation role of an intact plant organism. The data obtained from non-numerous space and clinostat experiments with plant cells in vitro have demonstrated that their metabolism is sensitive to g-environment. The most experiments have shown a decrease in the biomass production and cell proliferation of spaceflight samples compared with ground controls, although there is study reporting of increased biomass production in an anise suspension culture and D. carota crown gall tissue culture. At the same time, results of experiments with single carrot cells and tomato callus culture demonstrated similarities in differentiation process in microgravity and in ground controls. Noted ultrastructural arrangement in cells, especially mitochondria and plastids, have been related to altered energy load and functions of organelles in microgravity, as well as changes in the lipid peroxidation and the content of malonic dyaldehyde in a haplopappus tissue culture under altered gravity supposed with modification of membrane structural-functional state. This article focuses on growth aspects of the cultured cells in microgravity and under clinostat conditions and considers those aspects that require further analysis.

  7. Contemporary human-altered landscapes and oceanic barriers reduce bumble bee gene flow.

    Science.gov (United States)

    Jha, S

    2015-03-01

    Much of the world's terrestrial landscapes are being altered by humans in the form of agriculture, urbanization and pastoral systems, with major implications for biodiversity. Bumble bees are one of the most effective pollinators in both natural and cultivated landscapes, but are often the first to be extirpated in human-altered habitats. Yet, little is known about the role of natural and human-altered habitats in promoting or limiting bumble bee gene flow. In this study, I closely examine the genetic structure of the yellow-faced bumble bee, Bombus vosnesenskii, across the southwestern US coast and find strong evidence that natural oceanic barriers, as well as contemporary human-altered habitats, limit bee gene flow. Heterozygosity and allelic richness were lower in island populations, while private allelic richness was higher in island populations compared to mainland populations. Genetic differentiation, measured for three indices across the 1000 km study region, was significantly greater than the null expectation (F(ST) = 0.041, F'(ST) = 0.044 and D(est) = 0.155) and correlated with geographic distance. Furthermore, genetic differentiation patterns were most strongly correlated with contemporary (2011) not past (2006, 2001) resistance maps calibrated for high dispersal limitation over oceans, impervious habitat and croplands. Despite the incorporation of dramatic elevation gradients, the analyses reveal that oceans and contemporary human land use, not mountains, are the primary dispersal barriers for B. vosnesenskii gene flow. These findings reinforce the importance of maintaining corridors of suitable habitat across the distribution range of native pollinators to promote their persistence and safeguard their ability to provide essential pollination services.

  8. Functional and structural alterations of epithelial barrier properties of rat ileum following X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Dublineau, I. [Inst. de Radioprotection et de Surete Nucleaire (IRSN), Direction de la RadioProtection de l' Homme, Service de Radiobiologie et d' Epidemiologie, Fontenay-aux-Roses, CEDEX (France)]. E-mail: isabelle.dublineau@irsn.fr; Lebrun, F. [Commissariat a l' Energie Atomique (CEA), Dept. de Radiopathologie et de Radiobiologie, Fontenay-aux-Roses, CEDEX (France); Grison, S.; Griffiths, N.M. [Inst. de Radioprotection et de Surete Nucleaire (IRSN), Direction de la RadioProtection de l' Homme, Service de Radiobiologie et d' Epidemiologie, Fontenay-aux-Roses, CEDEX (France)

    2004-02-01

    Irradiation of the digestive system leads to alterations of the small intestine. We have characterized the disruption of the barrier integrity in rat ileum from 1 to 14 days following irradiation ranging from 6 to 12 Gy. The intestinal permeability to {sup 14}C-mannitol and {sup 3}H-dextran 70,000 was measured in vitro in Ussing chambers. In parallel to these functional studies, immunohistochemical analyses of junctional proteins (ZO-1 and {beta}-catenin) of ileal epithelium were performed by confocal microscopy. Irradiation with 10 Gy induced a marked decrease in epithelial tissue resistance at three days and a fivefold increase in mannitol permeability, without modifications of dextran permeability. A disorganization of the localization for ZO-1 and {beta}-catenin was also observed. At 7 days after irradiation, we observed a recovery of the organization of junctional proteins in parallel to a return of intestinal permeability to control value. In addition to these time-dependent effects, a gradual effect on epithelial integrity of the radiation doses was observed 3 days after irradiation. This study shows a disruption of the integrity of the intestinal barrier in rat ileum following abdominal X-irradiation, depending on the time postirradiation and on the delivered dose. The loss of barrier integrity was characterized by a disorganization of proteins of tight and adherent junctions, leading to increased intestinal permeability to mannitol. (author)

  9. Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy.

    Directory of Open Access Journals (Sweden)

    Sampathkumar Rangasamy

    Full Text Available Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB. We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2 was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/- mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

  10. Radiofrequency treatment alters cancer cell phenotype

    Science.gov (United States)

    Ware, Matthew J.; Tinger, Sophia; Colbert, Kevin L.; Corr, Stuart J.; Rees, Paul; Koshkina, Nadezhda; Curley, Steven; Summers, H. D.; Godin, Biana

    2015-07-01

    The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment.

  11. Metabolic alterations in renal cell carcinoma.

    Science.gov (United States)

    Massari, Francesco; Ciccarese, Chiara; Santoni, Matteo; Brunelli, Matteo; Piva, Francesco; Modena, Alessandra; Bimbatti, Davide; Fantinel, Emanuela; Santini, Daniele; Cheng, Liang; Cascinu, Stefano; Montironi, Rodolfo; Tortora, Giampaolo

    2015-11-01

    Renal cell carcinoma (RCC) is a metabolic disease, being characterized by the dysregulation of metabolic pathways involved in oxygen sensing (VHL/HIF pathway alterations and the subsequent up-regulation of HIF-responsive genes such as VEGF, PDGF, EGF, and glucose transporters GLUT1 and GLUT4, which justify the RCC reliance on aerobic glycolysis), energy sensing (fumarate hydratase-deficient, succinate dehydrogenase-deficient RCC, mutations of HGF/MET pathway resulting in the metabolic Warburg shift marked by RCC increased dependence on aerobic glycolysis and the pentose phosphate shunt, augmented lipogenesis, and reduced AMPK and Krebs cycle activity) and/or nutrient sensing cascade (deregulation of AMPK-TSC1/2-mTOR and PI3K-Akt-mTOR pathways). We analyzed the key metabolic abnormalities underlying RCC carcinogenesis, highlighting those altered pathways that may represent potential targets for the development of more effective therapeutic strategies.

  12. Moderate dietary protein restriction alters the composition of gut microbiota and improves ileal barrier function in adult pig model

    Science.gov (United States)

    Fan, Peixin; Liu, Ping; Song, Peixia; Chen, Xiyue; Ma, Xi

    2017-01-01

    This study was conducted to investigate impacts of dietary protein levels on gut bacterial community and gut barrier. The intestinal microbiota of finishing pigs, fed with 16%, 13% and 10% crude protein (CP) in diets, respectively, were investigated using Illumina MiSeq sequencing. The ileal bacterial richness tended to decrease when the dietary protein concentration reduced from 16% to 10%. The proportion of Clostridium_sensu_stricto_1 in ileum significantly decreased, whereas Escherichia-Shigella increased with reduction of protein concentration. In colon, the proportion of Clostridium_sensu_stricto_1 and Turicibacter increased, while the proportion of RC9_gut_group significantly decreased with the dietary protein reduction. Notably, the proportion of Peptostreptococcaceae was higher in both ileum and colon of 13% CP group. As for metabolites, the intestinal concentrations of SCFAs and biogenic amines decreased with the dietary protein reduction. The 10% CP dietary treatment damaged ileal mucosal morphology, and decreased the expression of biomarks of intestinal cells (Lgr5 and Bmi1), whereas the expression of tight junction proteins (occludin and claudin) in 13% CP group were higher than the other two groups. In conclusion, moderate dietary protein restriction (13% CP) could alter the bacterial community and metabolites, promote colonization of beneficial bacteria in both ileum and colon, and improve gut barrier function. PMID:28252026

  13. Contributions of altered permeability of intestinal barrier and defecation behavior to toxicity formation from graphene oxide in nematode Caenorhabditis elegans

    Science.gov (United States)

    Wu, Qiuli; Yin, Li; Li, Xing; Tang, Meng; Zhang, Tao; Wang, Dayong

    2013-09-01

    Graphene oxide (GO) has been extensively studied for potential biomedical applications. Meanwhile, potential GO toxicity arises in both biomedical applications and non-biomedical products where environmental exposures may occur. In the present study, we examined the potential adverse effects of GO and the underlying mechanism using nematode Caenorhabditis elegans as the assay system. We compared the in vivo effects of GO between acute exposure and prolonged exposure, and found that prolonged exposure to 0.5-100 mg L-1 of GO caused damage on functions of both primary (intestine) and secondary (neuron and reproductive organ) targeted organs. In the intestine, ROS production was significantly correlated with the formation of adverse effects on functions of both primary and secondary targeted organs. GO could be translocated into intestinal cells with loss of microvilli, and distributed to be adjacent to or surrounding mitochondria. Prolonged exposure to GO resulted in a hyper-permeable state of the intestinal barrier, an increase in mean defecation cycle length, and alteration of genes required for intestinal development and defecation behavior. Thus, our data suggest that prolonged exposure to GO may cause potential risk to environmental organisms after release into the environment. GO toxicity may be due to the combinational effects of oxidative stress in the intestinal barrier, enhanced permeability of the biological barrier, and suppressed defecation behavior in C. elegans.Graphene oxide (GO) has been extensively studied for potential biomedical applications. Meanwhile, potential GO toxicity arises in both biomedical applications and non-biomedical products where environmental exposures may occur. In the present study, we examined the potential adverse effects of GO and the underlying mechanism using nematode Caenorhabditis elegans as the assay system. We compared the in vivo effects of GO between acute exposure and prolonged exposure, and found that prolonged

  14. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  15. Altered calcium signaling in cancer cells.

    Science.gov (United States)

    Stewart, Teneale A; Yapa, Kunsala T D S; Monteith, Gregory R

    2015-10-01

    It is the nature of the calcium signal, as determined by the coordinated activity of a suite of calcium channels, pumps, exchangers and binding proteins that ultimately guides a cell's fate. Deregulation of the calcium signal is often deleterious and has been linked to each of the 'cancer hallmarks'. Despite this, we do not yet have a full understanding of the remodeling of the calcium signal associated with cancer. Such an understanding could aid in guiding the development of therapies specifically targeting altered calcium signaling in cancer cells during tumorigenic progression. Findings from some of the studies that have assessed the remodeling of the calcium signal associated with tumorigenesis and/or processes important in invasion and metastasis are presented in this review. The potential of new methodologies is also discussed. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

  16. Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility.

    Science.gov (United States)

    Cipolat, Sara; Hoste, Esther; Natsuga, Ken; Quist, Sven R; Watt, Fiona M

    2014-05-05

    Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults.DOI: http://dx.doi.org/10.7554/eLife.01888.001.

  17. Cell proliferation alterations in Chlorella cells under stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: cid@udc.es [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)

    2009-09-14

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  18. C5a alters blood-brain barrier integrity in a human in vitro model of systemic lupus erythematosus.

    Science.gov (United States)

    Mahajan, Supriya D; Parikh, Neil U; Woodruff, Trent M; Jarvis, James N; Lopez, Molly; Hennon, Teresa; Cunningham, Patrick; Quigg, Richard J; Schwartz, Stanley A; Alexander, Jessy J

    2015-09-01

    The blood-brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5-50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor-κB translocation into nucleus and regulates the expression of the tight junction proteins, claudin-5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

  19. Hyperammonemia,brain edema and blood-brain barrier alterations in prehepatic portal hypertensive rats and paravrtamol intoxication

    Institute of Scientific and Technical Information of China (English)

    Camila Scorticati; Juan P. Prestifilippo; Francisco X. Eizayaga; José L. Castro; Salvador Romay; Maria A. Fernández; Abraham Lemberg; Juan C. Perazzo

    2004-01-01

    AIM: To study the blood-brain barrier integrity, brain edema,animal behavior and ammonia plasma levels in prehepatic portal hypertensive rats with and without acute liver intoxication.METHODS: Adults male Wistar rats were divided into four groups. Group Ⅰ: sham operation; Ⅱ: Prehepatic portal hypertension, produced by partial portal vein ligation; Ⅲ:Acetaminophen intoxication and Ⅳ: Prehepatic portal hypertension plus acetaminophen. Acetaminophen was administered to produce acute hepatic injury. Portal pressure, liver serum enzymes and ammonia plasma levels were determined. Brain cortex water content was registered and trypan blue was utilized to study blood brain barrier integrity. Reflexes and behavioral tests were recorded.RESULTS: Portal hypertension was significantly elevated in groups Ⅱ and Ⅳ. Liver enzymes and ammonia plasma levels were increased in groups Ⅱ, Ⅳ and Ⅳ. Prehepatic portal hypertension (group Ⅱ), acetaminophen intoxication (group Ⅲ) and both (group Ⅳ) had changes in the blood brain-barrier integrity (trypan blue) and hyperammonemia. Cortical edema was present in rats with acute hepatic injury in groups Ⅲ and Ⅳ. Behavioral test (rota rod) was altered in group Ⅳ.CONCLUSION: These results suggest the possibility of another pathway for cortical edema production because blood brain barrier was altered (vasogenic) and hyperammonemia was registered (cytotoxic). Group Ⅳ, with behavioral altered test, can be considered as a model for study at an early stage of portal-systemic encephalopathy.

  20. Altered blood-brain barrier permeability in rats with prehepatic portal hypertension turns to normal when portal pressure is lowered

    Institute of Scientific and Technical Information of China (English)

    Francisco Eizayaga; Camila Scorticati; Juan P Prestifilippo; Salvador Romay; Maria A Fernandez; José L Castro; Abraham Lemberg; Juan C Perazzo

    2006-01-01

    AIM: To study the blood-brain barrier integrity in prehepatic portal hypertensive rats induced by partial portal vein ligation, at 14 and 40 d after ligation when portal pressure is spontaneously normalized.METHODS: Adult male Wistar rats were divided into four groups: Group Ⅰ: Sham14d, sham operated; Group Ⅱ: PH14d, portal vein stenosis; (both groups were used 14 days after surgery); Group Ⅲ: Sham40d, Sham operated and Group Ⅳ: PH40d Portal vein stenosis (Groups Ⅱ and Ⅳ used 40 d after surgery). Plasma ammonia,plasma and cerebrospinal fluid protein and liver enzymes concentrations were determined. Trypan and Evans blue dyes, systemically injected, were investigated in hippocampus to study blood-brain barrier integrity. Portal pressure was periodically recorded.RESULTS: Forty days after stricture, portal pressure was normalized, plasma ammonia was moderately high,and both dyes were absent in central nervous system parenchyma. All other parameters were reestablished.When portal pressure was normalized and ammonia level was lowered, but not normal, the altered integrity of blood-brain barrier becomes reestablished.CONCLUSION: The impairment of blood-brain barrier and subsequent normalization could be a mechanism involved in hepatic encephalopathy reversibility. Hemodynamic changes and ammonia could trigger blood-brain barrier alterations and its reestablishment.

  1. Dietary inulin alters the intestinal absorptive and barrier function of piglet intestine after weaning.

    Science.gov (United States)

    Awad, Wageha A; Ghareeb, Khaled; Paßlack, Nadine; Zentek, Jürgen

    2013-08-01

    , suggesting that the inulin supplementation increased the electrogenic transport of glucose across the jejunal mucosa of weaned piglets indicating higher glucose absorption. Furthermore, the Gt was higher in the inulin-supplemented weaned piglets than in control piglets, which could be due to the increased paracellular permeability to Na(+). In conclusion, dietary inulin increased the glucose transport and altered the intestinal barrier by increasing the intestinal permeability in the jejunal mucosa of post-weaned piglets. Furthermore, the results indicated that inulin has a positive effect on glucose absorption in the piglet small intestine after weaning and subsequently the dietary inulin offers a promising approach to avoid post-weaning gastrointestinal tract disorders in pigs.

  2. InGaP Heterojunction Barrier Solar Cells

    Science.gov (United States)

    Welser, Roger E. (Inventor)

    2014-01-01

    A new solar cell structure called a heterojunction barrier solar cell is described. As with previously reported quantum-well and quantum-dot solar cell structures, a layer of narrow band-gap material, such as GaAs or indium-rich InGaP, is inserted into the depletion region of a wide band-gap PN junction. Rather than being thin, however, the layer of narrow band-gap material is about 400-430 nm wide and forms a single, ultrawide well in the depletion region. Thin (e.g., 20-50 nm), wide band-gap InGaP barrier layers in the depletion region reduce the diode dark current. Engineering the electric field and barrier profile of the absorber layer, barrier layer, and p-type layer of the PN junction maximizes photogenerated carrier escape. This new twist on nanostructured solar cell design allows the separate optimization of current and voltage to maximize conversion efficiency.

  3. Recent key technical barriers in solid oxide fuel cell technology

    Directory of Open Access Journals (Sweden)

    Milewski Jarosław

    2014-03-01

    Full Text Available High-temperature solid oxide fuel cells (SOFCs are considered as suitable components of future large-scale clean and efficient power generation systems. However, at its current stage of development some technical barriers exists which limit SOFC’s potential for rapid large-scale deployment. The present article aims at providing solutions to key technical barriers in SOFC technology. The focus is on the solutions addressing thermal resistance, fuel reforming, energy conversion efficiency, materials, design, and fuel utilisation issues.

  4. Deciding Which Way to Go: How Do Insects alter Movements to Negotiate Barriers?

    Directory of Open Access Journals (Sweden)

    Roy E. Ritzmann

    2012-07-01

    Full Text Available Animals must routinely deal with barriers as they move through their natural environment. These challenges require directed changes in leg movements and posture performed in the context of ever changing internal and external conditions. In particular, cockroaches use a combination of tactile and visual information to evaluate objects in their path in order to effectively guide their movements in complex terrain. When encountering a large block, the insect uses its antennae to evaluate the object’s height then rears upward accordingly before climbing. A shelf presents a choice between climbing and tunneling that depends on how the antennae strike the shelf; tapping from above yields climbing, while tapping from below causes tunneling. However, ambient light conditions detected by the ocelli can bias that decision. Similarly, in a T-maze turning is determined by antennal contact but influenced by visual cues. These multi-sensory behaviors led us to look at the central complex as a center for sensori-motor integration within the insect brain. Visual and antennal tactile cues are processed within the central complex and, in tethered preparations, several central complex units changed firing rates in tandem with or prior to altered step frequency or turning, while stimulation through the implanted electrodes evoked these same behavioral changes. To further test for a central complex role in these decisions, we examined behavioral effects of brain lesions. Electrolytic lesions in restricted regions of the central complex generated site specific behavioral deficits. Similar changes were also found in reversible effects of procaine injections in the brain. Finally, we are examining these kinds of decisions made in a large arena that more closely matches the conditions under which cockroaches forage. Overall, our studies suggest that CC circuits may indeed influence the descending commands associated with navigational decisions, thereby making them

  5. Benefits of agomelatine in behavioral, neurochemical and blood brain barrier alterations in prenatal valproic acid induced autism spectrum disorder.

    Science.gov (United States)

    Kumar, Hariom; Sharma, B M; Sharma, Bhupesh

    2015-12-01

    Valproic acid administration during gestational period causes behavior and biochemical deficits similar to those observed in humans with autism spectrum disorder. Although worldwide prevalence of autism spectrum disorder has been increased continuously, therapeutic agents to ameliorate the social impairment are very limited. The present study has been structured to investigate the therapeutic potential of melatonin receptor agonist, agomelatine in prenatal valproic acid (Pre-VPA) induced autism spectrum disorder in animals. Pre-VPA has produced reduction in social interaction (three chamber social behavior apparatus), spontaneous alteration (Y-Maze), exploratory activity (Hole board test), intestinal motility, serotonin levels (prefrontal cortex and ileum) and prefrontal cortex mitochondrial complex activity (complex I, II, IV). Furthermore, Pre-VPA has increased locomotor activity (actophotometer), anxiety, brain oxidative stress (thiobarbituric acid reactive species, glutathione, and catalase), nitrosative stress (nitrite/nitrate), inflammation (brain and ileum myeloperoxidase activity), calcium levels and blood brain barrier leakage in animals. Treatment with agomelatine has significantly attenuated Pre-VPA induced reduction in social interaction, spontaneous alteration, exploratory activity intestinal motility, serotonin levels and prefrontal cortex mitochondrial complex activity. Furthermore, agomelatine also attenuated Pre-VPA induced increase in locomotion, anxiety, brain oxidative stress, nitrosative stress, inflammation, calcium levels and blood brain barrier leakage. It is concluded that, Pre-VPA has induced autism spectrum disorder, which was attenuated by agomelatine. Agomelatine has shown ameliorative effect on behavioral, neurochemical and blood brain barrier alteration in Pre-VPA exposed animals. Thus melatonin receptor agonists may provide beneficial therapeutic strategy for managing autism spectrum disorder.

  6. Spaceflight alters immune cell function and distribution

    Science.gov (United States)

    Sonnenfeld, Gerald; Mandel, Adrian D.; Konstantinova, Irina V.; Berry, Wallace D.; Taylor, Gerald R.; Lesniak, A. T.; Fuchs, Boris B.; Rakhmilevich, Alexander L.

    1992-01-01

    Experiments are described which were performed onboard Cosmos 2044 to determine spaceflight effects on immunologically important cell function and distribution. Results indicate that bone marrow cells from flown and suspended rats exhibited a decreased response to a granulocyte/monocyte colony-stimulating factor compared with the bone marrow cells from control rats. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleulin-2 receptor bearing pan T- and helper T-cells in the lymphocytic population.

  7. Raman spectroscopic study of a genetically altered kidney cell

    Science.gov (United States)

    Joshi, Joel; Garcia, Francisco; Centeno, Silvia P.; Joshi, N. V.

    2008-02-01

    A Raman spectroscopic investigation of a genetically altered Human Embryonic Kidney Cell (HEK293) along with a pathologically normal cell has been carried out by a conventional method. The genetic alteration was carried out with a standard protocol by using a Green Fluorescence Protein (GFP). Raman spectra show that there are dramatic differences between the spectrum obtained from a genetically altered cell and that obtained from a pathologically normal cell. The former shows three broad bands; meanwhile the latter shows several sharp peaks corresponding to the ring vibrational modes of Phen, GFP and DNA. The present analysis provides an indication that the force field near Phen located at 64, 65 and 66 was altered during the genetic transformation. The Raman spectrum could be a direct experimental evidence for substantial modifications triggered due to the expression of specific genes.

  8. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    During the last decade, cell-penetrating peptides have been investigated for their ability to overcome the plasma membrane barrier of mammalian cells for the intracellular or transcellular delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides......-penetrating peptides as transmembrane drug delivery agents, according to the recent literature, and discusses critical issues and future challenges in relation to fully understanding the fundamental principles of the cell-penetrating peptide-mediated membrane translocation of cargoes and the exploitation......, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell...

  9. Non-neuronal Cells in ALS: Role of Glial, Immune cells and Blood-CNS Barriers.

    Science.gov (United States)

    Puentes, Fabiola; Malaspina, Andrea; van Noort, Johannes M; Amor, Sandra

    2016-03-01

    Neurological dysfunction and motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is strongly associated with neuroinflammation reflected by activated microglia and astrocytes in the CNS. In ALS endogenous triggers in the CNS such as aggregated protein and misfolded proteins activate a pathogenic response by innate immune cells. However, there is also strong evidence for a neuroprotective immune response in ALS. Emerging evidence also reveals changes in the peripheral adaptive immune responses as well as alterations in the blood brain barrier that may aid traffic of lymphocytes and antibodies into the CNS. Understanding the triggers of neuroinflammation is key to controlling neuronal loss. Here, we review the current knowledge regarding the roles of non-neuronal cells as well as the innate and adaptive immune responses in ALS. Existing ALS animal models, in particular genetic rodent models, are very useful to study the underlying pathogenic mechanisms of motor neuron degeneration. We also discuss the approaches used to target the pathogenic immune responses and boost the neuroprotective immune pathways as novel immunotherapies for ALS.

  10. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ailar Nakhlband

    2011-09-01

    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  11. Nylon wool purification alters the activation of T cells.

    Science.gov (United States)

    Wohler, Jillian E; Barnum, Scott R

    2009-02-01

    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method.

  12. Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function

    DEFF Research Database (Denmark)

    Bailey, D M; Evans, K A; James, P E

    2008-01-01

    (2)) and following 6 h passive exposure to hypoxia (12% O(2)). Blood flow velocity in the middle cerebral artery (MCAv) and mean arterial blood pressure (MAP) were measured for determination of CA following calculation of transfer function analysis and rate of regulation (RoR). Nine subjects......We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood-brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia-mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O...

  13. EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

    Science.gov (United States)

    Ding, Gui-Rong; Qiu, Lian-Bo; Wang, Xiao-Wu; Li, Kang-Chu; Zhou, Yong-Chun; Zhou, Yan; Zhang, Jie; Zhou, Jia-Xing; Li, Yu-Rong; Guo, Guo-Zhen

    2010-07-15

    The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.

  14. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  15. Calcium signaling in plant cells in altered gravity

    Science.gov (United States)

    Kordyum, E. L.

    2003-10-01

    Changes in the intracellular Ca 2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca 2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80 th, a review highlighting the performed research and the possible significance of such Ca 2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface

  16. Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

    Science.gov (United States)

    Gokhale, Paul J; Au-Young, Janice K; Dadi, SriVidya; Keys, David N; Harrison, Neil J; Jones, Mark; Soneji, Shamit; Enver, Tariq; Sherlock, Jon K; Andrews, Peter W

    2015-01-01

    We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

  17. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.

    Science.gov (United States)

    Lippmann, Ethan S; Azarin, Samira M; Kay, Jennifer E; Nessler, Randy A; Wilson, Hannah K; Al-Ahmad, Abraham; Palecek, Sean P; Shusta, Eric V

    2012-08-01

    The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

  18. Etk/Bmx activation modulates barrier function in epithelial cells.

    Science.gov (United States)

    Hamm-Alvarez, S F; Chang, A; Wang, Y; Jerdeva, G; Lin, H H; Kim, K J; Ann, D K

    2001-06-01

    Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O(2)) exposure, the TER and equivalent active ion transport rate (I(eq)) were reduced to <5% of the normoxia control in Pa-4 cells, whereas both TER and I(eq) were maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of beta-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of beta-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.

  19. Metabolic alterations in cancer cells and therapeutic implications

    Institute of Scientific and Technical Information of China (English)

    Naima Hammoudi; Kausar Begam Riaz Ahmed; Celia Garcia-Prieto; Peng Huang

    2011-01-01

    Cancer metabolism has emerged as an important area of research in recent years. Elucidation of the metabolic differences between cancer and normal cells and the underlying mechanisms will not only advance our understanding of fundamental cancer cell biology but also provide an important basis for the development of new therapeutic strategies and novel compounds to selectively eliminate cancer cells by targeting their unique metabolism. This article reviews several important metabolic alterations in cancer cells, with an emphasis on increased aerobic glycolysis (the Warburg effect) and glutamine addiction, and discusses the mechanisms that may contribute to such metabolic changes. In addition, metabolic alterations in cancer stem cells, mitochondrial metabolism and its influence on drug sensitivity, and potential therapeutic strategies and agents that target cancer metabolism are also discussed.

  20. Nylon Wool Purification Alters the Activation of T Cells

    Science.gov (United States)

    Wohler, Jillian E.; Barnum, Scott R.

    2009-01-01

    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method. PMID:18952296

  1. Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease.

    Directory of Open Access Journals (Sweden)

    Jay R Thiagarajah

    Full Text Available Hirschsprung disease-associated enterocolitis (HAEC leads to significant mortality and morbidity, but its pathogenesis remains unknown. Changes in the colonic epithelium related to goblet cells and the luminal mucus layer have been postulated to play a key role. Here we show that the colonic epithelium of both aganglionic and ganglionic segments are altered in patients and in mice with Hirschsprung disease (HSCR. Structurally, goblet cells were altered with increased goblet cell number and reduced intracellular mucins in the distal colon of biopsies from patients with HSCR. Endothelin receptor B (Ednrb mutant mice showed increased goblet cell number and size and increased cell proliferation compared to wild-type mice in aganglionic segments, and reduced goblet cell size and number in ganglionic segments. Functionally, compared to littermates, Ednrb-/- mice showed increased transepithelial resistance, reduced stool water content and similar chloride secretion in the distal colon. Transcript levels of goblet cell differentiation factors SPDEF and Math1 were increased in the distal colon of Ednrb-/- mice. Both distal colon from Ednrb mice and biopsies from HSCR patients showed reduced Muc4 expression as compared to controls, but similar expression of Muc2. Particle tracking studies showed that mucus from Ednrb-/- mice provided a more significant barrier to diffusion of 200 nm nanoparticles as compared to wild-type mice. These results suggest that aganglionosis is associated with increased goblet cell proliferation and differentiation and subsequent altered surface mucus properties, prior to the development of inflammation in the distal colon epithelium. Restoration of normal goblet cell function and mucus layer properties in the colonic epithelium may represent a therapeutic strategy for prevention of HAEC.

  2. Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

    Science.gov (United States)

    Heijink, I H; Brandenburg, S M; Postma, D S; van Oosterhout, A J M

    2012-02-01

    Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell-cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

  3. Alteration of the chronic wasting disease species barrier by in vitro prion amplification

    Science.gov (United States)

    Kurt, Timothy D.; Seelig, Davis M.; Schneider, Jay R.; Johnson, Christopher J.; Telling, Glenn C.; Heisey, Dennis M.; Hoover, Edward A.

    2011-01-01

    Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrPRES) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrPRES deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.

  4. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

    Science.gov (United States)

    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y

    2012-11-01

    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  5. Understanding and altering cell tropism of vesicular stomatitis virus

    Science.gov (United States)

    Hastie, Eric; Cataldi, Marcela; Marriott, Ian; Grdzelishvili, Valery Z.

    2013-01-01

    Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV’s broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV’s neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications. PMID:23796410

  6. Altered cytoskeletal structures in transformed cells exhibiting obviously metastatic capabilities

    Institute of Scientific and Technical Information of China (English)

    LINZHONGXIANG; WUBINGQUAN; 等

    1990-01-01

    Cytoskeletal changes in transformed cells (LM-51) eshibiting obviously metastatic capabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluorescence plus Rhodamine-phalloidin staining of F-actins;(2) indirect immunofluorescent staining with α-actinin polyclonal-and vinculin monoclonal antibodies.The LM-51 cells which showed metastatic index of >50% were derived from lung metastasis in nude mice after subcutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants.The parent NIH3T3 cells exhibited well-organized microtubules,prominent stress fibers and adhesion plaques while their transformants showed remarkable cytoskeletal alterations:(1)reduced microtubules but increased MTOC fluorescence;(2)disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm;(3)Factin-and α-actinin/vinculin aggregates appeared in the cytoplasm.These aggregates were dot-like,varied in size(0.1-0.4μm) and number,located near the ventral surface of the cells.TPA-induced actin/vinculin bodies were studied too.Indications that actin and α-actinin/vinculin redistribution might be important alterations involved in the expression of metastatic capabilities of LM-51 transformed cells were discussed.

  7. Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity.

    Science.gov (United States)

    Asakura, Hiroshi; Momose, Yoshika; Ryu, C-H; Kasuga, Fumiko; Yamamoto, Shigeki; Kumagai, Susumu; Igimi, Shizunobu

    2013-01-01

    Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

  8. Cell alterations induced by a biotherapic for influenza

    Directory of Open Access Journals (Sweden)

    José Nelson Couceiro

    2011-07-01

    Full Text Available Introduction: Influenza viruses have been responsible for highly contagious acute respiratory illnesses with high mortality, mainly in the elderly, which encourages the development of new drugs for the treatment of human flu. The biotherapics are medicines prepared from biological products, which are not chemically defined. They are compounded following the homeopathic procedures indicated for infectious diseases with known etiology [1]. Aim: The purpose of the present study is to verify cellular alterations induced by a biotherapic prepared from the infectious influenza A virus. Methodology: This biotherapic was prepared for this study in the homeopathic potency of 30X according to the Brazilian Homeopathic Pharmacopeia [2]. The concentration of 10% was not cytotoxic to cells, as verified by neutral red assay. The cellular alterations observed in MDCK cells were analyzed by optical microscopy for the quantification of mitosis, nucleoli and lipid bodies. The mitochondrial activity was assessed by MTT assay and the phosphosfructokinase-1 (PFK-1 enzyme activity was analyzed on the MDCK cells treated for 5, 10 and 30 days. Macrophages J778.G8 were treated with this biotherapic to evaluate the immunostimulatory cytokine release. Results: The cellular alterations observed in MDCK cells were verified by optical microscopy. The number of lipid bodies present in MDCK cells stimulated for 10 days was significantly lower (p <0.05 when compared to controls. The biotherapic significantly increased (p <0.05 the number of mitosis and the mitochondrial activity of MDCK cells stimulated for 10 and 30 days. These changes were confirmed by a significant reduction (p <0.05 on the PFK-1 activity. These results suggest that the biotherapic was able to activate the Krebs cycle and pentose-phosphate metabolism to the generation of amino acids and nucleotides, situations common to cells whose rate of mitosis is increased. The quantification of immunostimulatory

  9. Metabolic monosaccharides altered cell responses to anticancer drugs.

    Science.gov (United States)

    Chen, Long; Liang, Jun F

    2012-06-01

    Metabolic glycoengineering has been used to manipulate the glycochemistry of cell surfaces and thus the cell/cell interaction, cell adhesion, and cell migration. However, potential application of glycoengineering in pharmaceutical sciences has not been studied until recently. Here, we reported that Ac(4)ManNAc, an analog of N-acetyl-D-mannosamine (ManNAc), could affect cell responses to anticancer drugs. Although cells from different tissues and organs responded to Ac(4)ManNAc treatment differently, treated cells with increased sialic acid contents showed dramatically reduced sensitivity (up to 130 times) to anti-cancer drugs as tested on various drugs with distinct chemical structures and acting mechanisms. Neither increased P-glycoprotein activity nor decreased drug uptake was observed during the course of Ac(4)ManNAc treatment. However, greatly altered intracellular drug distributions were observed. Most intracellular daunorubicin was found in the perinuclear region, but not the expected nuclei in the Ac(4)ManNAc treated cells. Since sialoglycoproteins and gangliosides were synthesized in the Golgi, intracellular glycans affected intracellular signal transduction and drug distributions seem to be the main reason for Ac(4)ManNAc affected cell sensitivity to anticancer drugs. It was interesting to find that although Ac(4)ManNAc treated breast cancer cells (MDA-MB-231) maintained the same sensitivity to 5-Fluorouracil, the IC(50) value of 5-Fluorouracil to the same Ac(4)ManNAc treated normal cells (MCF-10A) was increased by more than 20 times. Thus, this Ac(4)ManNAc treatment enlarged drug response difference between normal and tumor cells provides a unique opportunity to further improve the selectivity and therapeutic efficiency of anticancer drugs.

  10. Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

    NARCIS (Netherlands)

    M. Janssens (Michelle); J. van Smeden (Jeroen); G.S. Gooris (Gert); W. Bras (Wim); G. Portale (Guiseppe); P.J. Caspers (Peter); R. Vreeken (Rob); T. Hankemeier (Thomas); S. Kezic (Sanja); R. Wolterbeek (Ron); A.P.M. Lavrijsen (Adriana); J.A. Bouwstra (Joke)

    2012-01-01

    textabstractA hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a cen

  11. Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells

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    Rout Ujjwal K

    2006-08-01

    Full Text Available Abstract Background Valproate, thalidomide and alcohol (ethanol exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requires adequate invasion of trophoblast into the maternal uterine tissues. Changes in the migratory behavior of trophoblast by maternal exposure to these teratogens during placentogenesis may therefore alter the structure and function of the placenta. Methods In the present study, the effects of sodium valproate, thalidomide and alcohol on the migration of human first trimester trophoblast cell line (HTR-8/SVneo were examined in vitro. Cells were cultured in the wells of 48-well culture plates as mono or multilayers. Circular patches of cells were removed from the center of the wells by suction, and the migration of cells into the wound was studied using microscopy. Effects of low and high concentrations of valproate, thalidomide and alcohol were examined on the healing of wounds and on the migration rate of cells by determining the wound areas at 0, 3, 6, 12, 24 and 48 h. Effects of drugs and alcohol on the proliferation and the expression levels of integrin subunits beta1 and alpha5 in cells were examined. Results The migration rates of trophoblast differed between wounds created in mono and multilayers of cells. Exposure to teratogens altered the migration of trophoblast into mono and multilayer wounds. The effects of valproate, thalidomide and alcohol on the proliferation of cells during the rapid migratory phase were mild. Drug exposure caused significant changes in the expression levels of beta1 and alpha5 integrin subunits. Conclusion Results suggest that

  12. Genetic alterations in head and neck squamous cell carcinomas

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    Nagai M.A.

    1999-01-01

    Full Text Available The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations and tumor suppressor gene inactivation (loss of function mutations, leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

  13. CD20(+) B Cell Depletion Alters T Cell Homing

    NARCIS (Netherlands)

    Kap, Yolanda S.; van Driel, Nikki; Laman, Jon D.; Tak, Paul P.; 't Hart, Bert A.

    2014-01-01

    Depleting mAbs against the pan B cell marker CD20 are remarkably effective in the treatment of autoimmune-mediated inflammatory disorders, but the underlying mechanisms are poorly defined. The primary objective of this study was to find a mechanistic explanation for the remarkable clinical effect of

  14. A microfluidic cell culture device with integrated microelectrodes for barrier studies

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin; Dufva, Martin; Kutter, Jörg P.

    We present an eight cell culture microfluidic device fabricated using thiol-ene ‘click’ chemistry with embedded microelectrodes for evaluating barrier properties of human intestinal epithelial cells. The capability of the microelectrodes for trans-epithelial electrical resistance (TEER) measureme......We present an eight cell culture microfluidic device fabricated using thiol-ene ‘click’ chemistry with embedded microelectrodes for evaluating barrier properties of human intestinal epithelial cells. The capability of the microelectrodes for trans-epithelial electrical resistance (TEER......) measurements was demonstrated by using confluent human colorectal epithelial cells (Caco-2) and rat fibroblast (CT 26) cells cultured in the microfluidic device....

  15. Alterations induced in Escherichia Coli cells by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kappke, J.; Schelin, H.R.; Paschuk, S.A.; Denyak, V.; Silva, E.R. da [Federal University of Technology of Parana (CPGEI/UTFPR), Curitiba, PR (Brazil)]. E-mails: jaquekap@yahoo.com.br; schelin@cpgei.cefetpr.br; sergei@utfpr.edu.br; Jesus, E.F.O. de; Lopes, R.T. [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia (COPPE). Lab. de Instrumentacao Nuclear]. E-mails: ricardo@lin.ufrj.br; edgar@lin.ufrj.br; Carlin, N.; Toledo, E.S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Fisica]. E-mail: nelson.carlin@dfn.if.usp.br

    2007-07-01

    Modifications occurred in Escherichia coli cells exposed to gamma radiation ({sup 60}Co source) were investigated. The irradiations were done at the LIN-COPPE laboratory of the UFRJ and the analysis at the Biology Department of the UTFPR. The E. coli cells were irradiated with 30, 60, 90, 120, 150, 180, 210, 240, 300, 480, 600 e 750 Gy doses. The samples were analyzed with Gram-stain, biochemical tests in EPM, MIO and Lysine Broth, Simmons Cytrate Medium and Rhamnose Broth, antibiogram and isolation of auxotrophic mutants. It was observed that for the received doses the E. coli did not show morphological alterations in the tests. Some E. Coli cells showed to be able to deaminade the L-tryptophan or they changed their sensibility for amoxillin and cephaloonine after the irradiation. The existence of aauxotrophic mutants after irradiation was also verified. (author)

  16. Altered B cell receptor signaling in human systemic lupus erythematosus

    Science.gov (United States)

    Jenks, Scott A.; Sanz, Iñaki

    2009-01-01

    Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcγRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal. PMID:18723129

  17. Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration

    Science.gov (United States)

    Eisenmann, Kathryn M.

    2017-01-01

    Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy. PMID:28243603

  18. Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

    Science.gov (United States)

    Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

    2016-07-01

    Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier.

  19. Metabolic flux prediction in cancer cells with altered substrate uptake.

    Science.gov (United States)

    Schwartz, Jean-Marc; Barber, Michael; Soons, Zita

    2015-12-01

    Proliferating cells, such as cancer cells, are known to have an unusual metabolism, characterized by an increased rate of glycolysis and amino acid metabolism. Our understanding of this phenomenon is limited but could potentially be used in order to develop new therapies. Computational modelling techniques, such as flux balance analysis (FBA), have been used to predict fluxes in various cell types, but remain of limited use to explain the unusual metabolic shifts and altered substrate uptake in human cancer cells. We implemented a new flux prediction method based on elementary modes (EMs) and structural flux (StruF) analysis and tested them against experimentally measured flux data obtained from (13)C-labelling in a cancer cell line. We assessed the quality of predictions using different objective functions along with different techniques in normalizing a metabolic network with more than one substrate input. Results show a good correlation between predicted and experimental values and indicate that the choice of cellular objective critically affects the quality of predictions. In particular, lactate gives an excellent correlation and correctly predicts the high flux through glycolysis, matching the observed characteristics of cancer cells. In contrast with FBA, which requires a priori definition of all uptake rates, often hard to measure, atomic StruFs (aStruFs) are able to predict uptake rates of multiple substrates.

  20. P-glycoprotein alters blood–brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

    Directory of Open Access Journals (Sweden)

    Ma A

    2013-12-01

    Full Text Available Aimei Ma,1,* Cuicui Wang,2,3,* Yinghui Chen,2,3 Weien Yuan4 1Department of Neurology, The People's Hospital of Shanxi Province, Taiyuan, 2Department of Neurology, Jinshan Hospital, Fudan University, 3Department of Neurology, Shanghai Medical College, Shanghai, 4School of Pharmacy, Shanghai JiaoTong University, Shanghai, People's Republic of China *These authors contributed equally to this work Abstract: P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood–brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy. Keywords: P-glycoprotein, medically intractable epilepsy, antiepileptic drugs, amygdala kindling, verapamil

  1. Genetic alterations in B-cell non-Hodgkin's lymphoma

    Directory of Open Access Journals (Sweden)

    Magić Zvonko

    2005-01-01

    Full Text Available Background. Although the patients with diagnosed B-NHL are classified into the same disease stage on the basis of clinical, histopathological, and immunological parameters, they respond significantly different to the applied treatment. This points out the possibility that within the same group of lymphoma there are different diseases at molecular level. For that reason many studies deal with the detection of gene alterations in lymphomas to provide a better framework for diagnosis and treatment of these hematological malignancies. Aim. To define genetic alterations in the B-NHL with highest possibilities for diagnostic purposes and molecular detection of MRD. Methods. Formalin fixed and paraffin embedded lymph node tissues from 45 patients were examined by different PCR techniques for the presence of IgH and TCR γ gene rearrangement; K-ras and H-ras mutations; c-myc amplification and bcl-2 translocation. There were 34 cases of B-cell non-Hodgkin’s lymphoma (B-NHL, 5 cases of T-cell non-Hodgkin’s lymphoma (T-NHL and 6 cases of chronic lymphadenitis (CL. The mononuclear cell fraction of the peripheral blood of 12 patients with B-NHL was analyzed for the presence of monoclonality at the time of diagnosis and in 3 to 6 months time intervals after an autologous bone marrow transplantation (BMT. Results. The monoclonality of B-lymphocytes, as evidenced by DNA fragment length homogeneity, was detected in 88 % (30/34 of B-NHL, but never in CL, T-NHL, or in normal PBL. Bcl-2 translocation was detected in 7/31 (22.6% B-NHL specimens, c-myc amplification 9/31 (29%, all were more than doubled, K-ras mutations in 1/31 (3.23% and H-ras mutations in 2/31 (6.45% of the examined B-NHL samples. In the case of LC and normal PBL, however, these gene alterations were not detected. All the patients (12 with B-NHL had dominant clone of B-lymphocyte in the peripheral blood at the time of diagnosis while only in 2 of 12 patients MRD was detected 3 or 6 months after

  2. Altered T cell costimulation during chronic hepatitis B infection.

    Science.gov (United States)

    Barboza, Luisa; Salmen, Siham; Peterson, Darrell L; Montes, Henry; Colmenares, Melisa; Hernández, Manuel; Berrueta-Carrillo, Leidith E; Berrueta, Lisbeth

    2009-01-01

    T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

  3. Ureaplasma parvum infection alters filamin a dynamics in host cells

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    Brown Mary B

    2011-04-01

    Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

  4. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    Science.gov (United States)

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.

  5. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

  6. Glycoprotein mucin molecular brush on cancer cell surface acting as mechanical barrier against drug delivery

    Science.gov (United States)

    Wang, Xin; Shah, Aalok A.; Campbell, Robert B.; Wan, Kai-tak

    2010-12-01

    Uptake of cytotoxic drugs by typical tumor cells is limited by the dense dendritic network of oligosaccharide mucin chains that forms a mechanical barrier. Atomic force microscopy is used to directly measure the force needed to pierce the mucin layer to reach the cell surface. Measurements are analyzed by de Gennes' steric reptation theory. Multidrug resistant ovarian tumor cells shows significantly larger penetration load compared to the wide type. A pool of pancreatic, lung, colorectal, and breast cells are also characterized. The chemotherapeutic agent, benzyl-α-GalNac, for inhibiting glycosylation is shown to be effective in reducing the mechanical barrier.

  7. Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.

    Science.gov (United States)

    Janssens, Michelle; van Smeden, Jeroen; Gooris, Gert S; Bras, Wim; Portale, Guiseppe; Caspers, Peter J; Vreeken, Rob J; Hankemeier, Thomas; Kezic, Sanja; Wolterbeek, Ron; Lavrijsen, Adriana P; Bouwstra, Joke A

    2012-12-01

    A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

  8. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

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    Lefeng Wang

    2017-01-01

    Full Text Available Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC, leading to barrier dysfunction and acute respiratory distress syndrome (ARDS. Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix] of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

  9. Alterations in integrin expression modulates invasion of pancreatic cancer cells.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2009-01-01

    BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

  10. Withaferin a alters intermediate filament organization, cell shape and behavior.

    Directory of Open Access Journals (Sweden)

    Boris Grin

    Full Text Available Withaferin A (WFA is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

  11. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

    Directory of Open Access Journals (Sweden)

    Sofie Stalmans

    Full Text Available Cell-penetrating peptides (CPPs are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB. However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10 and TP10-2, were determined. The results of the multiple time regression (MTR analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min, 5.63 μl/(g × min and 6.02 μl/(g × min, respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

  12. Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

    2015-10-15

    Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

  13. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells

    OpenAIRE

    Wei-Chuan Mo; Zi-Jian Zhang; Dong-Liang Wang; Ying Liu; Bartlett, Perry F.; Rong-Qiao He

    2016-01-01

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expres...

  14. Iron serves as diffusion barrier in thermally regenerative galvanic cell

    Science.gov (United States)

    Crouthamel, C. E.

    1967-01-01

    Pure iron or iron-coated diaphragm provides a hydrogen diffusion electrode for a thermally regenerative galvanic cell. It allows the gas to diffuse through its interatomic spaces and resists the corrosive action of the cell environment.

  15. Human oral isolate Lactobacillus fermentum AGR1487 reduces intestinal barrier integrity by increasing the turnover of microtubules in Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Rachel C Anderson

    Full Text Available Lactobacillus fermentum is found in fermented foods and thought to be harmless. In vivo and clinical studies indicate that some L. fermentum strains have beneficial properties, particularly for gastrointestinal health. However, L. fermentum AGR1487 decreases trans-epithelial electrical resistance (TEER, a measure of intestinal barrier integrity. The hypothesis was that L. fermentum AGR1487 decreases the expression of intestinal cell tight junction genes and proteins, thereby reducing barrier integrity. Transcriptomic and proteomic analyses of Caco-2 cells (model of human intestinal epithelial cells treated with L. fermentum AGR1487 were used to obtain a global view of the effect of the bacterium on intestinal epithelial cells. Specific functional characteristics by which L. fermentum AGR1487 reduces intestinal barrier integrity were examined using confocal microscopy, cell cycle progression and adherence bioassays. The effects of TEER-enhancing L. fermentum AGR1485 were investigated for comparison. L. fermentum AGR1487 did not alter the expression of Caco-2 cell tight junction genes (compared to L. fermentum AGR1485 and tight junction proteins were not able to be detected. However, L. fermentum AGR1487 increased the expression levels of seven tubulin genes and the abundance of three microtubule-associated proteins, which have been linked to tight junction disassembly. Additionally, Caco-2 cells treated with L. fermentum AGR1487 did not have defined and uniform borders of zona occludens 2 around each cell, unlike control or AGR1485 treated cells. L. fermentum AGR1487 cells were required for the negative effect on barrier integrity (bacterial supernatant did not cause a decrease in TEER, suggesting that a physical interaction may be necessary. Increased adherence of L. fermentum AGR1487 to Caco-2 cells (compared to L. fermentum AGR1485 was likely to facilitate this cell-to-cell interaction. These findings illustrate that bacterial strains of the

  16. Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

    Science.gov (United States)

    Yu, Miao; Chen, Zongzheng; Xiang, Cheng; Liu, Bo; Xie, Handi; Qin, Kairong

    2016-06-01

    Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier. The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system. It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

  17. Psychological Stress-Derived Prolactin Modulates Occludin Expression in Vaginal Epithelial Cells to Compromise Barrier Function

    Directory of Open Access Journals (Sweden)

    Xueyan Li

    2015-08-01

    Full Text Available Background/Aims: The causative factors of the vaginitis are not fully understood yet. Epithelial barrier dysfunction plays a critical role in the pathogenesis of vaginitis. This study aims to investigate the role of prolactin (PRL in the causing the vaginal epithelial barrier dysfunction. Methods: Adult rats were treated with water-avoid-stress. The serum levels of PRL were determined by ELISA. T84 cell (T84 cells; a vaginal epithelial cell line monolayers were prepared to be used assessing the epithelial barrier functions. The expression of occludin in T84 cells was assessed by Chromatin immunoprecipitation assay, methylation specifIc PCR, real time quantitative RT-PCR and Western blotting. Results: The results showed that psychological stress markedly increased the serum levels of PRL in the rat vaginal epithelia. Exposure of T84 cells to PRL in the culture markedly increased the phosphorylation of STAT3 and suppressed the expression of occludin in the cells; the transepithelial electric resistance was decreased and the permeability to a macromolecular tracer was increased in the T84 monolayers, which was mimicked by blocking STAT3, or abolished by over expression of occludin in the epithelial cells. Conclusions: Psychological stress-derived PRL induces vaginal epithelial barrier dysfunction by inhibiting the expression of occludin.

  18. Tandem-structured, hot electron based photovoltaic cell with double Schottky barriers.

    Science.gov (United States)

    Lee, Young Keun; Lee, Hyosun; Park, Jeong Young

    2014-04-03

    We demonstrate a tandem-structured, hot electron based photovoltaic cell with double Schottky barriers. The tandem-structured, hot electron based photovoltaic cell is composed of two metal/semiconductor interfaces. Two types of tandem cells were fabricated using TiO2/Au/Si and TiO2/Au/TiO2, and photocurrent enhancement was detected. The double Schottky barriers lead to an additional pathway for harvesting hot electrons, which is enhanced through multiple reflections between the two barriers with different energy ranges. In addition, light absorption is improved by the band-to-band excitation of both semiconductors with different band gaps. Short-circuit current and energy conversion efficiency of the tandem-structured TiO2/Au/Si increased by 86% and 70%, respectively, compared with Au/Si metal/semiconductor nanodiodes, showing an overall solar energy conversion efficiency of 5.3%.

  19. Lipopolysaccharide impairs amyloid beta efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier

    Directory of Open Access Journals (Sweden)

    Erickson Michelle A

    2012-06-01

    Full Text Available Abstract Background Defects in the low density lipoprotein receptor-related protein-1 (LRP-1 and p-glycoprotein (Pgp clearance of amyloid beta (Aβ from brain are thought to contribute to Alzheimer’s disease (AD. We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood–brain barrier. Methods CD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV or into the jugular vein (intravenous (IV was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF. Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine. Results We found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain

  20. Blood-Brain Barrier Disruption and Oxidative Stress in Guinea Pig after Systemic Exposure to Modified Cell-Free Hemoglobin

    Science.gov (United States)

    Butt, Omer I.; Buehler, Paul W.; D'Agnillo, Felice

    2011-01-01

    Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis. PMID:21356382

  1. Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E.

    Science.gov (United States)

    Palusinska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Cytryńska, Małgorzata; Wdowiak-Wróbel, Sylwia; Chmiel, Elżbieta; Gruszecki, Wiesław I

    2015-03-01

    Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection.

  2. Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS): A Pilot Study

    Science.gov (United States)

    Bashir, Mina; Münzker, Julia; Trummer, Christian; Zachhuber, Verena; Leber, Bettina; Horvath, Angela; Pieber, Thomas R.; Gorkiewicz, Gregor; Stadlbauer, Vanessa; Obermayer-Pietsch, Barbara

    2017-01-01

    Background Polycystic ovary syndrome (PCOS) is a common female endocrinopathy of unclear origin characterized by hyperandrogenism, oligo-/anovulation, and ovarian cysts. Women with PCOS frequently display overweight, insulin resistance, and systemic low-grade inflammation. We hypothesized that endotoxemia resulting from a leaky gut is associated with inflammation, insulin resistance, fat accumulation, and hyperandrogenemia in PCOS. In this pilot study, we compared the stool microbiome, gut permeability, and inflammatory status of women with PCOS and healthy controls. Methods 16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analysis were conducted in mothur and QIIME using different relative abundance cut-offs. Gut barrier integrity, endotoxemia, and inflammatory status were evaluated using serum and stool markers and associations with reproductive, metabolic, and anthropometric parameters were investigated. Results The stool microbiome of PCOS patients showed a lower diversity and an altered phylogenetic composition compared to controls. We did not observe significant differences in any taxa with a relative abundance>1%. When looking at rare taxa, the relative abundance of bacteria from the phylum Tenericutes, the order ML615J-28 (phylum Tenericutes) and the family S24-7 (phylum Bacteroidetes) was significantly lower and associated with reproductive parameters in PCOS patients. Patients showed alterations in some, but not all markers of gut barrier function and endotoxemia. Conclusion Patients with PCOS have a lower diversity and an altered phylogenetic profile in their stool microbiome, which is associated with clinical parameters. Gut barrier dysfunction and endotoxemia were not driving factors in this patient cohort, but may contribute to the clinical phenotype in certain PCOS patients. PMID:28045919

  3. BARRIERS TO COMMERCIALIZATION OF PASSIVE DIRECT METHANOL FUEL CELLS: A REVEIW

    Directory of Open Access Journals (Sweden)

    N. K. SHRIVASTAVA

    2011-07-01

    Full Text Available Fuel cells are electro-chemical reactors which realize the direct conversion of the chemical energy of reactants to electrical energy, with high efficiency and high environmental compatibility. This article is concerned with one of the most advance fuel cells- direct methanol fuel cells (DMFCs. We present a comprehensive review on the commercialization barriers of passive DMFCs. The paper also summarizes past research efforts and possible future directions towards these problems.

  4. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    Science.gov (United States)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  5. Membrane alterations in irreversibly sickled cells: hemoglobin--membrane interaction.

    Science.gov (United States)

    Lessin, L S; Kurantsin-Mills, J; Wallas, C; Weems, H

    1978-01-01

    Irreversibly sickled cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation and owe their biophysical abnormalities to acquired membrane alterations. Freeze-etched membranes both of ISCs produced in vitro and ISCs isolated in vivo reveal microbodies fixed to the internal (PS) surface which obscure spectrin filaments. Intramembranous particles (IMPs) on the intramembrane (PF) surface aggregate over regions of subsurface microbodies. Electron microscopy of diaminobenzidine-treated of ISC ghosts show the microbodies to contain hemoglobin and/or hemoglobin derivatives. Scanning electron microscopy and freeze-etching demonstrate that membrane--hemoglobin S interaction in ISCs enhances the membrane loss by microspherulation. Membrane-bound hemoglobin is five times greater in in vivo ISCs than non-ISCs, and increases during ISC production, parallelling depletion of adenosine triphosphate. Polyacrylamide gel electrophoresis of ISC membranes shows the presence of high-molecular-weight heteropolymers in the pre--band 1 region, a decrease in band 4.1 and an increase in bands 7, 8, and globin. The role of cross-linked membrane protein polymers in the generation of ISCs is discussed and is synthesized in terms of a unified concept for the determinants of the genesis of ISCs.

  6. Intestinal barrier gene variants may not explain the increased levels of antigliadin antibodies, suggesting other mechanisms than altered permeability

    NARCIS (Netherlands)

    Wolters, Victorien M.; Alizadeh, Behrooz Z.; Weijerman, Michel E.; Zhernakova, Alexandra; van Hoogstraten, Ingrid M. W.; Mearin, M. Luisa; Wapenaar, Martin C.; Wijmenga, Cisca; Schreurs, Marco W. J.

    2010-01-01

    Various genes may influence intestinal barrier function, including MAGI2, MY09B, and PARD3, which are associated with celiac disease. Because direct measurement of intestinal permeability is difficult, antibodies against gliadin (AGA) and Baker's yeast (anti-Saccharomyces cerevisiae antibodies [ASCA

  7. Effect of heat stress on intestinal barrier function of human intestinal epithelial Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Gui-zhen XIAO

    2013-07-01

    Full Text Available Objective To investigate the heat stress-induced dysfunction of intestinal barrier including intestinal tight junction and apoptosis of epithelial cells. Methods Human intestinal epithelial Caco-2 cell monolayers, serving as the intestinal barrier model, were exposed to different temperature (37-43℃ for designated time. Transepithelial electrical resistance (TEER and horseradish peroxidase (HRP flux permeability were measured to evaluate barrier integrity. Level of tight junction (TJ protein occludin was analyzed by Western blotting. Cell apoptosis rate was determined using Annexin V-FITC/PI kit by flow cytometry. Results Compared with the 37℃ group, TEER lowered and the permeability for HRP increased significantly after heat exposure (P<0.01 in 39℃, 41℃ and 43℃ groups. The expression of occludin increased when the temperature was elevated from 37℃ to 41℃, and it reached the maximal level at 41℃. However, its expression gradually decreased with passage of time at 43℃. Cell apoptosis was enhanced with elevation of the temperature (P<0.05 or P<0.01. Conclusion Heat stress can induce damage to tight junction and enhance apoptosis of epithelial cells, thus causing dysfunction of intestinal epithelial barrier.

  8. Recombination barrier layers in solid-state quantum dot-sensitized solar cells

    KAUST Repository

    Roelofs, Katherine E.

    2012-06-01

    By replacing the dye in the dye-sensitized solar cell design with semiconductor quantum dots as the light-absorbing material, solid-state quantum dot-sensitized solar cells (ss-QDSSCs) were fabricated. Cadmium sulfide quantum dots (QDs) were grown in situ by successive ion layer adsorption and reaction (SILAR). Aluminum oxide recombination barrier layers were deposited by atomic layer deposition (ALD) at the TiO2/hole-conductor interface. For low numbers of ALD cycles, the Al2O3 barrier layer increased open circuit voltage, causing an increase in device efficiency. For thicker Al2O3 barrier layers, photocurrent decreased substantially, leading to a decrease in device efficiency. © 2012 IEEE.

  9. Effects of disorder in location and size of fence barriers on molecular motion in cell membranes

    CERN Document Server

    Kalay, Z; Kenkre, V M

    2008-01-01

    The effect of disorder in the energetic heights and in the physical locations of fence barriers encountered by transmembrane molecules such as proteins and lipids in their motion in cell membranes is studied theoretically. The investigation takes as its starting point a recent analysis of a periodic system with constant distances between barriers and constant values of barrier heights, and employs effective medium theory to treat the disorder. The calculations make possible, in principle, the extraction of confinement parameters such as mean compartment sizes and mean intercompartmental transition rates from experimentally reported published observations. The analysis should be helpful both as an unusual application of effective medium theory and as an investigation of observed molecular movements in cell membranes.

  10. Arctigenin from Fructus Arctii (Seed of Burdock Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

    Directory of Open Access Journals (Sweden)

    Hee Soon Shin

    2015-01-01

    Full Text Available Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER value (as an index of barrier function and ovalbumin (OVA permeation (as an index of permeability to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  11. Arctigenin from Fructus Arctii (Seed of Burdock) Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers.

    Science.gov (United States)

    Shin, Hee Soon; Jung, Sun Young; Back, Su Yeon; Do, Jeong-Ryong; Shon, Dong-Hwa

    2015-01-01

    Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) and ovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  12. Inflammatory response to mucosal barrier injury after myeloablative therapy in allogeneic stem cell transplant recipients.

    NARCIS (Netherlands)

    Blijlevens, N.M.A.; Donnelly, J.P.; Pauw, B.E. de

    2005-01-01

    We noted a significant increase of interleukin-8 (IL-8), LBP and CRP mirroring the pattern of mucosal barrier injury as measured by gut integrity (lactulose/rhamnose ratio), daily mucositis score (DMS) and serum citrulline concentrations of 32 haematopoietic stem cell transplant (HSCT) recipients fo

  13. Endothelial cells of the blood-brain barrier: a target for glucocorticoids and estrogens?

    Science.gov (United States)

    Dietrich, Jean-Bernard

    2004-01-01

    Adhesion molecules are involved in the leukocyte recruitment of leukocytes at the blood-brain barrier. For this reason, it is important to understand how the regulation of their gene expression controls lymphocyte adhesion to endothelial cells in microvessels. Indeed, due to their specificity and diversity, adhesion molecules involved in extravasation play an essential role in the recruitment of activated leukocytes and activation of inflammation. Multiple sclerosis results from a chronic inflammation of the CNS which is mediated by infiltration of inflammatory cells from the immune system. Administration of glucocorticoids is a routine method to control multiple sclerosis since naturally derived or synthetic glucocorticoids are potent immunosuppressive and anti-inflammatory agents. Glucocorticoids also have beneficial effects in stabilizing the blood-brain barrier, as steroid hormones regulate the expression of adhesion molecule genes in endothelial cells. Other hormones such as estrogens modulate many endothelial cell biological activities, among them adhesion to leukocytes. They regulate expression of adhesion molecules genes on endothelial cells and are useful for the treatment of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The effects of glucocorticoids and estrogens on the expression of adhesion molecules on endothelial cells, including microvascular endothelial cells of the blood-brain barrier, are reviewed in this paper, as well as the involvement of these hormones in the therapy of experimental autoimmune encephalomyelitis and multiple sclerosis.

  14. Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Smith, Ida Mosbech; Baker, A; Arneborg, Nils

    2015-01-01

    UNLABELLED: The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function....... In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability......). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. SIGNIFICANCE AND IMPACT...

  15. Particle-in-cell modeling of gas-confined barrier discharge

    Science.gov (United States)

    Levko, Dmitry; Raja, Laxminarayan L.

    2016-04-01

    Gas-confined barrier discharge is studied using the one-dimensional Particle-in-Cell Monte Carlo Collisions model for the conditions reported by Guerra-Garcia and Martinez-Sanchez [Appl. Phys. Lett. 106, 041601 (2015)]. Depending on the applied voltage, two modes of discharge are observed. In the first mode, the discharge develops in the entire interelectrode gap. In the second mode, the discharge is ignited and develops only in the gas layer having smaller breakdown voltage. The one-dimensional model shows that for the conditions considered, there is no streamer stage of breakdown as is typical for a traditional dielectric barrier discharge.

  16. Ring-Shaped Microlanes and Chemical Barriers as a Platform for Probing Single-Cell Migration

    Science.gov (United States)

    Schreiber, Christoph; Segerer, Felix J.; Wagner, Ernst; Roidl, Andreas; Rädler, Joachim O.

    2016-01-01

    Quantification and discrimination of pharmaceutical and disease-related effects on cell migration requires detailed characterization of single-cell motility. In this context, micropatterned substrates that constrain cells within defined geometries facilitate quantitative readout of locomotion. Here, we study quasi-one-dimensional cell migration in ring-shaped microlanes. We observe bimodal behavior in form of alternating states of directional migration (run state) and reorientation (rest state). Both states show exponential lifetime distributions with characteristic persistence times, which, together with the cell velocity in the run state, provide a set of parameters that succinctly describe cell motion. By introducing PEGylated barriers of different widths into the lane, we extend this description by quantifying the effects of abrupt changes in substrate chemistry on migrating cells. The transit probability decreases exponentially as a function of barrier width, thus specifying a characteristic penetration depth of the leading lamellipodia. Applying this fingerprint-like characterization of cell motion, we compare different cell lines, and demonstrate that the cancer drug candidate salinomycin affects transit probability and resting time, but not run time or run velocity. Hence, the presented assay allows to assess multiple migration-related parameters, permits detailed characterization of cell motility, and has potential applications in cell biology and advanced drug screening. PMID:27242099

  17. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells.

    Science.gov (United States)

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F; He, Rong-Qiao

    2016-03-31

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation.

  18. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  19. Altered intestinal microbial flora and impaired epithelial barrier structure and function in CKD: the nature, mechanisms, consequences and potential treatment.

    Science.gov (United States)

    Vaziri, Nosratola D; Zhao, Ying-Yong; Pahl, Madeleine V

    2016-05-01

    Chronic kidney disease (CKD) results in systemic inflammation and oxidative stress which play a central role in CKD progression and its adverse consequences. Although many of the causes and consequences of oxidative stress and inflammation in CKD have been extensively explored, little attention had been paid to the intestine and its microbial flora as a potential source of these problems. Our recent studies have revealed significant disruption of the colonic, ileal, jejunal and gastric epithelial tight junction in different models of CKD in rats. Moreover, the disruption of the epithelial barrier structure and function found in uremic animals was replicated in cultured human colonocytes exposed to uremic human plasma in vitro We have further found significant changes in the composition and function of colonic bacterial flora in humans and animals with advanced CKD. Together, uremia-induced impairment of the intestinal epithelial barrier structure and function and changes in composition of the gut microbiome contribute to the systemic inflammation and uremic toxicity by accommodating the translocation of endotoxin, microbial fragments and other noxious luminal products in the circulation. In addition, colonic bacteria are the main source of several well-known pro-inflammatory uremic toxins such as indoxyl sulfate, p-cresol sulfate, trimethylamine-N-oxide and many as-yet unidentified retained compounds in end-stage renal disease patients. This review is intended to provide an overview of the effects of CKD on the gut microbiome and intestinal epithelial barrier structure and their role in the pathogenesis of systemic inflammation and uremic toxicity. In addition, potential interventions aimed at mitigating these abnormalities are briefly discussed.

  20. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

    Directory of Open Access Journals (Sweden)

    David M Gravano

    Full Text Available BACKGROUND: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. METHODOLOGY/PRINCIPAL FINDINGS: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. CONCLUSIONS/SIGNIFICANCE: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and

  1. Preparation and characterization of TiO2 barrier layers for dye-sensitized solar cells.

    Science.gov (United States)

    Zheng, Yichen; Klankowski, Steven; Yang, Yiqun; Li, Jun

    2014-07-09

    A TiO2 barrier layer is critical in enhancing the performance of dye-sensitized solar cells (DSSCs). Two methods to prepare the TiO2 barrier layer on fluorine-doped tin dioxide (FTO) surface were systematically studied in order to minimize electron-hole recombination and electron backflow during photovoltaic processes of DSSCs. The film structure and materials properties were correlated with the photovoltaic characteristics and electrochemical properties. In the first approach, a porous TiO2 layer was deposited by wet chemical treatment of the sample with TiCl4 solution for time periods varying from 0 to 60 min. The N719 dye molecules were found to be able to insert into the porous barrier layers. The 20 min treatment formed a nonuniform but intact TiO2 layer of ∼100-300 nm in thickness, which gave the highest open-circuit voltage VOC, short-circuit photocurrent density JSC, and energy conversion efficiency. But thicker TiO2 barrier layers by this method caused a decrease in JSC, possibly limited by lower electrical conductance. In the second approach, a compact TiO2 barrier layer was created by sputter-coating 0-15 nm Ti metal films on FTO/glass and then oxidizing them into TiO2 with thermal treatment at 500 °C in the air for 30 min. The dye molecules were found to only attach at the outer surface of the barrier layer and slightly increased with the layer thickness. These two kinds of barrier layer showed different characteristics and may be tailored for different DSSC studies.

  2. Trypsin-induced proteome alteration during cell subculture in mammalian cells

    Directory of Open Access Journals (Sweden)

    Lin Cheng-Wen

    2010-05-01

    Full Text Available Abstract Background It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. Methods In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. Results 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. Conclusions In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

  3. Spray pyrolysis of doped-ceria barrier layers for solid oxide fuel cells

    DEFF Research Database (Denmark)

    Szymczewska, Dagmara; Chrzan, Aleksander; Karczewski, Jakub

    2017-01-01

    of elements. The parameters of the fabrication process are linked to the measured area specific resistances of the symmetrical cells and the efficiency of the fuel cells. Results show, that application of 800 nm thick barrier effectively hinder negative reactions, while 400 nm thick layer is sufficient......Gadolinium doped ceria (Ce0.8Gd0.2O2 − x-CGO) layer fabricated by spray pyrolysis is investigated as the diffusion barrier for solid oxide fuel cell. It is deposited between the La0.6Sr0.4FeO3 − δ cathode and the yttria stabilized zirconia electrolyte to mitigate harmful interdiffusion...

  4. Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

    Science.gov (United States)

    Parikh, Neil U; Aalinkeel, R; Reynolds, J L; Nair, B B; Sykes, D E; Mammen, M J; Schwartz, S A; Mahajan, S D

    2015-10-22

    Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

  5. Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells

    OpenAIRE

    Rout Ujjwal K

    2006-01-01

    Abstract Background Valproate, thalidomide and alcohol (ethanol) exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requi...

  6. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  7. Acute exposure to ergot alkaloids from endophyte-infected tall fescue does not alter absorptive or barrier function of the isolated bovine ruminal epithelium.

    Science.gov (United States)

    Foote, A P; Penner, G B; Walpole, M E; Klotz, J L; Brown, K R; Bush, L P; Harmon, D L

    2014-07-01

    Ergot alkaloids in endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum) have been shown to cause a reduction in blood flow to the rumen epithelium as well as a decrease in volatile fatty acids (VFA) absorption from the washed rumen of steers. Previous data also indicates that incubating an extract of endophyte-infected tall fescue seed causes an increase in the amount of VFA absorbed per unit of blood flow, which could result from an alteration in the absorptive or barrier function of the rumen epithelium. An experiment was conducted to determine the acute effects of an endophyte-infected tall fescue seed extract (EXT) on total, passive or facilitated acetate and butyrate flux across the isolated bovine rumen as well as the barrier function measured by inulin flux and tissue conductance (G t ). Flux of ergovaline across the rumen epithelium was also evaluated. Rumen tissue from the caudal dorsal sac of Holstein steers (n=6), fed a common diet, was collected and isolated shortly after slaughter and mounted between two halves of Ussing chambers. In vitro treatments included vehicle control (80% methanol, 0.5% of total volume), Low EXT (50 ng ergovaline/ml) and High EXT (250 ng ergovaline/ml). Results indicate that there is no effect of acute exposure to ergot alkaloids on total, passive or facilitated flux of acetate or butyrate across the isolate bovine rumen epithelium (P>0.51). Inulin flux (P=0.16) and G t (P>0.17) were not affected by EXT treatment, indicating no alteration in barrier function due to acute ergot alkaloid exposure. Ergovaline was detected in the serosal buffer of the High EXT treatment indicating that the flux rate is ~0.25 to 0.44 ng/cm2 per hour. Data indicate that specific pathways for VFA absorption and barrier function of the rumen epithelium are not affected by acute exposure to ergot alkaloids from tall fescue at the concentrations tested. Ergovaline has the potential to be absorbed from the rumen of cattle that

  8. Genetic alteration in notch pathway is associated with better prognosis in renal cell carcinoma.

    Science.gov (United States)

    Feng, Chenchen; Xiong, Zuquan; Jiang, Haowen; Ding, Qiang; Fang, Zujun; Hui, Wen

    2016-01-01

    Notch signaling was associated with a variety of cancers but was not comprehensively studied in clear-cell renal cell carcinoma (ccRCC). We have in this study studied the genetic alteration (mutation and copy number variance) of Notch gene set in the Cancer Genome Atlas (TCGA) Kidney Renal Clear Cell Carcinoma (KIRC) database. We found that Notch pathway was frequently altered in ccRCC. The Notch gene set was genetically altered in 182 (44%) of the 415 ccRCC patients. CNV was the predominant type of alteration in most genes. Alterations in KAT2B and MAML1 occurred in 13% and 19% of patients, respectively, both of which were functionally active in ccRCC. Deletion of VHL was exclusively found in cases with Notch alteration. Overall survival was longer in ccRCC patients with altered-Notch pathway. The median survival was 90.41 months in Notch-altered cases and 69.15 in Notch-unaltered cases (P = 0.0404). The median disease free time was 89.82 months in Notch-altered cases and 77.27 months in in Notch-unaltered cases (P = 0.935). Conclusively, Notch signaling was altered in almost half of the ccRCC patients and copy number variances in MAML1 and KAT2B were predominant changes. These findings broadened our understanding of the role of Notch in ccRCC.

  9. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

    Science.gov (United States)

    Xie, Lishi; Chiang, Eddie T.; Kelly, Gabriel T.; Kanteti, Prasad; Singleton, Patrick A.; Camp, Sara M.; Zhou, Tingting; Dudek, Steven M.; Natarajan, Viswanathan; Wang, Ting; Black, Steven M.; Garcia, Joe G. N.; Jacobson, Jeffrey R.

    2016-01-01

    Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis. PMID:27442243

  10. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

    Directory of Open Access Journals (Sweden)

    Lishi Xie

    Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

  11. The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells

    Institute of Scientific and Technical Information of China (English)

    Huanling Yao; Tianshi Wang; Jiexin Deng; Ding Liu; Xiaofei Li; Jinbo Deng

    2014-01-01

    Astrocytes are intimately involved in the formation and development of retinal vessels. Astrocyte dysfunction is a major cause of blood-retinal barrier injury and other retinal vascular diseases. In this study, the development of the retinal vascular system and the formation of the blood-ret-inal barrier in mice were investigated using immunolfuorescence staining, gelatin-ink perfusion, and transmission electron microscopy. The results showed that the retinal vascular system of mice develops from the optic disc after birth, and radiates out gradually to cover the entire retina, taking the papilla optica as the center. First, the superifcial vasculature is formed on the inner retinal layer;then, the vasculature extends into the inner and outer edges of the retinal inner nuclear layer, forming the deep vasculature that is parallel to the superifcial vasculature. The blood-retinal barrier is mainly composed of endothelium, basal lamina and the end-feet of astrocytes, which become mature during mouse development. Initially, the naive endothelial cells were immature with few organelles and many microvilli. The basal lamina was uniform in thickness, and the glial end-feet surrounded the outer basal lamina incompletely. In the end, the blood-retinal barrier matures with smooth endothelia connected through tight junctions, rela-tively thin and even basal lamina, and relatively thin glial cell end-feet. These ifndings indicate that the development of the vasculature in the retina follows the rules of“center to periphery”and“superifcial layer to deep layers”. Its development and maturation are spatially and tempo-rally consistent with the functional performance of retinal neurons and photosensitivity. The blood-retinal barrier gradually becomes mature via the process of interactions between astro-cytes and blood vessel cells.

  12. Tanycyte-Like Cells Form a Blood–Cerebrospinal Fluid Barrier in the Circumventricular Organs of the Mouse Brain

    OpenAIRE

    2013-01-01

    Tanycytes are highly specialized ependymal cells that form a blood–cerebrospinal fluid (CSF) barrier at the level of the median eminence (ME), a circumventricular organ (CVO) located in the tuberal region of the hypothalamus. This ependymal layer harbors well-organized tight junctions, a hallmark of central nervous system barriers that is lacking in the fenestrated portal vessels of the ME. The displacement of barrier properties from the vascular to the ventricular side allows the diffusion o...

  13. Altered status of glutathione and its metabolites in cystinotic cells.

    NARCIS (Netherlands)

    Levtchenko, E.N.; Graaf-Hess, A.C. de; Wilmer, M.J.G.; Heuvel, L.P.W.J. van den; Monnens, L.A.H.; Blom, H.J.

    2005-01-01

    BACKGROUND: Cystinosis is an autosomal recessive disorder, caused by mutations of the lysosomal cystine carrier cystinosin, encoded by the CTNS gene (17p13). The concomitant intralysosomal cystine accumulation leads to multi-organ damage, with kidneys being the first affected. Altered mitochondrial

  14. Virus Innexins induce alterations in insect cell and tissue function

    Science.gov (United States)

    Polydnaviruses are dsDNA viruses that induce immune and developmental alterations in their caterpillar hosts. Characterization of polydnavirus gene families and family members is necessary to understand mechanisms of pathology and evolution of these viruses, and may aid to elucidate the role of host...

  15. Alterations in the growth and adhesion pattern of Vero cells induced by nutritional stress conditions.

    Science.gov (United States)

    Genari, S C; Gomes, L; Wada, M L

    1998-01-01

    The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44-kDa protein was observed in stressed cells and was absent in non-stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell-cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

  16. Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

    Science.gov (United States)

    Park, Min Chul; Jeong, Hyobin; Son, Sung Hwa; Kim, YounHa; Han, Daeyoung; Goughnour, Peter C; Kang, Taehee; Kwon, Nam Hoon; Moon, Hyo Eun; Paek, Sun Ha; Hwang, Daehee; Seol, Ho Jun; Nam, Do-Hyun; Kim, Sunghoon

    2016-03-01

    Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

  17. Barrier potential design criteria in multiple-quantum-well-based solar-cell structures

    Science.gov (United States)

    Mohaidat, Jihad M.; Shum, Kai; Wang, W. B.; Alfano, R. R.

    1994-01-01

    The barrier potential design criteria in multiple-quantum-well (MQW)-based solar-cell structures is reported for the purpose of achieving maximum efficiency. The time-dependent short-circuit current density at the collector side of various MQW solar-cell structures under resonant condition was numerically calculated using the time-dependent Schroedinger equation. The energy efficiency of solar cells based on the InAs/Ga(y)In(1-y)As and GaAs/Al(x)Ga(1-x)As MQW structues were compared when carriers are excited at a particular solar-energy band. Using InAs/Ga(y)In(1-y)As MQW structures it is found that a maximum energy efficiency can be achieved if the structure is designed with barrier potential of about 450 meV. The efficiency is found to decline linearly as the barrier potential increases for GaAs/Al(x)Ga(1-x)As MQW-structure-based solar cells.

  18. Altering β-cell number through stable alteration of miR-21 and miR-34a expression

    DEFF Research Database (Denmark)

    Backe, Marie Balslev; Novotny, Guy Wayne; Christensen, Dan Ploug;

    2014-01-01

    RNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed...... miR-21 and knocked down miR-34a, and investigated essential cellular processes. Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were...... monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a...

  19. Alterations in Cell Signaling Pathways in Breast Cancer Cells after Environmental Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kulp, K; McCutcheon-Maloney, S M; Bennett, L M

    2003-02-01

    Recent human epidemiological studies suggest that up to 75% of human cancers can be attributed to environmental exposures. Understanding the biologic impact of being exposed to a lifetime of complex environmental mixtures that may not be fully characterized is currently a major challenge. Functional endpoints may be used to assess the gross health consequences of complex mixture exposures from groundwater contamination, superfund sites, biologic releases, or nutritional sources. Such endpoints include the stimulation of cell growth or the induction of a response in an animal model. An environmental exposure that upsets normal cell growth regulation may have important ramifications for cancer development. Stimulating cell growth may alter an individual's cancer risk by changing the expression of genes and proteins that have a role in growth regulatory pathways within cells. Modulating the regulation of these genes and their products may contribute to the initiation, promotion or progression of disease in response to environmental exposure. We are investigating diet-related compounds that induce cell proliferation in breast cancer cell lines. These compounds, PhIP, Flor-Essence{reg_sign} and Essiac{reg_sign}, may be part of an everyday diet. PhIP is a naturally occurring mutagen that is formed in well-cooked muscle meats. PhIP consistently causes dose-dependent breast tumor formation in rats and consumption of well-done meat has been linked to increased risk of breast cancer in women. Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics are complementary and alternative medicines used by women who have been diagnosed with breast cancer as an alternative therapy for disease treatment and prevention. The long-term goal of this work is to identify those cellular pathways that are altered by a chemical or biologic environmental exposure and understand how those changes correlate with and or predict changes in human health risk. This project addressed this goal

  20. c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes.

    Science.gov (United States)

    Xiao, Xiang; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2011-04-01

    During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites.

  1. High-efficiency silicon solar-cell design and practical barriers

    Science.gov (United States)

    Mokashi, A.

    1985-01-01

    A numerical evaluation technique is used to study the impact of practical barriers, such as heavy doping effects (Auger recombination, band gap narrowing), surface recombination, shadowing losses and minority-carrier lifetime (Tau), on a high efficiency silicon solar cell performance. Considering a high Tau of 1 ms, efficiency of a silicon solar cell of the hypothetical case is estimated to be around 29%. This is comparable with (detailed balance limit) maximum efficiency of a p-n junction solar cell of 30%. Value of Tau is varied from 1 second to 20 micro. Heavy doping effects, and realizable values of surface recombination velocities and shadowing, are then considered in succession and their influence on cell efficiency is evaluated and quantified. These practical barriers cause the cell efficiency to reduce from the maximum value of 29% to the experimentally achieved value of about 19%. Improvement in open circuit voltage V sub oc is required to achieve cell efficiency greater than 20%. Increased value of Tau reduces reverse saturation current and, hence, improves V sub oc. Control of surface recombination losses becomes critical at higher V sub oc. Substantial improvement in Tau and considerable reduction in surface recombination velocities is essential to achieve cell efficiencies greater than 20%.

  2. Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

    Institute of Scientific and Technical Information of China (English)

    Miao Yu; Zongzheng Chen; Cheng Xiang; Bo Liu; Handi Xie; Kairong Qin

    2016-01-01

    Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the rela-tionship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier. The microfluidic device overcomes the weakness of the tra-ditional ones, which have been only based upon either stag-nation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addi-tion, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system. It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and con-veniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

  3. Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

    Directory of Open Access Journals (Sweden)

    Ariane Willems

    Full Text Available The observation that mice with a selective ablation of the androgen receptor (AR in Sertoli cells (SC (SCARKO mice display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin. Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2. It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

  4. Oral collagen-derived dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine, ameliorate skin barrier dysfunction and alter gene expression profiles in the skin.

    Science.gov (United States)

    Shimizu, Jun; Asami, Naoto; Kataoka, Aya; Sugihara, Fumihito; Inoue, Naoki; Kimira, Yoshifumi; Wada, Masahiro; Mano, Hiroshi

    2015-01-09

    Oral supplementation with collagen hydrolysate (CH) has been shown to improve the condition of the skin in humans and experimental animals. Several hydroxyproline-containing oligo-peptides were previously detected in human peripheral blood after the ingestion of CH, and the two dipeptides, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG), have been proposed to have beneficial effects on human health. When HR-1 hairless mice were fed a HR-AD diet, which lacked magnesium and zinc, transepidermal water loss (TEWL) increased and water content of stratum corneum decreased. In the present study, we investigated the effects of dietary PO and OG on skin barrier dysfunction in HR-1 hairless mice. Mice were fed a HR-AD diet with or without PO (0.15%) and OG (0.15%) for 35 consecutive days. The administration of PO and OG significantly decreased TEWL, and significantly increased water content of stratum corneum. A DNA microarray analysis of the dorsal skin revealed differences in gene expression between the group administered PO and OG and the control group. We also identified muscle-related Gene Ontology as a result of analyzing the up-regulated genes. These results suggested that the administration of PO and OG improved skin barrier dysfunction and altered muscle-related gene expression.

  5. Development of microfluidic cell culture devices towards an in vitro human intestinal barrier model

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin

    to enable real-time detection of cell responses, adjustment of cellular stimulation etc. leading to establishment of conditional experiments. In this project, microfluidic systems engineering was leveraged to develop an eight chamber multi-layer microchip for intestinal barrier studies. Sandwiched between...... the layers was a modified Teflon porous membrane for cell culture. The novelty lies in modifying the surface of the porous Teflon support membrane using thiol-ene ‘click’ chemistry, thus allowing the modified Teflon membrane to be bonded between the chip layers to form an enclosed microchip. Successful...... application of the multi-layer microchip was demonstrated by integrating the microchip to an existing cell culture fluidic system to culture the human intestinal epithelial cells, Caco-2, for long term studies. Under the continuous low flow conditions, the cells differentiated into columnar cells displaying...

  6. Alemtuzumab treatment alters circulating innate immune cells in multiple sclerosis

    Science.gov (United States)

    Ahmetspahic, Diana; Ruck, Tobias; Schulte-Mecklenbeck, Andreas; Schwarte, Kathrin; Jörgens, Silke; Scheu, Stefanie; Windhagen, Susanne; Graefe, Bettina; Melzer, Nico; Klotz, Luisa; Arolt, Volker; Wiendl, Heinz; Meuth, Sven G.

    2016-01-01

    Objective: To characterize changes in myeloid and lymphoid innate immune cells in patients with relapsing-remitting multiple sclerosis (MS) during a 6-month follow-up after alemtuzumab treatment. Methods: Circulating innate immune cells including myeloid cells and innate lymphoid cells (ILCs) were analyzed before and 6 and 12 months after onset of alemtuzumab treatment. Furthermore, a potential effect on granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)–23 production by myeloid cells and natural killer (NK) cell cytolytic activity was determined. Results: In comparison to CD4+ T lymphocytes, myeloid and lymphoid innate cell subsets of patients with MS expressed significantly lower amounts of CD52 on their cell surface. Six months after CD52 depletion, numbers of circulating plasmacytoid dendritic cells (DCs) and conventional DCs were reduced compared to baseline. GM-CSF and IL-23 production in DCs remained unchanged. Within the ILC compartment, the subset of CD56bright NK cells specifically expanded under alemtuzumab treatment, but their cytolytic activity did not change. Conclusions: Our findings demonstrate that 6 months after alemtuzumab treatment, specific DC subsets are reduced, while CD56bright NK cells expanded in patients with MS. Thus, alemtuzumab specifically restricts the DC compartment and expands the CD56bright NK cell subset with potential immunoregulatory properties in MS. We suggest that remodeling of the innate immune compartment may promote long-term efficacy of alemtuzumab and preserve immunocompetence in patients with MS. PMID:27766281

  7. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp;

    Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming ...

  8. Altered effector function of peripheral cytotoxic cells in COPD

    Directory of Open Access Journals (Sweden)

    Corne Jonathan M

    2009-06-01

    Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

  9. Altered transcription levels of endocrine associated genes in two fisheries species collected from the Great Barrier Reef catchment and lagoon.

    Science.gov (United States)

    Kroon, Frederieke J; Hook, Sharon E; Jones, Dean; Metcalfe, Suzanne; Henderson, Brent; Smith, Rachael; Warne, Michael St J; Turner, Ryan D; McKeown, Adam; Westcott, David A

    2015-03-01

    The Great Barrier Reef (GBR) is chronically exposed to agricultural run-off containing pesticides, many of which are known endocrine disrupting chemicals (EDCs). Here, we measure mRNA transcript abundance of two EDC biomarkers in wild populations of barramundi (Lates calcarifer) and coral trout (Plectropomus leopardus and Plectropomus maculatus). Transcription levels of liver vitellogenin (vtg) differed significantly in both species amongst sites with different exposures to agricultural run-off; brain aromatase (cyp19a1b) revealed some differences for barramundi only. Exposure to run-off from sugarcane that contains pesticides is a likely pathway given (i) significant associations between barramundi vtg transcription levels, catchment sugarcane land use, and river pesticide concentrations, and (ii) consistency between patterns of coral trout vtg transcription levels and pesticide distribution in the GBR lagoon. Given the potential consequences of such exposure for reproductive fitness and population dynamics, these results are cause for concern for the sustainability of fisheries resources downstream from agricultural land uses.

  10. Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression of specific claudin isoforms: Protective effect of illite mineral clay.

    Science.gov (United States)

    Romero, Alejandro; Ares, Irma; Ramos, Eva; Castellano, Víctor; Martínez, Marta; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Martínez, María-Aránzazu

    2016-04-15

    Aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA) and T-2 toxin (T2) are mycotoxins that commonly contaminate the food chain and cause various toxicological effects. Their global occurrence is regarded as an important risk factor for human and animal health. In this study, the results demonstrate that, in human Caco-2 cells, AFB1, FB1, OTA and T2 origin cytotoxic effects, determining cell viability through MTT assay and LDH leakage, and decrease trans-epithelial electrical resistance (TEER). The decrease in barrier properties is concomitant with a reduction in the expression levels of the tight junction constituents claudin-3, claudin-4 and occludin. The protective effect of mineral clays (diosmectite, montmorillonite and illite) on alterations in cell viability and epithelial barrier function induced by the mycotoxins was also evaluated. Illite was the best clay to prevent the mycotoxin effects. Illite plus mycotoxin co-treatment completely abolished AFB1 and FB1-induced cytotoxicity. Also, the decreases in the gene expression of claudins and the reduction of TEER induced by mycotoxins were reversed by the illite plus mycotoxin co-treatment. In conclusion, these results demonstrated that mycotoxins AFB1, FB1, T2 and OTA disrupt the intestinal barrier permeability by a mechanism involving reduction of claudin isoform expressions, and illite counteracts this disruption.

  11. Studies on the potential neurotoxic and convulsant effects of increased blood levels of quinolinic acid in rats with altered blood-brain barrier permeability

    Energy Technology Data Exchange (ETDEWEB)

    Vezzani, A.; Stasi, M.A.; Wu, H.Q.; Castiglioni, M.; Weckermann, B.; Samanin, R. (Istituto di Ricerche Farmacologiche Mario Negri, Milano (Italy))

    1989-10-01

    Intravenous injection of 450 mg/kg quinolinic acid (Quin), an endogenous kynurenine metabolite with excitotoxic properties, induced only minor electroencephalographic (EEG) modifications and no neurotoxicity in rats with a mature blood-brain barrier (BBB). BBB permeability was altered in rats by focal unilateral irradiation of the cortex (7 mm in diameter and 5 mm in depth) with protons (60 Gy, 9 Gy/min). Three days after irradiation, Evans blue dye staining showed BBB breakdown in the dorsal hippocampus of the irradiated hemisphere. No neurotoxic or convulsant effects were observed as a consequence of the radiation itself. When BBB-lesioned rats were challenged with 225 mg/kg Quin iv, epileptiform activity was observed on EEG analysis. Tonic-clonic seizures were induced by 225-450 mg/kg Quin. Light microscopic analysis showed a dose-related excitotoxic type of lesion restricted to the hippocampus ipsilateral to the irradiated side. Neuro-degeneration was prevented by local injection of 120 nmol D(-)2-amino-7-phosphonoheptanoic acid, a selective N-methyl-D-aspartate receptor antagonist. No lesions or EEG or behavioral modifications occurred after 450 mg/kg nicotinic acid, an inactive analog of Quin. The potential neurotoxic and convulsant effects of increased blood levels of Quin under conditions of altered BBB permeability are discussed.

  12. Carbofuran alters centrosome and spindle organization, and delays cell division in oocytes and mitotic cells.

    Science.gov (United States)

    Cinar, Ozgur; Semiz, Olcay; Can, Alp

    2015-04-01

    Although many countries banned of its usage, carbofuran (CF) is still one of the most commonly used carbamate derivative insecticides against insects and nematodes in agriculture and household, threatening the human and animal health by contaminating air, water, and food. Our goal was to evaluate the potential toxic effects of CF on mammalian oocytes besides mitotic cells. Caspase-dependent apoptotic pathway was assessed by immunofluorescence and western blot techniques. Alterations in the meiotic spindle formation after CF exposure throughout the in vitro maturation of mice oocyte-cumulus complexes (COCs) were analyzed by using a 3D confocal laser microscope. Maturation efficiency and kinetics were assessed by direct observation of the COCs. Results indicated that the number of TUNEL-positive cells increased in CF-exposed groups, particularly higher doses (>250 µM) in a dose-dependent fashion. The ratio of anticleaved caspase-3 labeled cells in those groups positively correlated with TUNEL-positivity. Western blot analysis confirmed a significant increase in active caspase-3 activity. CF caused a dose-dependent accumulation of oocytes at prometaphase-I (PM-I) of meiosis. Partial loss of spindle microtubules (MTs) was noted, which consequently gave rise to a diamond shape spindle. Aberrant pericentrin foci were noted particularly in PM-I and metaphase-I (M-I) stages. Conclusively, CF (1) induces programmed cell death in a dose-dependent manner, and (2) alters spindle morphology most likely through a mechanism that interacts with MT assembly and/or disorientation of pericentriolar proteins. Overall, data suggest that CF could give rise to aneuploidy or cell death in higher doses, therefore reduce fertilization and implantation rates.

  13. Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC is high. It is well known that the epithelial mesenchymal transition (EMT and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface

  14. Changes in the permeability of blood brain barrier and endothelial cell damage after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Ke Liu; Jiansheng Li

    2006-01-01

    OBJECTIVE: To investigate the effect of endothelial cells on the permeability of blood brain barrier (BBB) after brain injury and its effect mechanism.DATA SOURCES: We searched for the articles of permeability of BBB and endothelial cell injury after brain ischemia, which were published between January 1982 and December 2005, with the key words of "cerebral ischemia damage,blood brain barrier ( BBB),permeability,effect of endothelial cell (EC) and its variation mechanism"in English.STUDY SELECTION: The materials were primarily selected. The articles related to the changes in the permeability of BBB and the effect of endothelial cells as well as the change mechanism after cerebral ischemia damage were chosen. Repetitive studies or review articles were excluded.DATA EXTRACTION: Totally 55 related articles were collected, and 35 were excluded due to repetitive or review articles, finally 20 articles were involved.DATA SYNTHESIS: The content or viewpoints of involved literatures were analyzed. Cerebral ischemia had damage for endothelial cells, such as the inflow of a lot of Ca2+, the production of nitrogen monoxide and oxygen free radical, and aggravated destruction of BBB. After acceptors of inflammatory mediators on cerebrovascular endothelial cell membrane, such as histamine, bradykinin , 5-hydroxytryptamine and so on are activated, endothelial cells shrink and the permeability of BBB increases. Its mechanism involves in the inflow of extracellular Ca2+and the release of intracellular Ca2+ in the cells. Glycocalyx molecule on the surface of endothelial cell, having structural polytropy, is the determinative factor of the permeability of BBB. VEGF, intensively increasing the vasopermeability and mainly effecting on postcapillary vein and veinlet, is the strongest known blood vessel permeation reagent. Its chronic overexpression in the brain can lead the destruction of BBB.CONCLUSION: The injury of endothelial cell participants in the pathological mechanism of BBB

  15. Alteration of skin hydration and its barrier function by vehicle and permeation enhancers: a study using TGA, FTIR, TEWL and drug permeation as markers.

    Science.gov (United States)

    Shah, D K; Khandavilli, S; Panchagnula, R

    2008-09-01

    Vehicles and permeation enhancers (PEs) used in transdermal drug delivery (TDD) of a drug can affect skin hydration, integrity and permeation of the solute administered. This investigation was designed to study the effect of the most commonly used vehicles and PEs on rat skin hydration, barrier function and permeation of an amphiphilic drug, imipramine hydrochloride (IMH). An array of well-established techniques were used to confirm the findings of the study. Thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy were used to determine changes in skin hydration. Alteration of the stratum corneum (SC) structure was investigated using FTIR studies. To monitor the barrier function alteration, transepidermal water loss (TEWL) measurement and permeation studies were performed. Our findings indicate that with hydration, there was an increase in the bound water content of the skin, and pseudoequilibrium of hydration (a drastic decrease in hydration rate) was achieved at around 12 h. Hydration increased the ratio between amide-I and amide-II peaks in FTIR and reduced the C-H stretching peak area. Both propylene glycol (PG) and ethanol (EtOH) dehydrated skin, with the latter showing a predominant effect. Furthermore, it was confirmed that PG and EtOH decreased the bound water content due to alteration in the protein domains and extraction of SC lipids, respectively. The effect of hydration on the SC was found to be similar to that reported for temperature. Permeation studies revealed that the dehydration caused by vehicles decreased IMH flux, whereas the flux was enhanced by PEs. The role of partition was predominant for the permeation of IMH through dehydrated skin. A synergistic effect was observed for PG and menthol in the enhancement of IMH. Further findings provided strong evidence that PG affects protein domains and EtOH extracts lipids from the bilayer. Both PG and EtOH, with or without PEs, increased TEWL. Initial TEWL was well

  16. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  17. Alterations in cell surface area and deformability of individual human red blood cells in stored blood

    CERN Document Server

    Park, HyunJoo; Lee, SangYun; Kim, Kyoohyun; Sohn, Yong-Hak; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusion. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called CPDA-1. With 3-D quantitative phase imaging techniques, the optical measurements of the 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and their progressive alterations in stored RBCs. Our results show that the stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within 2 weeks which was accompanied with significant ...

  18. Gap junction proteins in the blood-brain barrier control nutrient-dependent reactivation of Drosophila neural stem cells.

    Science.gov (United States)

    Spéder, Pauline; Brand, Andrea H

    2014-08-11

    Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion.

  19. Active barrier films of PET for solar cell application: Processing and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Rossi, Gabriella; Scarfato, Paola; Incarnato, Loredana [Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132 - 84084 Fisciano (Italy)

    2014-05-15

    A preliminary investigation was carried out on the possibility to improve the protective action offered by the standard multilayer structures used to encapsulate photovoltaic devices. With this aim, a commercial active barrier PET-based material, able to absorb oxygen when activated by liquid water, was used to produce flexible and transparent active barrier films, by means of a lab-scale film production plant. The obtained film, tested in terms of thermal, optical and oxygen absorption properties, shows a slow oxygen absorption kinetics, an acceptable transparency and an easy roll-to-roll processability, so proving itself as a good candidate for the development of protective coating for solar cells against the atmospheric degradation agents like the rain.

  20. Breaking down the barriers to commercialization of fuel cells in transportation through Government - industry R&D programs

    Energy Technology Data Exchange (ETDEWEB)

    Chalk, S.G. [Dept. of Energy, Washington, DC (United States); Venkateswaran, S.R. [Energetics, Inc., Columbia, MD (United States)

    1996-12-31

    PEM fuel cell technology is rapidly emerging as a viable propulsion alternative to the internal combustion engine. Fuel cells offer the advantages of low emissions, high efficiency, fuel flexibility, quiet and continuous operation, and modularity. Over the last decade, dramatic advances have been achieved in the performance and cost of PEM fuel cell technologies for automotive applications. However, significant technical barriers remain to making fuel cell propulsion systems viable alternatives to the internal combustion engine. This paper focuses on the progress achieved and remaining technical barriers while highlighting Government-industry R&D efforts that are accelerating fuel cell technology toward commercialization.

  1. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Science.gov (United States)

    Haskó, János; Fazakas, Csilla; Molnár, Judit; Nyúl-Tóth, Ádám; Herman, Hildegard; Hermenean, Anca; Wilhelm, Imola; Persidsky, Yuri; Krizbai, István A.

    2014-01-01

    During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma. PMID:24815068

  2. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    János Haskó

    2014-05-01

    Full Text Available During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB. The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2; therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A, GPR18 (transcriptional variant 1 and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A, GPR18 (transcriptional variants 1 and 2, GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

  3. The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

    Science.gov (United States)

    de Vallière, Cheryl; Vidal, Solange; Clay, Ieuan; Jurisic, Giorgia; Tcymbarevich, Irina; Lang, Silvia; Ludwig, Marie-Gabrielle; Okoniewski, Michal; Eloranta, Jyrki J; Kullak-Ublick, Gerd A; Wagner, Carsten A; Rogler, Gerhard; Seuwen, Klaus

    2015-09-15

    The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.

  4. Interface Engineering of Organic Schottky Barrier Solar Cells and Its Application in Enhancing Performances of Planar Heterojunction Solar Cells.

    Science.gov (United States)

    Jin, Fangming; Su, Zisheng; Chu, Bei; Cheng, Pengfei; Wang, Junbo; Zhao, Haifeng; Gao, Yuan; Yan, Xingwu; Li, Wenlian

    2016-05-17

    In this work, we describe the performance of organic Schottky barrier solar cells with the structure of ITO/molybdenum oxide (MoOx)/boron subphthalocyanine chloride (SubPc)/bathophenanthroline (BPhen)/Al. The SubPc-based Schottky barrier solar cells exhibited a short-circuit current density (Jsc) of 2.59 mA/cm(2), an open-circuit voltage (Voc) of 1.06 V, and a power conversion efficiency (PCE) of 0.82% under simulated AM1.5 G solar illumination at 100 mW/cm(2). Device performance was substantially enhanced by simply inserting thin organic hole transport material into the interface of MoOx and SubPc. The optimized devices realized a 180% increase in PCE of 2.30% and a peak Voc as high as 1.45 V was observed. We found that the improvement is due to the exciton and electron blocking effect of the interlayer and its thickness plays a vital role in balancing charge separation and suppressing quenching effect. Moreover, applying such interface engineering into MoOx/SubPc/C60 based planar heterojunction cells substantially enhanced the PCE of the device by 44%, from 3.48% to 5.03%. Finally, we also investigated the requirements of the interface material for Schottky barrier modification.

  5. Lipid body accumulation alters calcium signaling dynamics in immune cells.

    Science.gov (United States)

    Greineisen, William E; Speck, Mark; Shimoda, Lori M N; Sung, Carl; Phan, Nolwenn; Maaetoft-Udsen, Kristina; Stokes, Alexander J; Turner, Helen

    2014-09-01

    There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcɛRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signaling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcɛRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signaling pathway and its downstream targets.

  6. Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Christof Keller

    Full Text Available BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-β for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-β, even though TGF-β signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-β. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-β-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-β-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability

  7. Bactericidal Antibiotics Increase Hydroxyphenyl Fluorescein Signal by Altering Cell Morphology

    DEFF Research Database (Denmark)

    Paulander, Wilhelm; Wang, Ying; Folkesson, Sven Anders;

    2014-01-01

    It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH center dot) formation. Flow cytometric detection of OH center dot by hydroxyphenyl fluorescein (HPF) probe oxidation was used...... to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS) concentration. Independently of antibiotics, increased fluorescence was seen...... for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP). Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin...

  8. Bactericidal antibiotics increase hydroxyphenyl fluorescein signal by altering cell morphology.

    Directory of Open Access Journals (Sweden)

    Wilhelm Paulander

    Full Text Available It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH• formation. Flow cytometric detection of OH• by hydroxyphenyl fluorescein (HPF probe oxidation was used to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS concentration. Independently of antibiotics, increased fluorescence was seen for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP. Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin such relationships, methods for detecting bacterial metabolites at a cellular level are needed.

  9. Lowering extracellular chloride concentration alters outer hair cell shape.

    Science.gov (United States)

    Cecola, R P; Bobbin, R P

    1992-08-01

    In general, increasing external K+ concentration, as well as exposure to hypotonic medium, induces a shortening of outer hair cells (OHCs) accompanied by an increase in width and volume. One possible mechanism suggested for these changes is a movement of Cl- and/or water across the cell membrane. We therefore examined the role of Cl- in OHC volume maintenance by testing the effect of decreasing extracellular Cl- concentration on OHC length and shape. In addition, the effect of hypotonic medium was examined. OHCs were isolated from guinea pig cochleae, mechanically dissociated and dispersed, and placed in a modified Hanks balanced salt solution (HBS). Exposing the cells to a Cl(-)-free HBS produced an initial shortening, which was rapidly followed by an increase in length. After about 9 min of exposure to Cl(-)-free HBS, the cells appeared to lose all water and collapsed. Upon return to normal HBS, the OHCs returned to their normal shape. We speculate that the collapse of the OHCs may be due to the loss of intracellular Cl-, which, in turn, resulted in the loss of intracellular K+ and water. The results indicate that Cl- contributes greatly to the maintenance of OHC volume. In addition, we confirmed that isolated OHCs swell in hypotonic medium and maintain their swollen state until returned to normal medium. The mechanism for maintenance of the swollen state is unknown.

  10. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B;

    1996-01-01

    Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

  11. Spatial distributions of red blood cells significantly alter local haemodynamics.

    Directory of Open Access Journals (Sweden)

    Joseph M Sherwood

    Full Text Available Although bulk changes in red blood cell concentration between vessels have been well characterised, local distributions are generally overlooked. Red blood cells aggregate, deform and migrate within vessels, forming heterogeneous distributions which have considerable effect on local haemodynamics. The present study reports data on the local distribution of human red blood cells in a sequentially bifurcating microchannel, representing the branching geometry of the microvasculature. Imaging methodologies with simple extrapolations are used to infer three dimensional, time-averaged velocity and haematocrit distributions under a range of flow conditions. Strong correlation between the bluntness of the velocity and haematocrit profiles in the parent branch of the geometry is observed and red blood cell aggregation has a notable effect on the observed trends. The two branches of the first bifurcation show similar characteristics in terms of the shapes of the profiles and the extent of plasma skimming, despite the difference in geometric configuration. In the second bifurcation, considerable asymmetry between the branches in the plasma skimming relationship is observed, and elucidated by considering individual haematocrit profiles. The results of the study highlight the importance of considering local haematocrit distributions in the analysis of blood flow and could lead to more accurate computational models of blood flow in microvascular networks. The experimental approaches developed in this work provide a foundation for further examining the characteristics of microhaemodynamics.

  12. Alterations of red blood cell metabolome in overhydrated hereditary stomatocytosis.

    NARCIS (Netherlands)

    Darghouth, D.; Koehl, B.; Heilier, J.F.; Madalinski, G.; Bovee, P.H.; Bosman, G.J.C.G.M.; Delaunay, J.; Junot, C.; Romeo, P.H.

    2011-01-01

    Overhydrated hereditary stomatocytosis, clinically characterized by hemolytic anemia, is a rare disorder of the erythrocyte membrane permeability to monovalent cations, associated with mutations in the Rh-associated glycoprotein gene. We assessed the red blood cell metabolome of 4 patients with this

  13. Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery

    NARCIS (Netherlands)

    Heijink, I H; Brandenburg, S M; Postma, D S; van Oosterhout, A J M

    2012-01-01

    Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in

  14. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard

    2016-01-01

    Full Text Available Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  15. GM-CSF alters dendritic cells in autoimmune diseases.

    Science.gov (United States)

    Li, Bao-Zhu; Ye, Qian-Ling; Xu, Wang-Dong; Li, Jie-Hua; Ye, Dong-Qing; Xu, Yuekang

    2013-11-01

    Autoimmune diseases arise from an inappropriate immune response against self components, including macromolecules, cells, tissues, organs etc. They are often triggered or accompanied by inflammation, during which the levels of granulocyte macrophage colony-stimulating factor (GM-CSF) are elevated. GM-CSF is an inflammatory cytokine that has profound impact on the differentiation of immune system cells of myeloid lineage, especially dendritic cells (DCs) that play critical roles in immune initiation and tolerance, and is involved in the pathogenesis of autoimmune diseases. Although GM-CSF was discovered decades ago, recent studies with some new findings have shed an interesting light on the old hematopoietic growth factor. In the inflammatory autoimmune diseases, GM-CSF redirects the normal developmental pathway of DCs, conditions their antigen presentation capacities and endows them with unique cytokine signatures to affect autoimmune responses. Here we review the latest advances in the field, with the aim of demonstrating the effects of GM-CSF on DCs and their influences on autoimmune diseases. The summarized knowledge will help to design DC-based strategies for the treatment of autoimmune diseases.

  16. Butachlor, a suspected carcinogen, alters growth and transformation characteristics of mouse liver cells.

    Science.gov (United States)

    Ou, Y H; Chung, P C; Chang, Y C; Ngo, F Q; Hsu, K Y; Chen, F D

    2000-12-01

    Butachlor is a widely used herbicide in Asia and South America. Previous investigations have indicated that it is a suspected carcinogen. To understand more about the biological effects of butachlor on cultured cells and the mechanism(s) of its carcinogenicity, we studied the alteration of the growth characteristics that was induced by butachlor in normal mouse liver cells (BNL CL2). This study demonstrates that butachlor decreases the population-doubling time of BNL CL2 cells, suggesting that it stimulates cell proliferation. To support this finding, a thymidine incorporation assay was conducted and a similar result that butachlor stimulates cell proliferation was elucidated. In addition, we show that butachlor increases the saturation density of the BNL CL2 cells. When combined with the tumor initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), butachlor transforms cells efficiently, as demonstrated by loss of contact inhibition. These findings indicate that butachlor alters the growth characteristics of BNL CL2 cells and suggest that butachlor may induce malignant transformation through stimulation of cell proliferation, alteration of cell cycle regulation, and suppression of cell density-dependent inhibition of proliferation.

  17. Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Johnson

    2012-02-01

    Full Text Available Helicobacter pylori lives within the mucus layer of the human stomach, in close proximity to gastric epithelial cells. While a great deal is known about the effects of H. pylori on human cells and the specific bacterial products that mediate these effects, relatively little work has been done to investigate alterations in H. pylori that may be triggered by bacterial contact with human cells. In this review, we discuss the spectrum of changes in bacterial physiology and morphology that occur when H. pylori is in contact with gastric epithelial cells. Several studies have reported that cell contact causes alterations in H. pylori gene transcription. In addition, H. pylori contact with gastric epithelial cells promotes the formation of pilus-like structures at the bacteria-host cell interface. The formation of these structures requires multiple genes in the cag pathogenicity island, and these structures are proposed to have an important role in the type IV secretion system-dependent process through which CagA enters host cells. Finally, H. pylori contact with epithelial cells can promote bacterial replication and the formation of microcolonies, phenomena that are facilitated by the acquisition of iron and other nutrients from infected cells. In summary, the gastric epithelial cell surface represents an important niche for H. pylori, and upon entry into this niche, the bacteria alter their behavior in a manner that optimizes bacterial proliferation and persistent colonization of the host.

  18. Use of Genetically Altered Stem Cells for the Treatment of Huntington’s Disease

    Directory of Open Access Journals (Sweden)

    Andrew T. Crane

    2014-03-01

    Full Text Available Transplantation of stem cells for the treatment of Huntington’s disease (HD garnered much attention prior to the turn of the century. Several studies using mesenchymal stem cells (MSCs have indicated that these cells have enormous therapeutic potential in HD and other disorders. Advantages of using MSCs for cell therapies include their ease of isolation, rapid propagation in culture, and favorable immunomodulatory profiles. However, the lack of consistent neuronal differentiation of transplanted MSCs has limited their therapeutic efficacy to slowing the progression of HD-like symptoms in animal models of HD. The use of MSCs which have been genetically altered to overexpress brain derived neurotrophic factor to enhance support of surviving cells in a rodent model of HD provides proof-of-principle that these cells may provide such prophylactic benefits. New techniques that may prove useful for cell replacement therapies in HD include the use of genetically altering fate-restricted cells to produce induced pluripotent stem cells (iPSCs. These iPSCs appear to have certain advantages over the use of embryonic stem cells, including being readily available, easy to obtain, less evidence of tumor formation, and a reduced immune response following their transplantation. Recently, transplants of iPSCs have shown to differentiate into region-specific neurons in an animal model of HD. The overall successes of using genetically altered stem cells for reducing neuropathological and behavioral deficits in rodent models of HD suggest that these approaches have considerable potential for clinical use. However, the choice of what type of genetically altered stem cell to use for transplantation is dependent on the stage of HD and whether the end-goal is preserving endogenous neurons in early-stage HD, or replacing the lost neurons in late-stage HD. This review will discuss the current state of stem cell technology for treating the different stages of HD and

  19. Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

    Science.gov (United States)

    Sun, Yujie; Kaur, Kawaljit; Kanayama, Keiichi; Morinaga, Kenzo; Park, Sil; Hokugo, Akishige; Kozlowska, Anna; McBride, William H; Li, Jun; Jewett, Anahid; Nishimura, Ichiro

    2016-09-23

    Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

  20. Schottky barrier enhancement on n-InP solar cell applications

    DEFF Research Database (Denmark)

    Clausen, Thomas; Leistiko, Otto

    1994-01-01

    It is demonstrated that the Schottky barrier height on n-type InP can be enhanced to values close to the energy bandgap (1.35 eV) by employing a AuZnCr metallization. The process is simple and requires only mild and fast annealing sequences with temperatures not exceeding 500°C. Also, no critical...... epitaxial growth step of junctions is needed, making the process fairly cheap. Thus, prospects for an efficient and simple solar cell device structure for space application purposes based on highly radiant-resistant InP are greatly improved...

  1. Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

    Science.gov (United States)

    Lewis, Shanta

    Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4microg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4microg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.

  2. Prenatal cadmium exposure alters postnatal immune cell development and function

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Miranda L.; Holásková, Ida; Elliott, Meenal; Brundage, Kathleen M.; Schafer, Rosana; Barnett, John B., E-mail: jbarnett@hsc.wvu.edu

    2012-06-01

    Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl{sub 2} (10 ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7 weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7 weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2 weeks of age. At 7 weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4{sup +}FoxP3{sup +}CD25{sup +} (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8{sup +}CD223{sup +} T cells were markedly decreased in the spleens in all offspring at 7 weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can

  3. Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells.

    Directory of Open Access Journals (Sweden)

    Mehrnaz Nouri

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE, the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers. These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms and at 14 days (i.e., at the stage of paralysis after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.

  4. FGFR gene alterations in lung squamous cell carcinoma are potential targets for the multikinase inhibitor nintedanib.

    Science.gov (United States)

    Hibi, Masaaki; Kaneda, Hiroyasu; Tanizaki, Junko; Sakai, Kazuko; Togashi, Yosuke; Terashima, Masato; De Velasco, Marco Antonio; Fujita, Yoshihiko; Banno, Eri; Nakamura, Yu; Takeda, Masayuki; Ito, Akihiko; Mitsudomi, Tetsuya; Nakagawa, Kazuhiko; Okamoto, Isamu; Nishio, Kazuto

    2016-11-01

    Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non-small cell lung cancer. We have now applied next-generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined in vitro, and its effects on tumor formation were examined in vivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG-positive LSCC cell lines in association with attenuation of the FGFR1-ERK signaling pathway in vitro and in vivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy.

  5. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  6. Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    WEI-DONG JI; JIA-KUN CHEN; JIA-CHUN LU; ZHONG-LIANG WU; FEI YI; SU-MEI FENG

    2006-01-01

    To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immoral human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P<0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

  7. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    Science.gov (United States)

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S.; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam

    2016-01-01

    ABSTRACT The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV. IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level

  8. Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells

    DEFF Research Database (Denmark)

    Del Vecchio, Giovanna; Tscheik, Christian; Tenz, Kareen

    2012-01-01

    Claudin-5 is a tight junction (TJ) protein which limits the diffusion of small hydrophilic molecules. Thus, it represents a potential pharmacological target to improve drug delivery to the tissues protected by claudin-5-dependent barriers. Sodium caprate is known as an absorption enhancer which...... opens the paracellular space acting on TJ proteins and actin cytoskeleton. Its action on claudin-5 is not understood so far. Epithelial and endothelial systems were used to evaluate the effect of caprate on claudin-5 in TJ-free cells and on claudin-5 fully integrated in TJ. To this aim, confocal...... of endothelial and epithelial cells. In conclusion, the study further elucidates the cellular effects of caprate at the tight junctions....

  9. Impact of secondary barriers on copper-indium-gallium-selenide solar-cell operation

    Science.gov (United States)

    Pudov, Alexei O.

    Thin-film solar cells based on CuInSe2 (CIS) absorber with a band gap of Eg = 1.0 eV and also based on CuIn1-x GaxSe2 (CIGS) alloy absorbers with a band-gap range of Eg = 1.0--1.67 eV are investigated in this work. Intermediate "buffer" semiconductor layers in p-n junctions of CIGS solar cells often improve photodiode properties of the devices. The primary goal of the thesis is to study secondary barriers in the conduction band at the buffer/absorber interface, which may limit current transport and thus reduce the efficiency of the solar cells. The secondary goal is to explore alternative wide-bandgap buffers in CIGS cell structures. CIGS cells with standard CdS buffer layers, and alternative ZnS(O,OH) and InS(O,OH) buffer layers were studied. CdS/CuIn1-xGaxSe2 solar cells with variable Ga content have a range of conduction-band offsets (DeltaEc) in the junction from moderately positive (spike offsets) in CdS/CuInSe2 to moderately negative (cliff offsets) in CdS/CuGaSe 2. Moderate conduction-band spikes in CdS/CIS and low-Ga CdS/CIGS are expected to cause distortions in diode current-voltage (J-V) curves of such solar cells under "red" illumination (hnu < Eg(buffer)); no J-V distortions are expected for high-Ga CdS/CIGS with cliff offsets. These predictions were confirmed in experiments: the distortions were absent for cells with Eg above 1.2--1.3 eV, at which CdS/CIGS DeltaE c is near zero. Experiments and numerical simulations showed that one approach to reduce secondary barriers and J-V distortions in low-Ga high-spike cells is to thin the buffer layer(s). Blue photons (hnu above Eg(buffer)) in the solar spectrum induce photoconductivity in the otherwise compensated buffers, which also results in lowering of the secondary barriers. It was shown that CIGS cells with CdS, InS(O,OH), and ZnS(O,OH) buffers have a similar response to "blue" photons: J-V distortion, if present under red light, is reduced or entirely disappears with blue-light exposure within minutes

  10. Migration of tumor cells and leukocytes in extracellular matrix : proteolytic and nonproteolytic strategies for overcoming tissue barriers

    OpenAIRE

    Wolf, Katarina

    2003-01-01

    The extracellular matrix within connective tissues represents a structural scaffold as well as a barrier for motile cells, such as invading tumor cells or passenger leukocytes. It remains unclear how different cell types utilize matrix-degrading enzymes for proteolytic migration strategies and, on the other hand, non-proteolytic strategies to overcome 3D fibrillar matrix networks. To monitor cell migration, a 3D collagen model in vitro or the mouse dermis in vivo were used, in combination wit...

  11. Manipulating Somatic Cells to Remove Barriers in Induced Pluripotent Stem Cell Reprogramming

    OpenAIRE

    Chung, Julia

    2013-01-01

    Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells...

  12. Induction of apoptosis in human myeloid leukemia cells by remote exposure of resistive barrier cold plasma.

    Science.gov (United States)

    Thiyagarajan, Magesh; Anderson, Heather; Gonzales, Xavier F

    2014-03-01

    Cold atmospheric plasma (CAP), an ambient temperature ionized gas, is gaining extensive interest as a promising addition to anti-tumor therapy primarily due to the ability to generate and control delivery of electrons, ions, excited molecules, UV photons, and reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) to a specific site. The heterogeneous composition of CAP offers the opportunity to mediate several signaling pathways that regulate tumor cells. Consequently, the array of CAP generated products has limited the identification of the mechanisms of action on tumor cells. The aim of this work is to assess the cell death response of human myeloid leukemia cells by remote exposure to CAP generated RNS by utilizing a novel resistive barrier discharge system that primarily produces RNS. The effect of variable treatments of CAP generated RNS was tested in THP-1 cell (human monocytic leukemia cell line), a model for hematological malignancy. The number of viable cells was evaluated with erythrosine-B staining, while apoptosis and necrosis was assessed by endonuclease cleavage observed by agarose gel electrophoresis and detection of cells with the exclusionary dye propidium iodide and fluorescently labeled annexin-V by flow cytometry and fluorescent microscopy. Our observations indicate that treatment dosage levels of 45 s of exposure to CAP emitted RNS-induced apoptotic cell death and for higher dosage conditions of ≥50 s of exposure to CAP induced necrosis. Overall the results suggest that CAP emitted RNS play a significant role in the anti-tumor potential of CAP.

  13. Altered expression of glycosaminoglycans in metastatic 13762NF rat mammary adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Steck, P.A.; Cheong, P.H.; Nakajima, M.; Yung, W.K.A.; Moser, R.P.; Nicolson, G.L.

    1987-02-24

    A difference in the expression and metabolism of (/sup 35/S)sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate. These results suggested that altered glycosaminoglycan expression and metabolism may be associated with the metastatic process in 13762NF rat mammary tumor cells.

  14. Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis.

    Science.gov (United States)

    Viola, Katharina; Kopf, Sabine; Rarova, Lucie; Jarukamjorn, Kanokwan; Kretschy, Nicole; Teichmann, Mathias; Vonach, Caroline; Atanasov, Atanas G; Giessrigl, Benedikt; Huttary, Nicole; Raab, Ingrid; Krieger, Sigurd; Strnad, Miroslav; de Martin, Rainer; Saiko, Philipp; Szekeres, Thomas; Knasmüller, Siegfried; Dirsch, Verena M; Jäger, Walter; Grusch, Michael; Dolznig, Helmut; Mikulits, Wolfgang; Krupitza, Georg

    2013-07-01

    Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.

  15. Cell invasion through basement membrane: the anchor cell breaches the barrier.

    Science.gov (United States)

    Hagedorn, Elliott J; Sherwood, David R

    2011-10-01

    Cell invasion through basement membrane (BM) is a specialized cellular behavior critical to many normal developmental events, immune surveillance, and cancer metastasis. A highly dynamic process, cell invasion involves a complex interplay between cell-intrinsic elements that promote the invasive phenotype, and cell-cell and cell-BM interactions that regulate the timing and targeting of BM transmigration. The intricate nature of these interactions has made it challenging to study cell invasion in vivo and model in vitro. Anchor cell invasion in Caenorhabditis elegans is emerging as an important experimental paradigm for comprehensive analysis of BM invasion, revealing the gene networks that specify invasive behavior and the interactions that occur at the cell-BM interface.

  16. Effects of Chemotherapy-Induced Alterations in Cell Mechanical Properties on Cancer Metastasis

    Science.gov (United States)

    Prathivadhi, Sruti; Ekpenyong, Andrew; Nichols, Michael; Taylor, Carolyn; Ning, Jianhao

    Biological cells can modulate their mechanical properties to suit their functions and in response to changes in their environment. Thus, mechanical phenotyping of cells has been employed for tracking stem cell differentiation, bacterial infection, cell death, etc. Malignant transformation of cells also involves changes in mechanical properties. However, the extent to which mechanical properties of cancer cells contribute to metastasis is not well understood. Yet, more than 90% of all cancer deaths are directly related to metastasis. Transit of cells through the microcirculation is one of the key features of metastasis. We hypothesize that cancer treatment regimens do inadvertently alter cell mechanical properties in ways that might promote cancer metastasis. We use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation to determine if in-vivo-like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that cancer cells treated with chemotherapeutic drugs such as daunorubicin, become more deformable at short timescales (0.1 s) and transit faster through the device. Our results are first steps in evaluating the pro- or anti-metastatic effects of chemotherapeutic drugs based on their induced alterations in cell mechanical properties.

  17. Gadd45a inhibits cell migration and invasion by altering the global RNA expression.

    Science.gov (United States)

    Shan, Zhanhai; Li, Guiyuan; Zhan, Qimin; Li, Dan

    2012-09-01

    Gadd45a, the first well-defined p53 downstream gene, can be induced by multiple DNA-damaging agents, which plays important roles in the control of cell cycle checkpoint, DNA repair process and signaling transduction. Our previous findings suggested that Gadd45a maintains cell-cell adhesion and cell contact inhibition. However, little is known about how Gadd45a participates in the suppression of malignancy in human cancer cells. To examine the functions of Gadd45a in cell invasion and metastasis, we performed the adhesion, wound-healing and transwell assays in Gadd45a (+/+) and Gadd45a (-/-) MEF cell lines. We found the adhesion, migration and invasive abilities were much higher in Gadd45a deficient cells. We furthermore applied high-throughput cDNA microarray analysis and bioinformatics analysis to analyze the mechanisms of Gadd45a gene in invasion and metastasis. Compared with the Gadd45a wild type cells, the Gadd45a deficient cells showed a wide range of transcripts alterations. The altered gene pathways were predicted by the MAS software, which indicated focal adhesion,cell communication,ECM-receptor interaction as the three main pathways. Real-time PCR was employed to validate the differentially expressed genes. Interestingly, we figured out that the deregulations of these genes are caused neither by genomic aberrations nor methylation status. These findings provided a novel insight that Gadd45a may involve in tumor progression by regulating related genes expressions.

  18. Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high let radiation

    Science.gov (United States)

    Hei, T. K.; Zhao, Y. L.; Roy, D.; Piao, C. Q.; Calaf, G.; Hall, E. J.

    Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/μm alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three ( DCC, DNA-PK and p21 CIPI) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.

  19. Effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Zheng Wu; Yi Lüi; Bo Wang; Chang Liu; Zuo-Ren Wang,

    2003-01-01

    AIM: Pancreatic cancer in the head is frequently accompanied by jaundice and high bile acid level in serum. This study focused on the direct effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer.METHODS: Pancreatic cancer cell lines PANC-1, MIA PaCa2 and PGHAM-1 were explored in this study. The cell lines were cultured in media supplemented with certain bile acids,CA, DCA, LCA, TCDC, TDCA and GCA. Their influence on cell growth was measured with MTT assay after 72 h of incubation. Cell cycles of PANC-1 cells in 40 μM of bile acids media were analyzed by flow cytometry. Ultrastructural alteration of PANC-1 cells induced by DCA was observed using scanning and transmission electron microscope (SEM and TEM).RESULTS: At various concentrations of bile acids and incubation time, no enhanced effects of bile acids on cell proliferation were observed. Significant inhibitory effects were obtained in almost all media with bile acids. DCA and CA increased the percentage of G0+G1 phase cells, while GCA and TDCA elevated the S phase cell number. After 48 h of incubation in DCA medium, PANC-1 cells showed some structural damages such as loss of their microvilli and vacuolization of organelles in cytoplasm.CONCLUSION: Bile acids can reduce proliferation of pancreatic cancer cells due to their direct cytotoxicity. This result implies that elevation of bile acids in jaundiced serum may inhibit pancreatic cancer progression.

  20. Pericellular matrix formation alters the efficiency of intracellular uptake of oligonucleotides in osteosarcoma cells.

    Science.gov (United States)

    Suzuki, Yoshitaka; Nishida, Yoshihiro; Naruse, Takahiro; Gemba, Takefumi; Ishiguro, Naoki

    2009-03-01

    One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.

  1. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    Science.gov (United States)

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+).

  2. Tumor-altered dendritic cell function: implications for anti-tumor immunity

    Directory of Open Access Journals (Sweden)

    Kristian Michael Hargadon

    2013-07-01

    Full Text Available Dendritic cells are key regulators of both innate and adaptive immunity, and the array of immunoregulatory functions exhibited by these cells is dictated by their differentiation, maturation, and activation status. Although a major role for these cells in the induction of immunity to pathogens has long been appreciated, data accumulated over the last several years has demonstrated that DC are also critical regulators of anti-tumor immune responses. However, despite the potential for stimulation of robust anti-tumor immunity by DC, tumor-altered DC function has been observed in many cancer patients and tumor-bearing animals and is often associated with tumor immune escape. Such dysfunction has significant implications for both the induction of natural anti-tumor immune responses as well as the efficacy of immunotherapeutic strategies that target endogenous DC in situ or that employ exogenous DC as part of anti-cancer immunization maneuvers. In this review, the major types of tumor-altered DC function will be described, with emphasis on recent insights into the mechanistic bases for the inhibition of DC differentiation from hematopoietic precursors, the altered programming of DC precursors to differentiate into myeloid-derived suppressor cells or tumor-associated macrophages, the suppression of DC maturation and activation, and the induction of immunoregulatory DC by tumors, tumor-derived factors, and tumor-associated cells within the milieu of the tumor microenvironment. The impact of these tumor-altered cells on the quality of the overall anti-tumor immune response will also be discussed. Finally, this review will also highlight questions concerning tumor-altered DC function that remain unanswered, and it will address factors that have limited advances in the study of this phenomenon in order to focus future research efforts in the field on identifying strategies for interfering with tumor-associated DC dysfunction and improving DC-mediated anti

  3. Altered cerebral vascular volumes and solute transport at the blood-brain barriers of two transgenic mouse models of Alzheimer's disease.

    Science.gov (United States)

    Do, Tuan Minh; Alata, Wael; Dodacki, Agnès; Traversy, Marie-Thérèse; Chacun, Hélène; Pradier, Laurent; Scherrmann, Jean-Michel; Farinotti, Robert; Calon, Frédéric; Bourasset, Fanchon

    2014-06-01

    We evaluated the integrity and function of the blood-brain barrier in 3xTg-AD mice aged 3-18 months and in APP/PS1 mice aged 8-months to determine the impacts of changes in amyloid and tau proteins on the brain vascular changes. The vascular volume (Vvasc) was sub-normal in 3xTg-AD mice aged from 6 to 18 months, but not in the APP/PS1 mice. The uptakes of [(3)H]-diazepam by the brains of 3xTg-AD, APP/PS1 and their age-matched control mice were similar at all the times studied, suggesting that the simple diffusion of small solutes is unchanged in transgenic animals. The uptake of d-glucose by the brains of 18-month old 3xTg-AD mice, but not by those of 8-month old APP/PS1 mice, was reduced compared to their age-matched controls. Accordingly, the amount of Glut-1 protein was 1.4 times lower in the brain capillaries of 18 month-old 3xTg-AD mice than in those of age-matched control mice. We conclude that the brain vascular volume is reduced early in 3xTg-AD mice, 6 months before the appearance of pathological lesions, and that this reduction persists until they are at least 18 months old. The absence of alterations in the BBB of APP/PS1 mice suggests that hyperphosphorylated tau proteins contribute to the vascular changes that occur in AD.

  4. Endothelial cells derived from the blood-brain barrier and islets of Langerhans differ in their response to the effects of bilirubin on oxidative stress under hyperglycemic conditions

    Directory of Open Access Journals (Sweden)

    Jaime eKapitulnik

    2012-07-01

    Full Text Available Unconjugated bilirubin (UCB is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS. High glucose levels (hyperglycemia generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB. In the current study we show that UCB (1-40 M induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langherans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM glucose levels. While UCB (0.1-40 M did not alter ROS production in cells exposed to normal glucose, relatively low ('physiological' UCB concentrations (0.1-5 M attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20-40 M increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions.

  5. Altered expression of talin 1 in peripheral immune cells points to a significant role of the innate immune system in spontaneous autoimmune uveitis.

    Science.gov (United States)

    Degroote, Roxane L; Hauck, Stefanie M; Kremmer, Elisabeth; Amann, Barbara; Ueffing, Marius; Deeg, Cornelia A

    2012-07-19

    The molecular mechanism which enables activated immune cells to cross the blood-retinal barrier in spontaneous autoimmune uveitis is yet to be unraveled. Equine recurrent uveitis is the only spontaneous animal model allowing us to investigate the autoimmune mediated transformation of leukocytes in the course of this sight threatening disease. Hypothesizing that peripheral blood immune cells change their protein expression pattern in spontaneous autoimmune uveitis, we used DIGE to detect proteins with altered abundance comparing peripheral immune cells of healthy and ERU diseased horses. Among others, we found a significant downregulation of talin 1 in peripheral blood granulocytes of ERU specimen, pointing to changes in β integrin activation and indicating a significant role of the innate immune system in spontaneous autoimmune diseases.

  6. Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death.

    Science.gov (United States)

    Lantéri, Marion; Giordanengo, Valérie; Hiraoka, Nobuyoshi; Fuzibet, Jean-Gabriel; Auberger, Patrick; Fukuda, Minoru; Baum, Linda G; Lefebvre, Jean-Claude

    2003-12-01

    The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.

  7. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure

    OpenAIRE

    Coleman, Heather D.; Yan, Jimmy; Mansfield, Shawn D.

    2009-01-01

    Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba × grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in...

  8. Disodium Cromoglycate, A Mast-Cell Stabilizer, Alters Postradiation Regional Cerebral Blood Flow in Primates

    Science.gov (United States)

    1986-01-01

    ranD RISEARCH INSTITUTI SCIENTIFIC REPORTTC SR86-14 ELECTE JUL0186_ _ ~D DISODIUM CROMOGLYCATE , A MAST-CELL STABILIZER, ALTERS POSTRADIATION...were given the mast-cell stabilizers disodium cromoglycate (DSCC) or BRL 22321 (Beecham Phar- maceuticals, Research Division) before exposure to 100 Gy...flow could be mitigated by the pre- radiation administration of either disodium cromoglycate (DSCG) (Fisons Corporation, ledford, Mass.) or BRL 22321

  9. Flavonoid transport across RBE4 cells: A blood-brain barrier model.

    Science.gov (United States)

    Faria, Ana; Pestana, Diogo; Teixeira, Diana; Azevedo, Joana; De Freitas, Victor; Mateus, Nuno; Calhau, Conceição

    2010-06-01

    There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer's and Parkinson's diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.

  10. δ-Catenin promotes prostate cancer cell growth and progression by altering cell cycle and survival gene profiles

    Directory of Open Access Journals (Sweden)

    Chen Yan-Hua

    2009-03-01

    Full Text Available Abstract Background δ-Catenin is a unique member of β-catenin/armadillo domain superfamily proteins and its primary expression is restricted to the brain. However, δ-catenin is upregulated in human prostatic adenocarcinomas, although the effects of δ-catenin overexpression in prostate cancer are unclear. We hypothesized that δ-catenin plays a direct role in prostate cancer progression by altering gene profiles of cell cycle regulation and cell survival. Results We employed gene transfection and small interfering RNA to demonstrate that increased δ-catenin expression promoted, whereas its knockdown suppressed prostate cancer cell viability. δ-Catenin promoted prostate cancer cell colony formation in soft agar as well as tumor xenograft growth in nude mice. Deletion of either the amino-terminal or carboxyl-terminal sequences outside the armadillo domains abolished the tumor promoting effects of δ-catenin. Quantitative RT2 Profiler™ PCR Arrays demonstrated gene alterations involved in cell cycle and survival regulation. δ-Catenin overexpression upregulated cyclin D1 and cdc34, increased phosphorylated histone-H3, and promoted the entry of mitosis. In addition, δ-catenin overexpression resulted in increased expression of cell survival genes Bcl-2 and survivin while reducing the cell cycle inhibitor p21Cip1. Conclusion Taken together, our studies suggest that at least one consequence of an increased expression of δ-catenin in human prostate cancer is the alteration of cell cycle and survival gene profiles, thereby promoting tumor progression.

  11. Dyslipidemia-associated alterations in B cell subpopulation frequency and phenotype during experimental atherosclerosis.

    Science.gov (United States)

    Rincón-Arévalo, Héctor; Castaño, Diana; Villa-Pulgarín, Janny; Rojas, Mauricio; Vásquez, Gloria; Correa, Luis A; Ramírez-Pineda, José R; Yassin, Lina M

    2016-04-01

    Lymphocytes, the cellular effectors of adaptive immunity, are involved in the chronic inflammatory process known as atherosclerosis. Proatherogenic and atheroprotective properties have been ascribed to B cells. However, information regarding the role of B cells during atherosclerosis is scarce. Both the frequency and the phenotype of B cell subpopulations were studied by flow cytometry in wild type and apolipoprotein-E-deficient (apoE(-/-)) mice fed a high-fat (HFD) or control diet. Whereas the proportion of follicular cells was decreased, transitional 1-like cells were increased in mice with advanced atherosclerotic lesions (apoE(-/-) HFD). B cells in atherosclerotic mice were more activated, indicated by their higher surface expression of CD80, CD86, CD40 and CD95 and increased serum IgG1 levels. In the aorta, a decreased frequency of B cells was observed in mice with advanced atherosclerosis. Low expression of CD19 was observed on B cells from the spleen, aorta and lymph nodes of apoE(-/-) HFD mice. This alteration correlated with serum levels of IgG1 and cholesterol. A reduction in CD19 expression was induced in splenic cells from young apoE(-/-) mice cultured with lipemic serum. These results show that mice with advanced atherosclerosis display a variety of alterations in the frequency and phenotype of B lymphocytes, most of which are associated with dyslipidemia.

  12. Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Grøn, B; Mandel, U

    1998-01-01

    Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate......-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both...... epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells....

  13. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  14. Alteration of cadherin isoform expression and inhibition of gap junctions in stomach carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To explore cell malignant phenotype correlated changes of cell surface adhesion molecules and cell-cell communication in carcinogenesis, human stomach transformed and cancer cell lines were investigated. Expressions of E-cadherin, N-cadherin, ?-catenin, ?-catenin as well as gap junction (GJ) protein Cx32 were studied by utilization of immunoblotting, immunocytochemical and fluorescent dye transfer methods. Mammalian normal stomach mucosal cells expressed E-cadherin but not N-cadherin. E-cadherin immunofluorescence was detected at cell membranous adherens junctions (AJ) where colocalization with immunofluorescent staining of inner surface adhesion plaque proteins ?- and ?-catenins was observed. The existence of E-cadherin/ catenin (?-, ?-) protein complexes as AJ was suggested. In transformed and stomach cancer cells E-cadherin was inhibited, instead, N-cadherin was expressed and localized at membranous AJ where co-staining with ?- and ?-catenin fluorescence was observed. Formation of N-cadherin/catenin (?-, ?-) protein complex at AJs of transformed and cancer cells was suggested. The above observations were further supported by immunoblotting results. Normal stomach muscosal and transformed cells expressed Cx32 at membranous GJ and were competent of gap junction communication (GJIC). In stomach cancer cells, Cx32 was inhibited and GJIC was defective. The results suggested that changes of signal pathways mediated by both cell adhesion and cell communication systems are associated intracellular events of stomach carcinogenesis. The alteration of cadherin isoform from E- to N-cadherin in transformed and stomach cancer cells is the first report.

  15. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Jing; Wang; Ting-Jun; Fan; Xiu-Xia; Yang; Shi-Min; Chang

    2014-01-01

    AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P <0.01) and the length of F-actin,reduced the mean optical density(P <0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  16. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M; Gupta, Goutam; McCleskey, T Mark; Chaudhary, Anu

    2009-12-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

  17. Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells.

    Science.gov (United States)

    Lee, Hye Jeong; Choi, Na Young; Lee, Seung-Won; Ko, Kisung; Hwang, Tae Sook; Han, Dong Wook; Lim, Jisun; Schöler, Hans R; Ko, Kinarm

    2016-03-01

    Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.

  18. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

    Science.gov (United States)

    Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

    2017-02-14

    Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  19. Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability

    Directory of Open Access Journals (Sweden)

    Emma L. Wilkinson

    2016-10-01

    Full Text Available Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1 levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.

  20. Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema.

    Science.gov (United States)

    Yu, Jinyan; Ma, Zhongsen; Shetty, Sreerama; Ma, Mengshi; Fu, Jian

    2016-07-01

    Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

  1. Altered Pattern of Naive and Memory B cells and B1 Cells in Patients with Chronic Granulomatous Disease

    NARCIS (Netherlands)

    Mohsenzadegan, Monireh; Fattahi, Fahimeh; Fattahi, Fatemeh; Mirshafiey, Abbas; Fazlollahi, Mohammad Reza; Beni, Fariba Naderi; Movahedi, Masoud; Pourpak, Zahra

    2014-01-01

    Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder characterized by a greatly increased susceptibility to severe fungal and bacterial infections caused by defects in NADPH oxidase of phagocytic cells. We aimed to investigate immunophenotype alterations of naive and memor

  2. V(D)J recombination in mature B cells: a mechanism for altering antibody responses.

    Science.gov (United States)

    Papavasiliou, F; Casellas, R; Suh, H; Qin, X F; Besmer, E; Pelanda, R; Nemazee, D; Rajewsky, K; Nussenzweig, M C

    1997-10-10

    The clonal selection theory states that B lymphocytes producing high-affinity immunoglobulins are selected from a pool of cells undergoing antibody gene mutation. Somatic hypermutation is a well-documented mechanism for achieving diversification of immune responses in mature B cells. Antibody genes were also found to be modified in such cells in germinal centers by recombination of the variable (V), diversity (D), and joining (J) segments. The ability to alter immunoglobulin expression by V(D)J recombination in the selective environment of the germinal center may be an additional mechanism for inactivation or diversification of immune responses.

  3. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  4. Retinoic acid and hydrocortisone strengthen the barrier function of human RPMI 2650 cells, a model for nasal epithelial permeability.

    Science.gov (United States)

    Kürti, Levente; Veszelka, Szilvia; Bocsik, Alexandra; Ozsvári, Béla; Puskás, László G; Kittel, Agnes; Szabó-Révész, Piroska; Deli, Mária A

    2013-05-01

    The nasal pathway represents an alternative route for non-invasive systemic administration of drugs. The main advantages of nasal drug delivery are the rapid onset of action, the avoidance of the first-pass metabolism in the liver and the easy applicability. In vitro cell culture systems offer an opportunity to model biological barriers. Our aim was to develop and characterize an in vitro model based on confluent layers of the human RPMI 2650 cell line. Retinoic acid, hydrocortisone and cyclic adenosine monophosphate, which influence cell attachment, growth and differentiation have been investigated on the barrier formation and function of the nasal epithelial cell layers. Real-time cell microelectronic sensing, a novel label-free technique was used for dynamic monitoring of cell growth and barrier properties of RPMI 2650 cells. Treatments enhanced the formation of adherens and tight intercellular junctions visualized by electron microscopy, the presence and localization of junctional proteins ZO-1 and β-catenin demonstrated by fluorescent immunohistochemistry, and the barrier function of nasal epithelial cell layers. The transepithelial resistance of the RPMI 2650 cell model reached 50 to 200 Ω × cm(2), the permeability coefficient for 4.4 kDa FITC-dextran was 9.3 to 17 × 10(-6) cm/s, in agreement with values measured on nasal mucosa from in vivo and ex vivo experiments. Based on these results human RPMI 2650 cells seem to be a suitable nasal epithelial model to test different pharmaceutical excipients and various novel formulations, such as nanoparticles for toxicity and permeability.

  5. Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

    Directory of Open Access Journals (Sweden)

    Brown Theodore J

    2008-08-01

    Full Text Available Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol, which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis.

  6. Altered development of NKT cells, γδ T cells, CD8 T cells and NK cells in a PLZF deficient patient.

    Directory of Open Access Journals (Sweden)

    Maggie Eidson

    Full Text Available In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease.

  7. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

    Science.gov (United States)

    Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

    2016-01-06

    Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly.

  8. Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth

    Directory of Open Access Journals (Sweden)

    Jonathan M. Page

    2015-09-01

    Full Text Available The contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2, poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

  9. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

  10. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Lindblad, Maiken Marie; Jakobsen, Jannik E.;

    2015-01-01

    onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model...

  11. Histological alterations of intestinal villi and epithelial cells after feeding dietary sugar cane extract in piglets

    Directory of Open Access Journals (Sweden)

    Toshikazu Kawai

    2012-07-01

    Full Text Available Effects of sugar cane extract (SCE on the piglet intestinal histology were observed. Twelve castrated male piglets weaned at the age of 26 days were allotted to three groups fed diets containing 0, 0.05 or 0.10% SCE. At the end of feeding experiment, each intestinal segment was taken for light or scanning electron microscopy. Feed intake, body weight gain and feed efficiency did not show a difference among groups. Most of the values for villus height, villus area, cell area and cell mitosis numbers were not different among groups, except for that the villus area of the 0.10% SCE group and the cell area of both SCE groups increased significantly at the jejunum compared to the control (P<0.05. For cell mitosis numbers, the 0.10% SCE group was higher than the 0.05% SCE group at the jejunum. Compared with the majority of flat cells of each intestinal segment in the control, the SCE groups had protuberated cells. In the 0.05% SCE group, deeper cells at the sites of recently exfoliated cells in the duodenum, cell clusters aggregated by protuberated cells in the jejunum and much more protuberant cells in the ileum were observed. These histological intestinal alterations suggest that SCE could raise the functions of intestinal villi and epithelial cells, especially at the 0.05%.

  12. Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Cynthia C.T. Sprenger

    2008-12-01

    Full Text Available Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer.

  13. Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants

    Directory of Open Access Journals (Sweden)

    Pankaj Dhonukshe

    2012-01-01

    Full Text Available Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration.

  14. Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

    DEFF Research Database (Denmark)

    Orfila, C.; Huisman, M.M.H.; Willats, William George Tycho;

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic...... polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were...... that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained...

  15. Hydrochloric acid alters the effect of L-glutamic acid on cell viability in human neuroblastoma cell cultures.

    Science.gov (United States)

    Croce, Nicoletta; Bernardini, Sergio; Di Cecca, Stefano; Caltagirone, Carlo; Angelucci, Francesco

    2013-07-15

    l-Glutamic acid (l-glutamate) is used to induce excitotoxicity and test neuroprotective compounds in cell cultures. However, because l-glutamate powder is nearly insoluble in water, many manufacturers recommend reconstituting l-glutamate in hydrochloric acid (HCl) prior to successive dilutions. Nevertheless, HCl, even at low concentrations, may alter the pH of the cell culture medium and interfere with cell activity. Thus, the aim of this study was to evaluate whether the reconstitution of l-glutamate powder in HCl alters its capacity to induce neurotoxicity in different human neuroblastoma cell lines. SH-SY5Y, IMR-32 and SK-N-BE(2) cells were exposed to various concentrations of l-glutamate, which was either reconstituted in HCl (1M) or post re-equilibrated to the pH of the culture medium (7.5). After 24 and 48h of incubation, changes in the cell viability of treated versus untreated cells were evaluated. The effect of an identical amount of HCl present in the l-glutamate dilutions on neuroblastoma cell survival was also investigated. Our data showed that the neurotoxicity of glutamate reconstituted in HCl was comparable to that of HCl alone. Moreover, the pH variations induced by glutamate or HCl in the culture medium were similar. When the pH of the glutamate stock solution was re-equilibrated, l-glutamate induced variation in cell viability to a lower extent and after a longer incubation time. This study demonstrated that HCl used to reconstitute l-glutamate powder might alter the effect of glutamate itself in neuroblastoma cell cultures. Thus, this information might be useful to scientists who use l-glutamate to induce excitotoxicity or to test neuroprotective agents.

  16. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure.

    Science.gov (United States)

    Coleman, Heather D; Yan, Jimmy; Mansfield, Shawn D

    2009-08-04

    Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba x grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in all lines, with increases of 2% to 6% over control levels, without influencing plant growth. The elevated concentration of cellulose was associated with an increase in cell wall crystallinity but did not alter secondary wall microfibril angle. This finding suggests that the observed increase in crystallinity is a function of altered carbon partitioning to cellulose biosynthesis rather than the result of tension wood formation. Furthermore, the augmented deposition of cellulose in the transgenic lines resulted in thicker xylem secondary cell wall and consequently improved wood density. These findings clearly implicate SuSy as a key regulator of sink strength in poplar trees and demonstrate the tight association of SuSy with cellulose synthesis and secondary wall formation.

  17. Cardiac Arrest Alters Regional Ubiquitin Levels in Association with the Blood-Brain Barrier Breakdown and Neuronal Damages in the Porcine Brain.

    Science.gov (United States)

    Sharma, Hari S; Patnaik, Ranjana; Sharma, Aruna; Lafuente, José Vicente; Miclescu, Adriana; Wiklund, Lars

    2015-10-01

    The possibility that ubiquitin expression is altered in cardiac arrest-associated neuropathology was examined in a porcine model using immunohistochemical and biochemical methods. Our observations show that cardiac arrest induces progressive increase in ubiquitin expression in the cortex and hippocampus in a selective and specific manner as compared to corresponding control brains using enzyme-linked immunoassay technique (enzyme-linked immunosorbent assay (ELISA)). Furthermore, immunohistochemical studies showed ubiquitin expression in the neurons exhibiting immunoreaction in the cytoplasm and karyoplasm of distorted or damaged cells. Separate Nissl and ubiquitin staining showed damaged and distorted neurons and in the same cortical region ubiquitin expression indicating that ubiquitin expression after cardiac arrest represents dying neurons. The finding that methylene blue treatment markedly induced neuroprotection following identical cardiac arrest and reduced ubiquitin expression strengthens this view. Taken together, our observations are the first to show that cardiac arrest enhanced ubiquitin expression in the brain that is related to the magnitude of neuronal injury and the finding that methylene blue reduced ubiquitin expression points to its role in cell damage, not reported earlier.

  18. The challenges of translating stem cells for spinal cord injury and related disorders: what are the barriers and opportunities?

    Science.gov (United States)

    Hewson, Stephanie M; Fehlings, Lauren N; Messih, Mark; Fehlings, Michael G

    2013-02-01

    Stem cell therapies have significant potential to treat spinal cord injury (SCI), but it remains difficult to translate these therapies from 'bench to bedside'. Identifying barriers to translation and understanding how these barriers are viewed by stakeholders in the field of stem cell research are key steps to clinical translation. The Stem Cell Global Blueprint Conference, held in Toronto (ON, Canada) presented a unique opportunity to analyze the perspectives of multiple stakeholders on the future of stem cell therapies for SCI treatment. This article is an analysis of data collected at the conference, including a consensus-building process and pre- and in-conference questionnaires. The authors used these data to assess current perceptions of stem cell research and compared the findings with the literature. The authors identified the major barriers according to a wide range of stakeholders and what strategies they suggested to overcome these obstacles, with the aim of forwarding discussion on stem cell research. It is not a systematic review of the area, but rather a presentation of expert opinion with literature citations to give context and support to their arguments and suggestions. The authors believe that the international SCI community is ready for larger-scale clinical translation, which will require the continued cooperation of all stakeholders in the stem cell and SCI communities.

  19. WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wen Jiang

    Full Text Available Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.

  20. Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened.

    Science.gov (United States)

    Visbal, Adriana P; LaMarca, Heather L; Villanueva, Hugo; Toneff, Michael J; Li, Yi; Rosen, Jeffrey M; Lewis, Michael T

    2011-04-01

    The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.

  1. Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia.

    Science.gov (United States)

    Díez, Paula; Lorenzo, Seila; Dégano, Rosa M; Ibarrola, Nieves; González-González, María; Nieto, Wendy; Almeida, Julia; González, Marcos; Orfao, Alberto; Fuentes, Manuel

    2016-04-01

    Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.

  2. Magnetron sputtered gadolinia-doped ceria diffusion barriers for metal-supported solid oxide fuel cells

    DEFF Research Database (Denmark)

    Sønderby, Steffen; Klemensø, Trine; Christensen, Bjarke H.;

    2014-01-01

    and substrate bias voltage. A GDC layer thickness of 0.6 μm is found to effectively block Sr diffusion when bias voltage and deposition temperature is tuned to promote dense coatings. The adatom mobility has a large influence on the film density. Low temperature and bias voltage result in underdense column......Gadolinia-doped ceria (GDC) thin films are deposited by reactive magnetron sputtering in an industrial-scale setup and implemented as barrier layers between the cathode and electrolyte in metal-based solid oxide fuel cells consisting of a metal support, an electrolyte of ZrO2 co-doped with Sc2O3...... and Y2O3 (ScYSZ) and a Sr-doped lanthanum cobalt oxide cathode. In order to optimize the deposition of GDC to obtain high electrochemical performance of the cells, the influence of film thickness and adatom mobility is studied. The adatom mobility is varied by tuning the deposition temperature...

  3. ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier

    Science.gov (United States)

    Drolez, Aurore; Vandenhaute, Elodie; Delannoy, Clément Philippe; Dewald, Justine Hélène; Gosselet, Fabien; Cecchelli, Romeo; Julien, Sylvain; Dehouck, Marie-Pierre; Delannoy, Philippe; Mysiorek, Caroline

    2016-01-01

    The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of GD1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34+ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier. PMID:27529215

  4. Study on AlxNiy Alloys as Diffusion Barriers in Flexible Thin Film Solar Cells%Study on AlxNiy Alloys as Diffusion Barriers in Flexible Thin Film Solar Cells

    Institute of Scientific and Technical Information of China (English)

    岳红云; 吴爱民; 秦福文; 李廷举

    2011-01-01

    Co-sputtered AlxNiy thin films were used as diffusion barriers between aluminum and hydrogenated microcrystalline silicon (μc-Si:H) for flexible thin film solar cells. The stoichiometric ratio of AlxNiy showed a significant effect on the structures of the films. The obtained Al3Ni2 film was amorphous, while polycrystalline films were obtained when the ratio of aluminum to nickel was 1:1 and 2:3. An auger electron spectroscope and four-point probe system were applied to test the resistance to the interdiffusion between aluminum and silicon, as well as the conductivities of the AlxNiy barriers. The data of auger depth profile showed that the content of silicon was the minimum in the aluminum layer after sputtering for 4 min using AlNi thin film as the barrier layer. Compared to other AlxNiy alloys, the AlNi thin film possessed the lowest sheet resistance.

  5. MicroRNA and DNA methylation alterations mediating retinoic acid induced neuroblastoma cell differentiation.

    Science.gov (United States)

    Stallings, Raymond L; Foley, Niamh H; Bray, Isabella M; Das, Sudipto; Buckley, Patrick G

    2011-10-01

    Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

  6. Chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish.

    Science.gov (United States)

    Yu, Kaimin; Li, Guochao; Feng, Weimin; Liu, Lili; Zhang, Jiayu; Wu, Wei; Xu, Lei; Yan, Yanchun

    2015-09-05

    The potential interference of endocrine disrupting chemicals (EDCs) on aquatic animals and humans has drawn wide attention in recent years. Reports have shown that some organophosphorus pesticides were a kind of EDCs, but their effects on fish species are still under research. In present study, flow cytometry data of HEC-1B cell line showed that chlorpyrifos (CPF) could increase cell proliferation index like 17β-estradiol (E2), but the effect of CPF was weaker than of E2 in the same concentration. Moreover, CPF altered the expression pattern of estrogen-responsive gene VTG and ERα in zebrafish embryos. When exposed to CPF at various concentrations (0, 0.10, 0.25, 0.50, 0.75 and 1.00mg/L) for 48h during the embryo stage, compared with controls, the hatching rate of treated groups significantly increased at the same time and the hatching rate of embryos was proportional to CPF concentration. The mRNA expression levels of c-myc, cyclin D1, Bax and Bcl-2, which are closely related to cell proliferation and cell apoptosis, were disturbed by CPF in zebrafish embryos after exposure treated for 48h. In addition, acridine orange (AO) staining of zebrafish embryos showed that cell apoptosis was appeared in the 0.75, 1.00mg/L CPF treated groups. Taken together, the results obtained in the present study indicated that chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish.

  7. Selective alterations of transcription factors in MPP+-induced neurotoxicity in PC12 cells.

    Science.gov (United States)

    Xu, Z; Cawthon, D; McCastlain, K A; Duhart, H M; Newport, G D; Fang, H; Patterson, T A; Slikker, W; Ali, S F

    2005-08-01

    MPP(+) (1-methyl-4-phenylpyridinium; the active metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine)) depletes dopamine (DA) content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. In this study, the dose response and time-course of MPP(+)-induced DA depletion and decreased cell viability were determined in nerve growth factor (NGF)-differentiated PC12 cells. The alteration of transcription factors (TFs) induced by MPP(+) from a selected dose level and time point was then evaluated using protein/DNA-binding arrays. K-means clustering analysis identified four patterns of protein/DNA-binding changes. Three of the 28 TFs identified in PC12 cells increased by 100% (p53, PRE, Smad SBE) and 2 decreased by 50% (HSE, RXR(DR1)) of control with MPP(+) treatment. In addition, three TFs decreased within the range of 33-50% (TFIID, E2F1, CREB) and two TFs increased within the range of 50-100% (PAX-5, Stat4). An electrophoretic mobility shift assay (EMSA) was used to confirm the changes of p53 and HSE. The observed changes in TFs correlated with the alterations of DA and cell viability. The data indicates that selective transcription factors are involved in MPP(+)-induced neurotoxicity and it provides mechanistic information that may be applicable to animal studies with MPTP and clinical studies of Parkinson's disease.

  8. Targeted alteration of real and imaginary refractive index of biological cells by histological staining

    OpenAIRE

    Cherkezyan, Lusik; Subramanian, Hariharan; Stoyneva, Valentina; Rogers, Jeremy D.; Yang, Seungmoo; Damania, Dhwanil; Taflove, Allen; Backman, Vadim

    2012-01-01

    Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers-Kronig relationship. We present a method for creating 2-D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of sta...

  9. An altered endometrial CD8 tissue resident memory T cell population in recurrent miscarriage.

    Science.gov (United States)

    Southcombe, J H; Mounce, G; McGee, K; Elghajiji, A; Brosens, J; Quenby, S; Child, T; Granne, I

    2017-01-23

    When trying to conceive 1% of couples have recurrent miscarriages, defined as three or more consecutive pregnancy losses. This is not accounted for by the known incidence of chromosomal aneuploidy in miscarriage, and it has been suggested that there is an immunological aetiology. The endometrial mucosa is populated by a variety of immune cells which in addition to providing host pathogen immunity must facilitate pregnancy. Here we characterise the endometrial CD8-T cell population during the embryonic window of implantation and find that the majority of cells are tissue resident memory T cells with high levels of CD69 and CD103 expression, proteins that prevent cells egress. We demonstrate that unexplained recurrent miscarriage is associated with significantly decreased expression of the T-cell co-receptor CD8 and tissue residency marker CD69. These cells differ from those found in control women, with less expression of CD127 indicating a lack of homeostatic cell control through IL-7 signalling. Nevertheless this population is resident in the endometrium of women who have RM, more than three months after the last miscarriage, indicating that the memory CD8-T cell population is altered in RM patients. This is the first evidence of a differing pre-pregnancy phenotype in endometrial immune cells in RM.

  10. Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines.

    Science.gov (United States)

    Blanchère, M; Saunier, E; Mestayer, C; Broshuis, M; Mowszowicz, I

    2002-11-01

    TGF beta can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGF beta function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGF beta 1 and TGF beta 2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGF beta 1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGF beta secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGF beta which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGF beta secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGF beta, and to regulate this secretion through stromal-epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.

  11. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  12. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

    Science.gov (United States)

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A; Wertheimer, Joshua; Mullin, James M

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

  13. Menstrual cycle distribution of uterine natural killer cells is altered in heavy menstrual bleeding.

    Science.gov (United States)

    Biswas Shivhare, Sourima; Bulmer, Judith N; Innes, Barbara A; Hapangama, Dharani K; Lash, Gendie E

    2015-11-01

    Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation.

  14. Efficient, air-stable colloidal quantum dot solar cells encapsulated using atomic layer deposition of a nanolaminate barrier

    KAUST Repository

    Ip, Alexander H.

    2013-12-23

    Atomic layer deposition was used to encapsulate colloidal quantum dot solar cells. A nanolaminate layer consisting of alternating alumina and zirconia films provided a robust gas permeation barrier which prevented device performance degradation over a period of multiple weeks. Unencapsulated cells stored in ambient and nitrogen environments demonstrated significant performance losses over the same period. The encapsulated cell also exhibited stable performance under constant simulated solar illumination without filtration of harsh ultraviolet photons. This monolithically integrated thin film encapsulation method is promising for roll-to-roll processed high efficiency nanocrystal solar cells. © 2013 AIP Publishing LLC.

  15. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  16. Positional and expressive alteration of prohibitin during the induced differentiation of human hepatocarcinoma SMMC-7721 cells

    Institute of Scientific and Technical Information of China (English)

    Dong-Hui Xu; Jian Tang; Qi-Fu Li; Song-Lin Shi; Xiang-Feng Chen; Ying Liang

    2008-01-01

    AIM: To explore the existence and distribution of prohibitin (PHB) in nuclear matrix and its co-localization with products of some related genes during the differentiation of human hepatocarcinoma SMMC-7721cells.METHODS: The nuclear matrix of the SHHC-7721 cells cultured with or without 5 x 10-3 mmol/L hexamethylene bisacetamide (HMBA) was selectively extracted.Western blot was used to analyze the expression of PHB in nuclear matrix; imrnunofluorescence microscope observation was used to analyze the distribution of PHB in cell. LCSM was used to observe the co-localization of PHB with products of oncogenes and tumor suppressor genes.RESULTS: Western blot analysis showed that PHB existed in the composition of nuclear matrix proteins and was down-regulated by HMBA treatment.Immunofluorescence observation revealed that PHB existed in the nuclear matrix, and its distribution regions and expression levels were altered after HMBA treatment. Laser scanning confocal microscopy revealed the co-localization between PHB and the products of oncogenes or tumor repression genes including c-fos, c-myc, p53 and Rb and its alteration of distributive area in the cells treated by HMBA.CONCLUSION: These data confirm that PHB is a nuclear matrix protein, which is located in the nuclear matrix, and the distribution and expression of PHB and its relation with associated genes may play significant roles during the differentiation of SMHC-7721 cells.

  17. Low oxygen alters mitochondrial function and response to oxidative stress in human neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Yury M. Lages

    2015-12-01

    Full Text Available Oxygen concentration should be carefully regulated in all living tissues, beginning at the early embryonic stages. Unbalances in oxygen regulation can lead to cell death and disease. However, to date, few studies have investigated the consequences of variations in oxygen levels for fetal-like cells. Therefore, in the present work, human neural progenitor cells (NPCs derived from pluripotent stem cells grown in 3% oxygen (v/v were compared with NPCs cultured in 21% (v/v oxygen. Low oxygen concentrations altered the mitochondrial content and oxidative functions of the cells, which led to improved ATP production, while reducing generation of reactive oxygen species (ROS. NPCs cultured in both conditions showed no differences in proliferation and glucose metabolism. Furthermore, antioxidant enzymatic activity was not altered in NPCs cultured in 3% oxygen under normal conditions, however, when exposed to external agents known to induce oxidative stress, greater susceptibility to DNA damage was observed. Our findings indicate that the management of oxygen levels should be considered for in vitro models of neuronal development and drug screening.

  18. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  19. Hydrogen sulfide improves colonic barrier integrity in DSS-induced inflammation in Caco-2 cells and mice.

    Science.gov (United States)

    Zhao, Hongyu; Yan, Rui; Zhou, Xiaogang; Ji, Fang; Zhang, Bing

    2016-10-01

    Intestinal barrier involves in the pathogeny of inflammatory bowel disease (IBD) and hydrogen sulfide (H2S) has been reported to improve intestinal barrier integrity. Thus, this study investigated the effects of GYY4137, a slow-release H2S donor, on DSS-induced inflammation and intestinal dysfunction. In vitro model, cellular permeability was significantly increased and expression of tight junctions (ZO-1, Cauldin4, and Occludin) was downregulated in Caco-2 cells. GYY4137 treatment markedly attenuated DSS-induced inflammation and barrier dysfunction. Cystathionine β-synthase (CBS)-siRNA transfection further demonstrated that endogenous H2S system involves in DSS-induced inflammation and mediates barrier function. In vivo model, DSS exposure caused colonic inflammation and injury in mice and GYY4137 injection alleviated inflammatory response and improved intestinal barrier via reducing intestinal permeability and upregulating of tight junctions. In conclusion, endogenous H2S system involves in DSS-induced inflammation and H2S addition alleviated inflammation and intestinal dysfunction in vitro and in vivo.

  20. Phenotypic alterations in breast cancer cells overexpressing the nuclear receptor co-activator AIB1

    Directory of Open Access Journals (Sweden)

    Azorsa David O

    2003-09-01

    Full Text Available Abstract Background Estrogen signaling plays a critical role in a number of normal physiological processes and has important implications in the treatment of breast cancer. The p160 nuclear receptor coactivator, AIB1 (amplified in breast cancer 1, is frequently amplified and overexpressed in human breast cancer and has been shown to enhance estrogen-dependent transactivation. Methods To better understand the molecular and physiological consequences of AIB1 overexpression in breast cancer cells, an AIB1 cDNA was transfected into the low AIB1 expressing, estrogen-receptor (ER negative breast cancer cell line, MDA-MB-436. The features of a derivative cell line, designated 436.1, which expresses high levels of AIB1, are described and compared with the parental cell line. Results A significant increase in the levels of CREB binding protein (CBP was observed in 436.1 cells and immunofluorescent staining revealed altered AIB1 and CBP staining patterns compared to the parental cells. Further, transient transfection assays demonstrated that the overall estrogen-dependent transactivation in 436.1 cells is approximately 20-fold higher than the parental cells and the estrogen dose-response curve is repositioned to the right. Finally, cDNA microarray analysis of approximately 7,100 cDNAs identified a number of differentially expressed genes in the 436.1 cells. Conclusion These observations lend insight into downstream signaling pathways that are influenced by AIB1.

  1. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A;

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...

  2. Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver

    Directory of Open Access Journals (Sweden)

    Saura C. Sahu

    2016-01-01

    Full Text Available Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 μg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver.

  3. Ceramic barrier layers for flexible thin film solar cells on metallic substrates: a laboratory scale study for process optimization and barrier layer properties.

    Science.gov (United States)

    Delgado-Sanchez, Jose-Maria; Guilera, Nuria; Francesch, Laia; Alba, Maria D; Lopez, Laura; Sanchez, Emilio

    2014-11-12

    Flexible thin film solar cells are an alternative to both utility-scale and building integrated photovoltaic installations. The fabrication of these devices over electrically conducting low-cost foils requires the deposition of dielectric barrier layers to flatten the substrate surface, provide electrical isolation between the substrate and the device, and avoid the diffusion of metal impurities during the relatively high temperatures required to deposit the rest of the solar cell device layers. The typical roughness of low-cost stainless-steel foils is in the hundred-nanometer range, which is comparable or larger than the thin film layers comprising the device and this may result in electrical shunts that decrease solar cell performance. This manuscript assesses the properties of different single-layer and bilayer structures containing ceramics inks formulations based on Al2O3, AlN, or Si3N4 nanoparticles and deposited over stainless-steel foils using a rotogravure printing process. The best control of the substrate roughness was achieved for bilayers of Al2O3 or AlN with mixed particle size, which reduced the roughness and prevented the diffusion of metals impurities but AlN bilayers exhibited as well the best electrical insulation properties.

  4. Restricted maternal nutrition alters myogenic regulatory factor expression in satellite cells of ovine offspring.

    Science.gov (United States)

    Raja, J S; Hoffman, M L; Govoni, K E; Zinn, S A; Reed, S A

    2016-07-01

    Poor maternal nutrition inhibits muscle development and postnatal muscle growth. Satellite cells are myogenic precursor cells that contribute to postnatal muscle growth, and their activity can be evaluated by the expression of several transcription factors. Paired-box (Pax)7 is expressed in quiescent and active satellite cells. MyoD is expressed in activated and proliferating satellite cells and myogenin is expressed in terminally differentiating cells. Disruption in the expression pattern or timing of expression of myogenic regulatory factors negatively affects muscle development and growth. We hypothesized that poor maternal nutrition during gestation would alter the in vitro temporal expression of MyoD and myogenin in satellite cells from offspring at birth and 3 months of age. Ewes were fed 100% or 60% of NRC requirements from day 31±1.3 of gestation. Lambs from control-fed (CON) or restricted-fed (RES) ewes were euthanized within 24 h of birth (birth; n=5) or were fed a control diet until 3 months of age (n=5). Satellite cells isolated from the semitendinosus muscle were used for gene expression analysis or cultured for 24, 48 or 72 h and immunostained for Pax7, MyoD or myogenin. Fusion index was calculated from a subset of cells allowed to differentiate. Compared with CON, temporal expression of MyoD and myogenin was altered in cultured satellite cells isolated from RES lambs at birth. The percent of cells expressing MyoD was greater in RES than CON (P=0.03) after 24 h in culture. After 48 h of culture, there was a greater percent of cells expressing myogenin in RES compared with CON (P0.05). In satellite cells from RES lambs at 3 months of age, the percent of cells expressing MyoD and myogenin were greater than CON after 72 h in culture (Psatellite cells of the offspring, which may reduce the pool of myoblasts, decrease myoblast fusion and contribute to the poor postnatal muscle growth previously observed in these animals.

  5. Systemic sclerosis patients present alterations in the expression of molecules involved in B cell regulation

    Directory of Open Access Journals (Sweden)

    Lilian eSoto

    2015-09-01

    Full Text Available The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors, in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naïve and memory B cell sub-populations from systemic sclerosis patients. To achieve this, blood samples were drawn from thirty one systemic sclerosis patients and fifty three healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcgammaRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naïve B cells related to memory B cells, compared to healthy controls. Transitional and naïve B cells from patients express higher levels of CD86 and FcgammaRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, while memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate to different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B cell regulation. These abnormalities may be determinant in the B cell hyperactivation observed in systemic sclerosis.

  6. Microgravity-induced alterations in signal transduction in cells of the immune system

    Science.gov (United States)

    Paulsen, Katrin; Thiel, Cora; Timm, Johanna; Schmidt, Peter M.; Huber, Kathrin; Tauber, Svantje; Hemmersbach, Ruth; Seibt, Dieter; Kroll, Hartmut; Grote, Karl-Heinrich; Zipp, Frauke; Schneider-Stock, Regine; Cogoli, Augusto; Hilliger, Andre; Engelmann, Frank; Ullrich, Oliver

    2010-11-01

    Since decades it is known that the activity of cells of the immune system is severely dysregulated in microgravity, however, the underlying molecular aspects have not been elucidated yet. The identification of gravity-sensitive molecular mechanisms in cells of the immune system is an important and indispensable prerequisite for the development of counteractive measures to prevent or treat disturbed immune cell function of astronauts during long-term space missions. Moreover, their sensitivity to altered gravity renders immune cells an ideal model system to understand if and how gravity on Earth is required for normal mammalian cell function and signal transduction. We investigated the effect of simulated weightlessness (2D clinostat) and of real microgravity (parabolic flights) on key signal pathways in a human monocytic and a T lymphocyte cell line. We found that cellular responses to microgravity strongly depend on the cell-type and the conditions in which the cells are subjected to microgravity. In Jurkat T cells, enhanced phosphorylation of the MAP kinases ERK-1/2, MEK and p38 and inhibition of nuclear translocation of NF-kB were the predominant responses to simulated weightlessness, in either stimulated or non-stimulated cells. In contrast, non-stimulated monocytic U937 cells responded to simulated weightlessness with enhanced overall tyrosine-phosphorylation and activation of c-jun, whereas PMA-stimulated U937 cells responded the opposite way with reduced tyrosine-phosphorylation and reduced activation of c-jun, compared with PMA-stimulated 1 g controls. P53 protein was phosphorylated rapidly in microgravity. The identification of gravi-sensitive mechanisms in cells of the immune system will not only enable us to understand and prevent the negative effects of long time exposure to microgravity on Astronauts, but could also lead to novel therapeutic targets in general.

  7. Temperature-dependent Schottky barrier in high-performance organic solar cells

    Science.gov (United States)

    Li, Hui; He, Dan; Zhou, Qing; Mao, Peng; Cao, Jiamin; Ding, Liming; Wang, Jizheng

    2017-01-01

    Organic solar cells (OSCs) have attracted great attention in the past 30 years, and the power conversion efficiency (PCE) now reaches around 10%, largely owning to the rapid material developments. Meanwhile with the progress in the device performance, more and more interests are turning to understanding the fundamental physics inside the OSCs. In the conventional bulk-heterojunction architecture, only recently it is realized that the blend/cathode Schottky junction serves as the fundamental diode for the photovoltaic function. However, few researches have focused on such junctions, and their physical properties are far from being well-understood. In this paper based on PThBDTP:PC71BM blend, we fabricated OSCs with PCE exceeding 10%, and investigated temperature-dependent behaviors of the junction diodes by various characterization including current-voltage, capacitance-voltage and impedance measurements between 70 to 290 K. We found the Schottky barrier height exhibits large inhomogeneity, which can be described by two sets of Gaussian distributions.

  8. Alterations in expression, proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hong-Zhi He; Xiao-Hang Zhao; Zhen-Mei Song; Kun Wang; Liang-Hong Teng; Fang Liu; You-Sheng Mao; Ning Lu; Shang-Zhong Zhang; Min Wu

    2004-01-01

    AIM: To investigate biogenesis and intracellular localizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa.METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular localizations of clusterin were carried out in both tissues and cell lines of ESCC.RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform,were the major alterations in cancer cells of esophagus.Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia.CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

  9. F-cell levels are altered with erythrocyte density in sickle cell disease.

    Science.gov (United States)

    Basu, Sumanta; Dash, Bisnu Prasad; Patel, Dilip Kumar; Chakravarty, Sudipa; Chakravarty, Amit; Banerjee, Debashis; Chakrabarti, Abhijit

    2011-08-15

    Lighter cells from density fractionated erythrocytes of sickle cell disease (SCD) patients carry higher amount of externalized phosphatidylserine (PS) and cell surface glycophorins compared to the denser counterparts. Further analysis also revealed that the denser cells contained higher levels of fetal hemoglobin (HbF) compared to the lighter cells, supported by the presence of larger number of F-cells in these populations. In this report, we have found direct evidence on the higher survival of the HbF rich erythrocytes in SCD.

  10. Nicotine alters MicroRNA expression and hinders human adult stem cell regenerative potential.

    Science.gov (United States)

    Ng, Tsz Kin; Carballosa, Carlos M; Pelaez, Daniel; Wong, Hoi Kin; Choy, Kwong Wai; Pang, Chi Pui; Cheung, Herman S

    2013-03-01

    Adult stem cells are critical for the healing process in regenerative medicine. However, cigarette smoking inhibits stem cell recruitment to tissues and delays the wound-healing process. This study investigated the effect of nicotine, a major constituent in the cigarette smoke, on the regenerative potentials of human mesenchymal stem cells (MSC) and periodontal ligament-derived stem cells (PDLSC). The cell proliferation of 1.0 μM nicotine-treated MSC and PDLSC was significantly reduced when compared to the untreated control. Moreover, nicotine also retarded the locomotion of these adult stem cells. Furthermore, their osteogenic differentiation capabilities were reduced in the presence of nicotine as evidenced by gene expression (RUNX2, ALPL, BGLAP, COL1A1, and COL1A2), calcium deposition, and alkaline phosphatase activity analyses. In addition, the microRNA (miRNA) profile of nicotine-treated PDLSC was altered; suggesting miRNAs might play an important role in the nicotine effects on stem cells. This study provided the possible mechanistic explanations on stem cell-associated healing delay in cigarette smoking.

  11. Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity[OPEN

    Science.gov (United States)

    Rebocho, Alexandra B.

    2016-01-01

    Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth. PMID:27553356

  12. Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity.

    Science.gov (United States)

    Richardson, Annis Elizabeth; Rebocho, Alexandra B; Coen, Enrico S

    2016-08-23

    Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth.

  13. Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

    Science.gov (United States)

    Ramesh, Govindarajan; Wu, Honglu

    2012-01-01

    Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

  14. Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade.

    Science.gov (United States)

    Bagley, J; Yuan, J; Chandrakar, A; Iacomini, J

    2015-09-01

    Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti-donor Th17 reactivity and production of IL-17. Here, we show that hyperlipidemia also affects FoxP3(+) regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25(low) Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3(+) , CD25(high) , CD4(+) Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti-CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3(+) CD25(low) CD4(+) T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti-CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance.

  15. Chronic ketamine produces altered distribution of parvalbumin-positive cells in the hippocampus of adult rats.

    Science.gov (United States)

    Sabbagh, Jonathan J; Murtishaw, Andrew S; Bolton, Monica M; Heaney, Chelcie F; Langhardt, Michael; Kinney, Jefferson W

    2013-08-29

    The underlying mechanisms of schizophrenia pathogenesis are not well understood. Increasing evidence supports the glutamatergic hypothesis that posits a hypofunction of the N-methyl D-aspartate (NMDA) receptor on specific gamma amino-butyric acid (GABA)-ergic neurons may be responsible for the disorder. Alterations in the GABAergic system have been observed in schizophrenia, most notably a change in the expression of parvalbumin (PV) in the cortex and hippocampus. Several reports also suggest abnormal neuronal migration may play a role in the etiology of schizophrenia. The current study examined the positioning and distribution of PV-positive cells in the hippocampus following chronic treatment with the NMDA receptor antagonist ketamine. A robust increase was found in the number of PV-positive interneurons located outside the stratum oriens (SO), the layer where most of these cells are normally localized, as well as an overall numerical increase in CA3 PV cells. These results suggest ketamine leads to an abnormal distribution of PV-positive cells, which may be indicative of aberrant migratory activity and possibly related to the Morris water maze deficits observed. These findings may also be relevant to alterations observed in schizophrenia populations.

  16. Canine Adipose Derived Mesenchymal Stem Cells Transcriptome Composition Alterations: A Step towards Standardizing Therapeutic

    Directory of Open Access Journals (Sweden)

    Nina Krešić

    2017-01-01

    Full Text Available Although canine adipose derived stem cells (cASCs morphology characteristics and differentiation ability are well documented, transcriptome alterations of undifferentiated cASCs during ex vivo cultivation remain unknown. Here we demonstrate, for the first time, the transcriptome composition of isolated cASCs in undifferentiated state originating from six donors. Transcriptome changes were monitored during ex vivo cultivation between passage 3 (P3 and P5, which are mostly used in therapy. Influence of donors’ age in given passage number on transcriptome composition was also investigated. Cultivation from P3 to P5 resulted in 16 differentially expressed genes with cooverexpression of pluripotency and self-renewal transcription factors genes SOX2 and POU5F1 dominant in old donors’ cells. Furthermore, cASCs demonstrated upregulation of IL-6 in young and old donors’ cells. In addition, ex vivo cultivation of cASCs revealed well-known morphological alterations accompanied with decrease in expression of CD90 and CD44 markers in P4 and higher monitored by flow cytometry and successful osteo- and chondrodifferentiation but inefficient adipodifferentiation in P3. Our results revealed the impact of ex vivo cultivation on nature of cells. Correlation of transcriptome changes with secretome composition is needed and its further impact on therapeutic potential of cASCs remains to be evaluated in clinical trials.

  17. Bone cell expression on titanium surfaces is altered by sterilization treatments.

    Science.gov (United States)

    Stanford, C M; Keller, J C; Solursh, M

    1994-05-01

    Phenotypic responses of rat calvarial osteoblast-like cells (RCOB) were evaluated on commercially pure titanium (cpTi) surfaces when cultured at high density (5100 cells/mm2). These surfaces were prepared to three different clinically relevant surface preparations (1-micron, 600-grit, and 50-microns-grit sand-blast), followed by sterilization with either ultraviolet light, ethylene oxide, argon plasma-cleaning, or routine clinical autoclaving. Osteocalcin and alkaline phosphatase, but not collagen expression, were significantly affected by surface roughness when these surfaces were altered by argon plasma-cleaning. In general, plasma-cleaned cpTi surfaces demonstrated an inverse relationship between surface roughness and phenotypic markers for a bone-like response. On a per-cell basis, levels of the bone-specific protein, osteocalcin, and the enzymatic activity of alkaline phosphatase were highest on the smooth 1-micron polished surface and lowest on the roughest surfaces for the plasma-cleaned cpTi. Detectable bone cell expression can be altered by clinically relevant surfaces prepared by standard dental implant preparation techniques.

  18. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    Science.gov (United States)

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  19. Efficiency enhancement of solid-state PbS quantum dot-sensitized solar cells with Al2O3 barrier layer

    KAUST Repository

    Brennan, Thomas P.

    2013-01-01

    Atomic layer deposition (ALD) was used to grow both PbS quantum dots and Al2O3 barrier layers in a solid-state quantum dot-sensitized solar cell (QDSSC). Barrier layers grown prior to quantum dots resulted in a near-doubling of device efficiency (0.30% to 0.57%) whereas barrier layers grown after quantum dots did not improve efficiency, indicating the importance of quantum dots in recombination processes. © 2013 The Royal Society of Chemistry.

  20. Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

    Science.gov (United States)

    Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

    2012-07-01

    Spaceflight factors, including microgravity and space radiation, have many detrimental short-term effects on human physiology, including muscle and bone degradation, and immune system dysfunction. The long-term progression of these physiological effects is still poorly understood, and a serious concern for long duration spaceflight missions. We hypothesized that some of the degenerative effects of spaceflight may be caused in part by an inability of stem cells to proliferate and differentiate normally resulting in an impairment of tissue regenerative processes. Furthermore, we hypothesized that long-term bone tissue degeneration in space may be mediated by activation of the p53 signaling network resulting in cell cycle arrest and/or apoptosis in osteoprogenitors. In our analyses we found that spaceflight caused significant bone loss in the weight-bearing bones of mice with a 6.3% reduction in bone volume and 11.9% decrease in bone thickness associated with increased osteoclastic activity. Along with this rapid bone loss we also observed alterations in the cell cycle characterized by an increase in the Cdkn1a/p21 cell cycle arrest molecule independent of Trp53. Overexpression of Cdkn1a/p21 was localized to osteoblasts lining the periosteal surface of the femur and chondrocytes in the head of the femur, suggesting an inhibition of proliferation in two key regenerative cell types of the femur in response to spaceflight. Additionally we found overexpression of several matrix degradation molecules including MMP-1a, 3 and 10, of which MMP-10 was localized to osteocytes within the shaft of the femur. This, in conjunction with 40 nm resolution synchrotron nano-Computed Tomography (nano-CT) observations of an increase in osteocyte lacunae cross-sectional area, perimeter and a decrease in circularity indicates a potential role for osteocytic osteolysis in the observed bone degeneration in spaceflight. To further investigate the genetic response of bone to mechanical

  1. Liver cell adenoma showing sequential alteration of radiological findings suggestive of well-differentiated hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Takayuki Kogure; Yoshiyuki Ueno; Satoshi Sekiguchi; Kazuyuki Ishida; Takehiko Igarashi; Yuta Wakui; Takao Iwasaki; Tooru Shimosegawa

    2009-01-01

    A liver tumor 35 mm in diameter was found incidentally in a 40-year-old woman who had no history of liver diseases or the use of oral contraceptives. Radiological diagnostics showed the typical findings of liver cell adenoma (LCA). Dynamic computed tomography revealed that the tumor showed a homogenous enhancement in the arterial phase and almost the same enhancement as the surrounding liver parenchyma in the delayed phase. The tumor was found to contain fat on magnetic resonance imaging. A benign fat containing liver tumor was suggested. However, radiological findings altered, which caused us to suspect that a welldifferentiated hepatocellular carcinoma (HCC) containing fat was becoming dedifferentiated. Partial hepatectomy was performed and the pathological findings showed the typical findings of LCA. This case was an extremely rare LCA, which had no background of risk for LCA and developed the sequential alteration of the radiological findings to suspect well-differentiated HCC.

  2. Baicalein induces CD4+Foxp3+ T cells and enhances intestinal barrier function in a mouse model of food allergy

    Science.gov (United States)

    Bae, Min-Jung; Shin, Hee Soon; See, Hye-Jeong; Jung, Sun Young; Kwon, Da-Ae; Shon, Dong-Hwa

    2016-01-01

    The incidence of food allergy, which is triggered by allergen permeation of the gastrointestinal tract followed by a T-helper (Th) 2-mediated immune response, has been increasing annually worldwide. We examined the effects of baicalein (5,6,7-trihydroxyflavone), a flavonoid from Scutellaria baicalensis used in oriental herbal medicine, on regulatory T (Treg) cell induction and intestinal barrier function through the regulation of tight junctions in a mouse model of food allergy. An allergic response was induced by oral challenge with ovalbumin, and the incidence of allergic symptoms and T cell-related activity in the mesenteric lymph nodes were analyzed with and without the presence of baicalein. Our results demonstrated that the administration of baicalein ameliorated the symptoms of food allergy and attenuated serum IgE and effector T cells. However, Treg-related factors were up-regulated by baicalein. Furthermore, baicalein was shown to enhance intestinal barrier function through the regulation of tight junctions. We also found that baicalein treatment induced the differentiation of Treg cells via aryl hydrocarbon receptors (AhRs). Thus, the action of baicalein as an agonist of AhR can induce Treg differentiation and enhance barrier function, suggesting that baicalein might serve as an effective immune regulator derived from foods for the treatment of food allergy. PMID:27561877

  3. Imatinib alters cell viability but not growth factors levels in TM4 Sertoli cells

    Science.gov (United States)

    Hashemnia, Seyyed Mohammad Reza; Atari-Hajipirloo, Somayeh; Roshan-Milani, Shiva; Valizadeh, Nasim; Mahabadi, Sonya; Kheradmand, Fatemeh

    2016-01-01

    Background: The anticancer agent imatinib (IM) is a small molecular analog of ATP that inhibits tyrosine kinase activity of platelet derived growth factors (PDGFs) and stem cell factor (SCF) receptor in cancer cells. However these factors have a key role in regulating growth and development of normal Sertoli, Leydig and germ cells. Objective: The aim of this study was to determine cell viability, PDGF and SCF levels in mouse normal Sertoli cells exposed to IM. Materials and Methods: In this experimental study, the mouse TM4 Sertoli cells were treated with 0, 2.5, 5, 10 and 20 μM IM for 2, 4 or 6 days. The cell viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, One-Way ANOVA was performed. Results: IM showed significant decrease in Sertoli cell viability compared to control group (p=0.001). However, IM increased PDGF and SCF level insignificantly (p>0.05). Conclusion: Results suggested that IM treatment induced a dose dependent reduction of cell viability in Sertoli cells. It seems that treatment with this anticancer drug is involved in the fertility process. Further studies are needed to evaluate the role of PDGF and SCF in this cell. PMID:27738659

  4. Knockdown of a Laccase in Populus deltoides Confers Altered Cell Wall Chemistry and Increased Sugar Release

    Energy Technology Data Exchange (ETDEWEB)

    Bryan, Anthony C.; Jawdy, Sara; Gunter, Lee; Gjersing, Erica; Sykes, Robert; Hinchee, Maud A. W.; Winkeler, Kimberly A.; Collins, Cassandra M.; Engle, Nancy; Tschaplinski, Timothy J.; Yang, Xiaohan; Tuskan, Gerald A.; Muchero, Wellington; Chen, Jin-Gui

    2016-10-01

    Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl (S/G) ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.

  5. Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release.

    Science.gov (United States)

    Bryan, Anthony C; Jawdy, Sara; Gunter, Lee; Gjersing, Erica; Sykes, Robert; Hinchee, Maud A W; Winkeler, Kimberly A; Collins, Cassandra M; Engle, Nancy; Tschaplinski, Timothy J; Yang, Xiaohan; Tuskan, Gerald A; Muchero, Wellington; Chen, Jin-Gui

    2016-10-01

    Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.

  6. Apoptosis and morphological alterations after UVA irradiation in red blood cells of p53 deficient Japanese medaka (Oryzias latipes).

    Science.gov (United States)

    Sayed, Alla El-Din Hamid; Watanabe-Asaka, Tomomi; Oda, Shoji; Mitani, Hiroshi

    2016-08-01

    Morphological alterations in red blood cells were described as hematological bioindicators of UVA exposure to investigate the sensitivity to UVA in wild type Japanese medaka (Oryzias latipes) and a p53 deficient mutant. The fewer abnormal red blood cells were observed in the p53 mutant fish under the control conditions. After exposure to different doses of UVA radiation (15min, 30min and 60min/day for 3days), cellular and nuclear alterations in red blood cells were analyzed in the UVA exposed fish compared with non-exposed controls and those alterations included acanthocytes, cell membrane lysis, swollen cells, teardrop-like cell, hemolyzed cells and sickle cells. Those alterations were increased after the UVA exposure both in wild type and the p53 deficient fish. Moreover, apoptosis analyzed by acridine orange assay showed increased number of apoptosis in red blood cells at the higher UVA exposure dose. No micronuclei but nuclear abnormalities as eccentric nucleus, nuclear budding, deformed nucleus, and bilobed nucleus were observed in each group. These results suggested that UVA exposure induced both p53 dependent and independent apoptosis and morphological alterations in red blood cells but less sensitive to UVA than Wild type in medaka fish.

  7. Herbicide effects on freshwater benthic diatoms: Induction of nucleus alterations and silica cell wall abnormalities

    Energy Technology Data Exchange (ETDEWEB)

    Debenest, T. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Silvestre, J. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Coste, M.; Delmas, F. [Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Pinelli, E. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France)], E-mail: pinelli@ensat.fr

    2008-06-02

    Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5 x 10{sup -6}, 10{sup -6}, 10{sup -7} M) for 6 h followed by a 24 h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per mille), S.D. = 4.23) and the highest concentrations (29.40 per mille, S.D. = 8.49 for 10{sup -6} M and 35.96 per mille , S.D. = 3.71 for 5 x 10{sup -6} M) was statistically significant (Tukey test, P < 0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10{sup -6} and 5 x 10{sup -6} M; Tukey test, P < 0.05). These two parameters tended to increase together (Pearson correlation = 0.702, P < 0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic

  8. Activation of secretion and surface alteration of cytolytic T-lymphocytes interacting with target cells.

    Science.gov (United States)

    Bykovskaya, S N; Shevelev, A A; Kupriyanova, T A

    1988-01-01

    Cells obtained in mixed lymphocyte culture (MLC) and memory cells adsorbed on the surface of target cells (TC) were examined using scanning and transmission electron microscopy depending on the time of interaction with TC. Three types of lymphocytes were revealed: type I - cells of spherical shape with a smooth surface or an insignificant amount of microvilli; predominantly small and medium-sized lymphocytes contacting TC with non significant involvement of their surface or by several microvilli; type II - oval or round-shaped lymphocytes evenly covered with microvilli with considerably enlarged region of contact; type III cells - predominantly large lymphocytes and lymphoblasts flattened (spread) on TC, with multiple microvilli, ridge-like projections, and ruffles on their surface. TEM revealed activation of the secretory apparatus in the cytoplasm of such lymphocytes. With increased time of interaction, type III cells increase in number (from 8.6% after 10 min to 90.2% after 60 min of incubation). Memory cells show no morphologic signs of secretion in correlation with the absence of lysis of TC on which they are adsorbed. The surface of the lymphocytes adsorbed on the substrate with poly-L-lysin is not noticeably altered. It is suggested that 3 morphological types of lymphocytes correspond to 3 stages of secretion activation. Lymphocyte contact with TC surface is evidently a specific stimulus for activating secretory apparatus of CTL. SEM can be used for quantitation of activated lymphocytes.

  9. A deficiency of uPAR alters endothelial angiogenic function and cell morphology

    Directory of Open Access Journals (Sweden)

    Balsara Rashna D

    2011-05-01

    Full Text Available Abstract The angiogenic potential of a cell requires dynamic reorganization of the cytoskeletal architecture that involves the interaction of urokinase-type plasminogen activator receptor (uPAR with the extracellular matrix. This study focuses on the effect of uPAR deficiency (uPAR-/- on angiogenic function and associated cytoskeletal organization. Utilizing murine endothelial cells, it was observed that adhesion, migration, proliferation, and capillary tube formation were altered in uPAR-/- cells compared to wild-type (WT cells. On a vitronectin (Vn matrix, uPAR-/- cells acquired a "fried egg" morphology characterized by circular actin organization and lack of lamellipodia formation. The up-regulation of β1 integrin, FAK(P-Tyr925, and paxillin (P-Tyr118, and decreased Rac1 activation, suggested increased focal adhesions, but delayed focal adhesion turnover in uPAR-/- cells. This accounted for the enhanced adhesion, but attenuated migration, on Vn. VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice. Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

  10. Calprotectin induces cell death in human prostate cancer cell (LNCaP) through survivin protein alteration.

    Science.gov (United States)

    Sattari, Mina; Pazhang, Yaghub; Imani, Mehdi

    2014-11-01

    Calprotectin (CP), an abundant heterodimeric cytosolic protein of neutrophils, conveys a variety of functions such as tumor cell growth arrest and antimicrobial activity. We investigated CP activity and its possible apoptosis-inducing mechanism of action against an antiandrogen therapy-resistance prostate cancer cell line LNCaP. Cell viability and Annexin V FITC assays were performed in order to investigate its cell death activity and apoptosis, respectively. In order to address cell death inducing mechanism(s), immunocytochemistry and immunobloting analysis, reactive oxygen species (ROS) and nitric oxide (NO) measurements were performed. The effective concentration of CP against LNCaP promoting LNCaP cell death was 200 µg/mL. ROS and NO levels of cells remarkably were enhanced following treatment with 50 and 100 µg/mL of CP, respectively. Protein expression of anti-apoptotic protein survivin was significantly decreased after administration of tumor cells with CP. Our data indicate that CP regulates the LNCaP cells viability via survivin-mediated pathway and ROS and NO enhancement. Thus, inhibition of survivin expression, enhancement of ROS and NO level by CP or other similar pharmaceutical agents might be effective in lowering the malignant proliferation of human prostate cancer cells.

  11. Genome wide transcriptome analysis of dendritic cells identifies genes with altered expression in psoriasis.

    Directory of Open Access Journals (Sweden)

    Kata Filkor

    Full Text Available Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS or peptidoglycan (PGN induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs upon PGN induced tolerance. Using SAGE-Seq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (Kegg analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified

  12. A viable circulating tumor cell isolation device with high retrieval efficiency using a reversibly deformable membrane barrier

    Science.gov (United States)

    Kim, Yoonji; Bu, Jiyoon; Cho, Young-Ho; Son, Il Tae; Kang, Sung-Bum

    2017-02-01

    Circulating tumor cells (CTCs) contain prognostic information of the tumor, since they shed from the primary tumor and invade into the bloodstream. Therefore, the viable isolation is necessary for a consequent analysis of CTCs. Here, we present a device for the viable isolation and efficient retrieval of CTCs using slanted slot filters, formed by a reversibly deformable membrane barrier. Conventional filters have difficulties in retrieving captured cells, since they easily clog the slots. Moreover, large stress concentration at the sharp edges of squared slots, causes cell lysis. In contrast, the present device shows over 94% of high retrieval efficiency, since the slots can be opened simply by relieving the pressure. Furthermore, the inflated membrane barrier naturally forms the slanted slots, thus reducing the cell damage. By using cancer cell lines, we verified that the present device successfully isolate targeted cells, even at an extremely low concentrations (~10 cells/0.1 ml). In the clinical study, 85.7% of patients initially showed CTC positive while the numbers generally decreased after the surgery. We have also proved that the number of CTCs were highly correlated with tumour invasiveness. Therefore, the present device has potential for use in cancer diagnosis, surgical validation, and invasiveness analysis.

  13. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells.

    Science.gov (United States)

    Carter, Charleata A; Hamm, Jonathan T

    2009-07-10

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-kappaB (NF-kappaB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-alpha and NF-kappaB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30mug/ml CSC significantly activated PKC-alpha, while CSC doses above 20mug/ml CSC significantly activated NF-kappaB. As NF-kappaB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30mug/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30mug/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-alpha, NF-kappaB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  14. Injury to the Spinal Cord Niche Alters the Engraftment Dynamics of Human Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Christopher J. Sontag

    2014-05-01

    Full Text Available The microenvironment is a critical mediator of stem cell survival, proliferation, migration, and differentiation. The majority of preclinical studies involving transplantation of neural stem cells (NSCs into the CNS have focused on injured or degenerating microenvironments, leaving a dearth of information as to how NSCs differentially respond to intact versus damaged CNS. Furthermore, single, terminal histological endpoints predominate, providing limited insight into the spatiotemporal dynamics of NSC engraftment and migration. We investigated the early and long-term engraftment dynamics of human CNS stem cells propagated as neurospheres (hCNS-SCns following transplantation into uninjured versus subacutely injured spinal cords of immunodeficient NOD-scid mice. We stereologically quantified engraftment, survival, proliferation, migration, and differentiation at 1, 7, 14, 28, and 98 days posttransplantation, and identified injury-dependent alterations. Notably, the injured microenvironment decreased hCNS-SCns survival, delayed and altered the location of proliferation, influenced both total and fate-specific migration, and promoted oligodendrocyte maturation.

  15. Acute high-intensity sound exposure alters responses of place cells in hippocampus.

    Science.gov (United States)

    Goble, T J; Møller, A R; Thompson, L T

    2009-07-01

    Overstimulation is known to activate neural plasticity in the auditory nervous system causing changes in function and re-organization. It has been shown earlier that overstimulation using high-intensity noise or tones can induce signs of tinnitus. Here we show in studies in rats that overstimulation causes changes in the way place cells of the hippocampus respond as rats search for rewards in a spatial maze. In familiar environments, a subset of hippocampal pyramidal neurons, known as place cells, respond when the animal moves through specific locations but are relatively silent in others. This place-field activity (i.e. location-specific firing) is stable in a fixed environment. The present study shows that activation of neural plasticity through overstimulation by sound can alter the response of these place cells. Rats implanted with chronic drivable dorsal hippocampal tetrodes (four microelectrodes) were assessed for stable single-unit place-field responses that were extracted from multiunit responses using NeuroExplorer computer spike-sorting software. Rats then underwent either 30 min exposure to a 4 kHz tone at 104 dB SPL or a control period in the same sound chamber. The place-field activity was significantly altered after sound exposure showing that plastic changes induced by overstimulation are not limited to the auditory nervous system but extend to other parts of the CNS, in this case to the hippocampus, a brain region often studied in the context of plasticity.

  16. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Esteban R Fernández

    Full Text Available Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  17. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Science.gov (United States)

    Fernández, Esteban R; Olivera, Gabriela C; Quebrada Palacio, Luz P; González, Mariela N; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L; Ledesma Patiño, Oscar S; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  18. Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

    Directory of Open Access Journals (Sweden)

    Konstantin V Salojin

    Full Text Available Mammalian sterile 20-like kinase 1 (Mst1 is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

  19. Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton

    NARCIS (Netherlands)

    Ulbrich, C.; Pietsch, J.; Grosse, J.; Wehland, M.; Schulz, H.; Saar, K.; Hübner, N.; Hauslage, J.; Hemmersbach, R.; Braun, M.; van Loon, J.; Vagt, N.; Egli, M.; Richter, P.; Einspanier, R.; Sharbati, S.; Baltz, T.; Infanger, M.; Ma, X.; Grimm, D.

    2011-01-01

    Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions

  20. In vitro organotin administration alters guinea pig cochlear outer hair cell shape and viability.

    Science.gov (United States)

    Clerici, W J; Chertoff, M E; Brownell, W E; Fechter, L D

    1993-06-01

    Trimethyltin (TMT) and triethyltin (TET) disrupt auditory function at doses far below those shown to be neurotoxic. In vivo studies suggest that the initial effect of TMT on hearing occurs at the inner hair cell/spiral ganglion cell synapse, while later, the outer hair cell (OHC) undergoes structural and functional damage. TET produces acute effects upon afferent neurotransmission similar to those observed following TMT, but TET's effects on OHC structure and function have not been examined. OHCs are motile elements within the cochlea, believed to modulate the sensitivity and tuning within the inner ear. Changes in OHC length may alter hearing function, and length changes have been reported following exposure to various ototoxic agents in vitro. In the present study, 77 OHCs from 45 pigmented male guinea pigs were isolated in primary culture and exposed for 90 min to concentrations between 30 microM and 1.0 mM of TMT or TET and then to bathing medium for 30 min to remove the toxicant. Significant shortening of the OHC cell body occurred at all doses to both organotins, with a mean reduction in length of 15.1 and 20.2% for 1.0 mM TMT and TET, respectively, at the end of testing; control cells were only 3.4% shorter at the end of 90 min of perfusion with bathing medium. The effect of organotin exposure on OHC volume was not consistently related to either TMT or TET concentration or altered cell length. In addition, disruption of the plasma membrane characterized by bleb formation, the forceful ejection of cytoplasm, or bursting was seen in 80% of cells exposed to 1.0 mM TET, although not TMT; lower concentrations of both organotins disrupted the cell membrane in 10-30% of cells. Membrane rupture was not reliably associated with either increased cell volume or decreased length, implicating a weakening of the plasma membrane or cortical lattice as the basis for this effect. Consistent with the irreversible structural weakening of the lateral wall, resorption of

  1. HYDROGEN-RICH MEDIUM AMELIORATES LIPOPOLYSACCHARIDE-INDUCED BARRIER DYSFUNCTION VIA RHOA-MDIA1 SIGNALING IN CACO-2 CELLS.

    Science.gov (United States)

    Yang, Tao; Wang, Lu; Sun, Ruiqiang; Chen, Hongguang; Zhang, Hongtao; Yu, Yang; Wang, Yanyan; Wang, Guolin; Yu, Yonghao; Xie, Keliang

    2016-02-01

    Gastrointestinal barrier dysfunction is associated with the severity and prognosis of sepsis. Hydrogen gas (H2) can ameliorate multiple organ damage in septic animals. Ras homolog gene family member A (RhoA) and mammalian diaphanous-related formin 1 (mDia1) are important to regulate tight junction (TJ) and adherens junction (AJ), both of which determine the integrity of the intestinal barrier. This study was aimed to investigate whether H2 could modulate lipopolysaccharide (LPS)-stimulated dysfunction of the intestinal barrier and whether RhoA-mDia1 signaling is involved. Caco-2 cells were exposed to different concentrations of LPS (1 μg/mL-1 mg/mL). The permeability of the intestinal barrier was evaluated by transepithelial resistance (TER) and fluorescein-isothiocyanate-dextran flux. Expression and distribution of occludin and E-cadherin were analyzed by Western blot and immunofluorescence. RhoA activity was measured by G-Lisa assay, and mDia1 expression was assessed by Western blot. LPS (100 μg/mL) decreased TER and increased fluorescein-isothiocyanate-dextran flux, which were alleviated by H2-rich medium. Also, H2 down-regulated LPS-induced oxidative stress. Moreover, H2 improved the down-regulated expression and redistribution of occludin and E-cadherin caused by LPS. Additionally, H2 alleviated LPS-caused RhoA activation, and the beneficial effects of H2 on barrier were counteracted by RhoA agonist CN03. Rho inhibitor C3 exoenzyme mitigated LPS-induced barrier breakdown. Furthermore, H2-rich medium increased mDia1 expression, and mDia1 knockdown abolished protections of H2 on barrier permeability. mDia1 knockdown eliminated H2-induced benefits for occludin and E-cadherin. These findings suggest that H2 improves LPS-induced hyperpermeability of the intestinal barrier and disruptions of TJ and AJ by moderating RhoA-mDia1 signaling.

  2. Ginsenoside Rg1-induced alterations in gene expression in TNF-α stimulated endothelial cells

    Institute of Scientific and Technical Information of China (English)

    吕俊萍; 马增春; 杨静; 黄坚; 王树人; 王升启

    2004-01-01

    Background In China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-α and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.Methods Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells(HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-α were detected by oligonucleotide microarray analysis.Results NO production in HUVECs was decreased significantly after TNF-α treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-αstimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-αstimulated HUVECs.Conclusions Ginsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-α stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-αactivation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.

  3. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

    Science.gov (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami

    2013-05-01

    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

  4. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas.

    Science.gov (United States)

    Campbell, Joshua D; Alexandrov, Anton; Kim, Jaegil; Wala, Jeremiah; Berger, Alice H; Pedamallu, Chandra Sekhar; Shukla, Sachet A; Guo, Guangwu; Brooks, Angela N; Murray, Bradley A; Imielinski, Marcin; Hu, Xin; Ling, Shiyun; Akbani, Rehan; Rosenberg, Mara; Cibulskis, Carrie; Ramachandran, Aruna; Collisson, Eric A; Kwiatkowski, David J; Lawrence, Michael S; Weinstein, John N; Verhaak, Roel G W; Wu, Catherine J; Hammerman, Peter S; Cherniack, Andrew D; Getz, Gad; Artyomov, Maxim N; Schreiber, Robert; Govindan, Ramaswamy; Meyerson, Matthew

    2016-06-01

    To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor-normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase-Ras-Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

  5. Correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Xiaoping Huang; Jun Qi; Cai Yan; Xin Xu; Yaling Han; Mingrong Wang

    2008-01-01

    Androgen-induced proliferation shutoff gene AS3, also known as APRIN, is a growth inhibitory gene that is in itially implicated inprostate cancer. This gene is required for androgen-dependent growth arrest and is a primary target for 1,25(OH)2D3 and androgens. Alle-lic loss at AS3 locus has been linked to a variety of cancers. However, the correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma (ESCC) is not well established. In this study, the genomic and expression alterations of AS3 in ESCC and their clinical significance are evaluated. Loss of beterozygosity (LOH) analysis using an AS3 intragenic mierosatellite marker D13S171 revealed 72% allelic loss at AS3 locus in ESCC, which is significantly correlated with higher pathological grade (P=0.042).RT-PCR examination showed that AS3 mRNA obviously decreased in 44% tumors and its down-regulation was correlated with the sex of patients (P=0.03). Furthermore, the correlation between genomic and expression alterations of AS3 gene was analyzed in 18 ESCC specimens, which indicated that the consistency between allelic loss and decreased mRNA expression of AS3 was relatively poor. The results of this study indicate that the aberrant expression of AS3 may be involved in the tumorigenesis of esophagus and is responsible for the male predominance of ESCC.

  6. Protective effects of Lactobacillus plantarum on epithelial barrier disruption caused by enterotoxigenic Escherichia coli in intestinal porcine epithelial cells.

    Science.gov (United States)

    Wu, Yunpeng; Zhu, Cui; Chen, Zhuang; Chen, Zhongjian; Zhang, Weina; Ma, Xianyong; Wang, Li; Yang, Xuefen; Jiang, Zongyong

    2016-04-01

    Tight junctions (TJs) play an important role in maintaining the mucosal barrier function and gastrointestinal health of animals. Lactobacillus plantarum (L. plantarum) was reported to protect the intestinal barrier function of early-weaned piglets against enterotoxigenic Escherichia coli (ETEC) K88 challenge; however, the underlying cellular mechanism of this protection was unclear. Here, an established intestinal porcine epithelia cell (IPEC-J2) model was used to investigate the protective effects and related mechanisms of L. plantarum on epithelial barrier damages induced by ETEC K88. Epithelial permeability, expression of inflammatory cytokines, and abundance of TJ proteins, were determined. Pre-treatment with L. plantarum for 6h prevented the reduction in transepithelial electrical resistance (TEER) (Pplantarum were higher (Pplantarum was shown to regulate proteins of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. These results indicated that L. plantarum may improve epithelial barrier function by maintenance of TEER, inhibiting the reduction of TJ proteins, and reducing the expression of proinflammatory cytokines induced by ETEC K88, possibly through modulation of TLRs, NF-κB and MAPK pathways.

  7. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells

    DEFF Research Database (Denmark)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A;

    2016-01-01

    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation......-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested...... the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant...

  8. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    OpenAIRE

    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

  9. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells.

    Science.gov (United States)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A; Mollenkopf, Hans-Joachim; Meyer, Thomas F; Brüggemann, Holger

    2016-11-01

    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

  10. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    Science.gov (United States)

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  11. Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

    Science.gov (United States)

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2016-05-01

    Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes.

  12. Breaking the Blood-Brain Barrier With Mannitol to Aid Stem Cell Therapeutics in the Chronic Stroke Brain.

    Science.gov (United States)

    Tajiri, Naoki; Lee, Jea Young; Acosta, Sandra; Sanberg, Paul R; Borlongan, Cesar V

    2016-01-01

    Blood-brain barrier (BBB) permeabilizers, such as mannitol, can facilitate peripherally delivered stem cells to exert therapeutic benefits on the stroke brain. Although this BBB permeation-aided stem cell therapy has been demonstrated in the acute stage of stroke, such BBB permeation in the chronic stage of the disease remains to be examined. Adult Sprague-Dawley rats initially received sham surgery or experimental stroke via the 1-h middle cerebral artery occlusion (MCAo) model. At 1 month after the MCAo surgery, stroke animals were randomly assigned to receive human umbilical cord stem cells only (2 million viable cells), mannitol only (1.1 mol/L mannitol at 4°C), combined human umbilical cord stem cells (200,000 viable cells) and mannitol (1.1 mol/L mannitol at 4°C), and vehicle (phosphate-buffered saline) only. Stroke animals that received human umbilical cord blood cells alone or combined human umbilical cord stem cells and mannitol exhibited significantly improved motor performance and significantly better brain cell survival in the peri-infarct area compared to stroke animals that received vehicle or mannitol alone, with mannitol treatment reducing the stem cell dose necessary to afford functional outcomes. Enhanced neurogenesis in the subventricular zone accompanied the combined treatment of human umbilical cord stem cells and mannitol. We showed that BBB permeation facilitates the therapeutic effects of a low dose of peripherally transplanted stem cells to effectively cause functional improvement and increase neurogenesis in chronic stroke.

  13. Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hilde Kvestad

    2014-01-01

    Full Text Available The combined use of the histone deacetylase inhibitor valproic acid (VPA, the retinoic acid receptor-α agonist all-trans retinoic acid (ATRA, and the deoxyribonucleic acid polymerase-α inhibitor cytarabine (Ara-C is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML. Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

  14. Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells.

    Science.gov (United States)

    Wang, Zhi-Ming; Zhou, Le-Yuan; Liu, Bin-Bin; Jia, Qin-An; Dong, Yin-Ying; Xia, Yun-Hong; Ye, Sheng-Long

    2014-10-01

    The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (Pprofile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

  15. T-cell intracellular antigen (TIA-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy.

    Directory of Open Access Journals (Sweden)

    Carmen Sánchez-Jiménez

    Full Text Available Mice lacking either T-cell intracellular antigen 1 (TIA1 or TIA1 related/like protein (TIAR/TIAL1 show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF. Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress.

  16. Uncivil engineers: Chlamydia, Salmonella and Shigella alter cytoskeleton architecture to invade epithelial cells.

    Science.gov (United States)

    Dunn, Joe Dan; Valdivia, Raphael H

    2010-08-01

    The obligate intracellular bacterial pathogen Chlamydia trachomatis is a major cause of blindness and sexually transmitted diseases. Like the enteric pathogens Salmonella and Shigella, Chlamydia injects effector proteins into epithelial cells to initiate extensive remodeling of the actin cytoskeleton at the bacterial attachment site, which culminates in the engulfment of the bacterium by plasma membrane extensions. Numerous Salmonella and Shigella effectors promote this remodeling by activating Rho GTPases and tyrosine kinase signaling cascades and by directly manipulating actin dynamics. Recent studies indicate that similar host-cell alterations occur during Chlamydia invasion, but few effectors are known. The identification of additional Chlamydia effectors and the elucidation of their modes of function are critical steps towards an understanding of how this clinically important pathogen breaches epithelial surfaces and causes infection.

  17. NF-Y activates genes of metabolic pathways altered in cancer cells.

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  18. Alteration of T cell function in healthy persons with a history of thymic x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Rieger, C.H.L.; Kraft, S.C.; Rothberg, R.M.

    1975-10-01

    The possible late effects of x irradiation to the infantile thymus were investigated by studying immune functions in 12 healthy persons with a history of thymic x irradiation and healthy control subjects. No differences were found in serum immunoglobulin values, humoral antibody levels, lymphocyte counts, and lymphocyte reactivity to phytohemagglutinin, vaccinia virus, purified protein derivative (PPD), and allogeneic cells. The irradiation group exhibited cellular hyperresponsiveness to streptokinase-streptodornase (SK-SD). In contrast, mean skin and in vitro lymphocyte responses to Candida albicans were depressed in the patients with thymic irradiation. A dissociation of these two Candida responses was found in only 1 of 14 healthy control subjects but in 7 of 12 irradiated individuals. While thymic irradiation did not result in impaired immunologic defenses leading to clinical disease, it caused alterations in T cell responses similar to those reported in patients with chronic mucocutaneous candidiasis.

  19. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    Science.gov (United States)

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  20. Psychoneuroendocrine immunology: perception of stress can alter body temperature and natural killer cell activity.

    Science.gov (United States)

    Hiramoto, R N; Solvason, H B; Hsueh, C M; Rogers, C F; Demissie, S; Hiramoto, N S; Gauthier, D K; Lorden, J F; Ghanta, V K

    1999-01-01

    Psychoimmunology has been credited with using the mind as a way to alter immunity. The problem with this concept is that many of the current psychoimmunology techniques in use are aimed at alleviating stress effects on the immune system rather than at direct augmentation of immunity by the brain. Studies in animals provide a model that permits us to approach the difficulties associated with gaining an understanding of the CNS-immune system connection. A particular advantage of using animals over humans is that psychological and social contributions play a less prominent role for animals than for human subjects, since the animals are all inbred and reared under identical controlled conditions. If the insightful information provided by animal studies is correct, then psychotherapy for the treatment of diseases might be made more effective if some aspect of this knowledge is included in the design of the treatment. We emphasize conditioning as a regimen and an acceptable way to train the brain to remember an output pathway to raise immunity. We propose that a specific drug or perception (mild stress, represented by rotation, total body heating or handling) could substitute and kindle the same output pathway without the need for conditioning. If this view is correct, then instead of using conditioning, it may be possible to use an antigen to activate desired immune cells, and substitute a drug or an external environmental sensory stimulus (perception) to energize the output pathway to these cells. Alternatively, monitoring alterations of body temperature in response to a drug or perception might allow us to follow how effectively the brain is performing in altering immunity. Studies with animals suggest that there are alternative ways to use the mind to raise natural or acquired immunity in man.

  1. Alteration of osteoblast arrangement via direct attack by cancer cells: New insights into bone metastasis

    Science.gov (United States)

    Kimura, Yumi; Matsugaki, Aira; Sekita, Aiko; Nakano, Takayoshi

    2017-01-01

    Intact bone tissue exhibits a characteristic anisotropic microstructure derived from collagen fiber alignment and the related c-axis orientation of apatite crystals, which govern the mechanical properties of bone tissue. In contrast, tumor-invaded bone exhibits a disorganized, less-aligned microstructure that results in severely disrupted mechanical function. Despite its importance both in basic principle and in therapeutic applications, the classical understanding of bone metastasis is limited to alterations in bone mass regulated by metastatic cancer cells. In this study, we demonstrate a novel mechanism underlying the disruption of bone tissue anisotropy in metastasized bone. We observed that direct attack by cancer cells on osteoblasts induces the less-organized osteoblast arrangement. Importantly, the crystallographic anisotropy of bone tissue is quantitatively determined by the level of osteoblast arrangement. Osteoblast arrangement was significantly disrupted by physical contact with cancer cells such as osteolytic melanoma B16F10, breast cancer MDA-MB-231, and osteoblastic prostate cancer MDA-PCa-2b cells. The present findings demonstrate that the abnormal arrangement of osteoblasts induced by physical contact with cancer cells facilitates the disorganized microstructure of metastasized bone. PMID:28303941

  2. Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance.

    Science.gov (United States)

    Kalluri, Udaya C; Yin, Hengfu; Yang, Xiaohan; Davison, Brian H

    2014-12-01

    Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance.

  3. Matrine alters microRNA expression profiles in SGC-7901 human gastric cancer cells.

    Science.gov (United States)

    Li, Hailong; Xie, Shoupin; Liu, Xiaojun; Wu, Hongyan; Lin, Xingyao; Gu, Jing; Wang, Huping; Duan, Yongqiang

    2014-11-01

    Matrine, a major alkaloid extracted from Sophora flavescens, has been reported to possess antitumor properties in several types of cancers, including gastric cancer. However, its mechanisms of action on gastric cancer remain poorly understood. Dysregulation of microRNAs, a class of small, non-coding, regulatory RNA molecules involved in gene expression, is strongly correlated with cancer. The aim of the present study was to demonstrate that matrine treatment altered miRNA expression in SGC7901 cells. Using miRCURY™ microarray analysis, we identified 128 miRNAs substantially exhibiting >2-fold expression changes in matrine-treated cells relative to their expression levels in untreated cells. RT-qPCR was used to show that the levels of 8 miRNAs whose target genes were clustered in the cell cycle pathway increased, while levels of 14 miRNAs whose target genes were clustered in the MAPK signaling pathway decreased. These results were consistent with those from the miRNA microarray experiment. Bioinformatical analysis revealed that the majority of 57 identified enrichment pathways were highly involved in tumorigenesis. In conclusion, the results demonstrated that matrine induces considerable changes in the miRNA expression profiles of SGC7901 cells, suggesting miRNA microarray combined with RT-qPCR validation and bioinformatical analysis provide a novel and promising approach to identify anticancer targets and the mechanisms of matrine involved.

  4. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

    Science.gov (United States)

    Blaya, Delia; Morales-Ibanez, Oriol; Coll, Mar; Millán, Cristina; Altamirano, José; Arroyo, Vicente; Caballería, Joan; Bataller, Ramón; Ginès, Pere; Sancho-Bru, Pau

    2015-01-01

    Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis. PMID:26691857

  5. Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The events of cell death and the expression of nuclear matrix protein(NMP)have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide.By means of TUNEL assay,the nuclei displayed a characteristic morphology change,and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h.Nucleosomal DNA fragmentation was observed after treatment for 4 h.The morphological change of HL-60 cells,thus,occurred earlier than the appearance of DNA ladder.Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis.Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells.Western blotting was then performed on three nuclear matrix acssociated proteins,PML,HSC70 and NuMA.The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment,while NuMA,a nuclear mitotic apparatus protein,was down regulated.These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

  6. Fingolimod alters the transcriptome profile of circulating CD4+ cells in multiple sclerosis

    Science.gov (United States)

    Friess, Jörg; Hecker, Michael; Roch, Luisa; Koczan, Dirk; Fitzner, Brit; Angerer, Ines Charlotte; Schröder, Ina; Flechtner, Kristin; Thiesen, Hans-Jürgen; Winkelmann, Alexander; Zettl, Uwe Klaus

    2017-01-01

    Multiple sclerosis is a demyelinating disease affecting the central nervous system. T cells are known to contribute to this immune-mediated condition. Fingolimod modulates sphingosine-1-phosphate receptors, thereby preventing the egress of lymphocytes, especially CCR7-expressing CD8+ and CD4+ T cells, from lymphoid tissues. Using Affymetrix Human Transcriptome Arrays (HTA 2.0), we performed a transcriptome profiling analysis of CD4+ cells obtained from the peripheral blood of patients with highly active relapsing-remitting multiple sclerosis. The samples were drawn before the first administration of fingolimod as well as 24 hours and 3 months after the start of therapy. Three months after treatment initiation, 890 genes were found to be differentially expressed with fold-change >2.0 and t-test p-value CCR7 as expected, while 618 genes showed an increase in expression, e.g., CCR2, CX3CR1, CD39, CD58 as well as LYN, PAK1 and TLR2. To conclude, we studied the gene expression of CD4+ cells to evaluate the effects of fingolimod treatment, and we identified 890 genes to be altered in expression after continuous drug administration. T helper cells circulating in the blood during fingolimod therapy present a distinct gene expression signature. PMID:28155899

  7. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis.

    Directory of Open Access Journals (Sweden)

    Silvia Affò

    Full Text Available Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+ and mature dendritic (MHCII+CD11c+ cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

  8. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis.

    Science.gov (United States)

    Affò, Silvia; Rodrigo-Torres, Daniel; Blaya, Delia; Morales-Ibanez, Oriol; Coll, Mar; Millán, Cristina; Altamirano, José; Arroyo, Vicente; Caballería, Joan; Bataller, Ramón; Ginès, Pere; Sancho-Bru, Pau

    2015-01-01

    Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

  9. Diet-induced obesity alters dendritic cell function in the presence and absence of tumor growth.

    Science.gov (United States)

    James, Britnie R; Tomanek-Chalkley, Ann; Askeland, Eric J; Kucaba, Tamara; Griffith, Thomas S; Norian, Lyse A

    2012-08-01

    Obesity is a mounting health concern in the United States and is associated with an increased risk for developing several cancers, including renal cell carcinoma (RCC). Despite this, little is known regarding the impact of obesity on antitumor immunity. Because dendritic cells (DC) are critical regulators of antitumor immunity, we examined the combined effects of obesity and tumor outgrowth on DC function. Using a diet-induced obesity (DIO) model, DC function was evaluated in mice bearing orthotopic RCC and in tumor-free controls. Tumor-free DIO mice had profoundly altered serum cytokine and chemokine profiles, with upregulation of 15 proteins, including IL-1α, IL-17, and LIF. Tumor-free DIO mice had elevated percentages of conventional splenic DC that were impaired in their ability to stimulate naive T cell expansion, although they were phenotypically similar to normal weight (NW) controls. In DIO mice, intrarenal RCC tumor challenge in the absence of therapy led to increased local infiltration by T cell-suppressive DC and accelerated early tumor outgrowth. Following administration of a DC-dependent immunotherapy, established RCC tumors regressed in normal weight mice. The same immunotherapy was ineffective in DIO mice and was characterized by an accumulation of regulatory DC in tumor-bearing kidneys, decreased local infiltration by IFN-γ-producing CD8 T cells, and progressive tumor outgrowth. Our results suggest that the presence of obesity as a comorbidity can impair the efficacy of DC-dependent antitumor immunotherapies.

  10. Genetic alteration of poly(ADP-ribose) polymerase-1 in human germ cell tumors.

    Science.gov (United States)

    Shiokawa, Motoko; Masutani, Mitsuko; Fujihara, Hisako; Ueki, Keisuke; Nishikawa, Ryo; Sugimura, Takashi; Kubo, Harumi; Nakagama, Hitoshi

    2005-02-01

    Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5'-UTR (untranslated region) were also detected.

  11. Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration.

    Science.gov (United States)

    Lechner, Judith; Chen, Mei; Hogg, Ruth E; Toth, Levente; Silvestri, Giuliana; Chakravarthy, Usha; Xu, Heping

    2015-11-17

    Neovascular age-related macular degeneration (nAMD) is the leading cause of irreversible blindness in developed countries. Recent advances have highlighted the essential role of inflammation in the development of the disease. In addition to local retinal chronic inflammatory response, systemic immune alterations have also been observed in AMD patients. In this study we investigated the association between the frequency of circulating leukocyte populations and the prevalence as well as clinical presentations of nAMD. Leukocyte subsets of 103 nAMD patients (most of them were receiving anti-VEGF therapy prior to enrolment) and 26 controls were analysed by flow cytometry by relative cell size, granularity and surface markers. Circulating CD11b(+) cells and CD16(hi)HLA-DR(-) neutrophils were significantly increased (P = 0.015 and 0.009 respectively) in nAMD when compared to controls. The percentage of circulating CD4(+) T-cells was reduced in nAMD patients without subretinal fibrosis (P = 0.026) compared to patients with subretinal fibrosis. There was no correlation between the percentage of circulating leukocytes and the responsiveness to anti-VEGF therapy in nAMD patients. Our results suggest that higher levels of circulating CD11b(+) cells and neutrophils are associated with nAMD and that reduced levels of CD4(+) T-cells are associated with the absence of subretinal fibrosis in nAMD.

  12. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    Directory of Open Access Journals (Sweden)

    Thiel Cora S

    2012-01-01

    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  13. Hepatitis C virus and ethanol alter antigen presentation in liver cells

    Institute of Scientific and Technical Information of China (English)

    Natalia A Osna

    2009-01-01

    Alcoholic patients have a high incidence of hepatitis Cvirus (HCV) infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCVinduced inability of the immune system to recognizeinfected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex (MHC) classⅠ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC classⅠand class Ⅱ in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) predominance,preventing cell maturation and allostimulation capacity.The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC classⅠ-restricted antigen presentation.Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.

  14. Alteration of CD44 expression in HIV type 1-infected T cell lines.

    Science.gov (United States)

    Giordanengo, V; Limouse, M; Doglio, A; Lesimple, J; Lefebvre, J C

    1996-11-20

    CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.

  15. Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

    Directory of Open Access Journals (Sweden)

    Damien Maggiorani

    Full Text Available Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2 were subjected to FSS (0.5 Pa for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1, Par polarity complex (Pard6, adherens junctions (E-Cadherin, β-Catenin and the primary cilium (α-acetylated Tubulin were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

  16. Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues

    Science.gov (United States)

    Akşit, Dilek; Yazıcı, Alper; Akşit, Hasan; Sarı, Esin S.; Yay, Arzu; Yıldız, Onur; Kılıç, Adil; Ermiş, Sıtkı S.; Seyrek, Kamil

    2016-01-01

    Background: Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. Aims: The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. Study Design: Animal experiment. Methods: Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. Results: Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA) strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues) specific Griffonia simplicifolia-1 (GSL–1) was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc) specific wheat germ agglutinin (WGA) was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. Conclusion: Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells. PMID:27606141

  17. Toward guided tissue and bone regeneration: morphology, attachment, proliferation, and migration of cells cultured on collagen barrier membranes. A systematic review.

    NARCIS (Netherlands)

    Behring, J.; Junker, R.; Walboomers, X.F.; Chessnut, B.; Jansen, J.A.

    2008-01-01

    Collagen barrier membranes are frequently used in both guided tissue regeneration (GTR) and guided bone regeneration (GBR). Collagen used for these devices is available from different species and is often processed to alter the properties of the final product. This is necessary because unprocessed c

  18. Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

    Institute of Scientific and Technical Information of China (English)

    Yan Gu; Chen-Fang Zhu; Hitoshi Iwamoto; Ji-Sheng Chen

    2005-01-01

    AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice.Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected.RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%)and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75± 1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807)was significantly lower than that in control group (22.330 ± 5.696, P< 0.01).CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.

  19. Genetic and Biochemical Alterations in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jackie L. Johnson

    2012-01-01

    Full Text Available Despite significant advances in the detection and treatment of lung cancer, it causes the highest number of cancer-related mortality. Recent advances in the detection of genetic alterations in patient samples along with physiologically relevant animal models has yielded a new understanding of the molecular etiology of lung cancer. This has facilitated the development of potent and specific targeted therapies, based on the genetic and biochemical alterations present in the tumor, especially non-small-cell lung cancer (NSCLC. It is now clear that heterogeneous cell signaling pathways are disrupted to promote NSCLC, including mutations in critical growth regulatory proteins (K-Ras, EGFR, B-RAF, MEK-1, HER2, MET, EML-4-ALK, KIF5B-RET, and NKX2.1 and inactivation of growth inhibitory pathways (TP53, PTEN, p16, and LKB-1. How these pathways differ between smokers and non-smokers is also important for clinical treatment strategies and development of targeted therapies. This paper describes these molecular targets in NSCLC, and describes the biological significance of each mutation and their potential to act as a therapeutic target.

  20. Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

    Science.gov (United States)

    Schröder, Anke; Rohrbeck, Astrid; Just, Ingo; Pich, Andreas

    2015-11-06

    C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

  1. Alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells.

    Science.gov (United States)

    Gaydos, Jeanette; McNally, Alicia; Guo, Ruixin; Vandivier, R William; Simonian, Philip L; Burnham, Ellen L

    2016-03-15

    Alcohol use disorders (AUDs) and tobacco smoking are associated with an increased predisposition for community-acquired pneumonia and the acute respiratory distress syndrome. Mechanisms are incompletely established but may include alterations in response to pathogens by immune cells, including alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs). We sought to determine the relationship of AUDs and smoking to expression of IFNγ, IL-1β, IL-6, and TNFα by AMs and PBMCs from human subjects after stimulation with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). AMs and PBMCs from healthy subjects with AUDs and controls, matched on smoking, were cultured with LPS (1 μg/ml) or LTA (5 μg/ml) in the presence and absence of the antioxidant precursor N-acetylcysteine (10 mM). Cytokines were measured in cell culture supernatants. Expression of IFNγ, IL-1β, IL-6, and TNFα in AMs and PBMCs was significantly increased in response to stimulation with LPS and LTA. AUDs were associated with augmented production of proinflammatory cytokines, particularly IFNγ and IL-1β, by AMs and PBMCs in response to LPS. Smoking diminished the impact of AUDs on AM cytokine expression. Expression of basal AM and PBMC Toll-like receptors-2 and -4 was not clearly related to differences in cytokine expression; however, addition of N-acetylcysteine with LPS or LTA led to diminished AM and PBMC cytokine secretion, especially among current smokers. Our findings suggest that AM and PBMC immune cell responses to LPS and LTA are influenced by AUDs and smoking through mechanisms that may include alterations in cellular oxidative stress.

  2. Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

    Directory of Open Access Journals (Sweden)

    Marcel A Schijf

    Full Text Available Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+CD25(+Foxp3(+ regulatory T-cells (Tregs in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+ (Th1 activated CD69(+CD4(+ T cells (p<0.001 and reduced percentage of Gata-3(+ (Th2 activated CD69(+CD4(+T cells (p<0.001 was detected in the mesenteric lymph nodes (MLN of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+Foxp3(+ could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+ /T-bet(+ (Th1-Tregs was significantly reduced (p<0.05 in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.

  3. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

    Science.gov (United States)

    FAUVELLE, CATHERINE; ZAHID, MUHAMMAD NAUMAN; TUREK, MARINE; HEYDMANN, LAURA; CURY, KARINE; HAYER, JULIETTE; COMBET, CHRISTOPHE; COSSET, FRANÇOIS–LOÏC; PIETSCHMANN, THOMAS; HIET, MARIE–SOPHIE; BARTENSCHLAGER, RALF; HABERSETZER, FRANÇOIS; DOFFOËL, MICHEL; KECK, ZHEN–YONG; FOUNG, STEVEN K. H.; ZEISEL, MIRJAM B.; STOLL–KELLER, FRANÇOISE; BAUMERT, THOMAS F.

    2017-01-01

    BACKGROUND & AIMS The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture–derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus–antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. PMID:22503792

  4. Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

    Directory of Open Access Journals (Sweden)

    Grotewold Erich

    2005-05-01

    Full Text Available Abstract Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin.

  5. Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

    Indian Academy of Sciences (India)

    Kelley Strohacker; Whitney L Breslin; Katie C Carpenter; Brian K McFarlin

    2012-03-01

    The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115+/Gr-1high) and non-classic (CD115+/Gr-1low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent -tests; significance was set at < 0.05. Old mice had a greater concentration of both classic (258%, =0.003) and non-classic (70%, =0.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (=0.006), indicating a pro-inflammatory shift. TLR4 ($\\downarrow$27%, =0.001) and CD80 ($\\downarrow$37%, =0.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 ($\\uparrow$24%, =0.002) and MHCII ($\\downarrow$21%, =0.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.

  6. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    Science.gov (United States)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  7. Auxin Transport and Ribosome Biogenesis Mutant/Reporter Lines to Study Plant Cell Growth and Proliferation under Altered Gravity

    Science.gov (United States)

    Valbuena, Miguel A.; Manzano, Ana I.; van Loon, Jack JWA.; Saez-Vasquez, Julio; Carnero-Diaz, Eugenie; Herranz, Raul; Medina, F. J.

    2013-02-01

    We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (μg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (μgRPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

  8. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

    Directory of Open Access Journals (Sweden)

    Sylvia Wagner

    Full Text Available BACKGROUND: The blood-brain barrier (BBB represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. CONCLUSIONS/SIGNIFICANCE: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

  9. Magnesium regulates neural stem cell proliferation in the mouse hippocampus by altering mitochondrial function.

    Science.gov (United States)

    Jia, Shanshan; Mou, Chengzhi; Ma, Yihe; Han, Ruijie; Li, Xue

    2016-04-01

    In the adult brain, neural stem cells from the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the cortex progress through the following five developmental stages: radial glia-like cells, neural progenitor cells, neuroblasts, immature neurons, and mature neurons. These developmental stages are linked to both neuronal microenvironments and energy metabolism. Neurogenesis is restricted and has been demonstrated to arise from tissue microenvironments. We determined that magnesium, a key nutrient in cellular energy metabolism, affects neural stem cell (NSC) proliferation in cells derived from the embryonic hippocampus by influencing mitochondrial function. Densities of proliferating cells and NSCs both showed their highest values at 0.8 mM [Mg(2+) ]o , whereas lower proliferation rates were observed at 0.4 and 1.4 mM [Mg(2+) ]o . The numbers and sizes of the neurospheres reached the maximum at 0.8 mM [Mg(2+) ]o and were weaker under both low (0.4 mM) and high (1.4 mM) concentrations of magnesium. In vitro experimental evidence demonstrates that extracellular magnesium regulates the number of cultured hippocampal NSCs, affecting both magnesium homeostasis and mitochondrial function. Our findings indicate that the effect of [Mg(2+) ]o on NSC proliferation may lie downstream of alterations in mitochondrial function because mitochondrial membrane potential was highest in the NSCs in the moderate [Mg(2+) ]o (0.8 mM) group and lower in both the low (0.4 mM) and high (1.4 mM) [Mg(2+) ]o groups. Overall, these findings demonstrate a new function for magnesium in the brain in the regulation of hippocampal neural stem cells: affecting their cellular energy metabolism.

  10. Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain.

    Directory of Open Access Journals (Sweden)

    Manohar B Mutnal

    Full Text Available BACKGROUND: Congenital cytomegalovirus (CMV brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV and the pattern of injury to the developing brain. METHODOLOGY/PRINCIPAL FINDINGS: We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection. CONCLUSIONS: MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

  11. Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Sagarika; Mukherji, Suparna [Indian Institute of Technology Bombay, Mumbai (India). Centre for Environmental Science and Engineering (CESE)

    2012-04-15

    Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50-52 ) was in the same range for both strains while zeta potential at neutral pH was -38 and -31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75 and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

  12. Clozapine-induced mitochondria alterations and inflammation in brain and insulin-responsive cells.

    Directory of Open Access Journals (Sweden)

    Verόnica Contreras-Shannon

    Full Text Available BACKGROUND: Metabolic syndrome (MetS is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined. METHODOLOGY/PRINCIPAL FINDINGS: Cultured mouse myoblasts (C2C12, adipocytes (3T3-L1, hepatocytes (FL-83B, and monocytes (RAW 264.7 were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line. CONCLUSIONS/SIGNIFICANCE: Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated

  13. Altered Morphology of Hippocampal Dentate Granule Cell Presynaptic and Postsynaptic Terminals Following Conditional Deletion of TrkB

    OpenAIRE

    Danzer, Steve C.; Kotloski, Robert J.; Walter, Cynthia; Hughes, Maya; McNamara, James O.

    2008-01-01

    Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand...

  14. Altered Cytokine Production By Specific Human Peripheral Blood Cell Subsets Immediately Following Spaceflight

    Science.gov (United States)

    Crucian, Brian E.; Cubbage, Michael L.; Sams, Clarence F.

    1999-01-01

    In this study, we have attempted to combine standard immunological assays with the cellular resolving power of the flow cytometer to positively identify the specific cell types involved in spaceflight-induced immune alterations. We have obtained whole blood samples from 27 astronauts collected at three timepoints (L-10, R+0 and R+3) surrounding four recent space shuttle missions. The duration of these missions ranged from 10 to 18 days. Assays performed included serum/urine cortisol, comprehensive subset phenotyping, assessment of cellular activation markers and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following spaceflight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated trends towards a decreased percentage of T cells and an increased percentage of B cells. Nearly all of the astronauts exhibited an increased CD4:CD8 ratio, which was dramatic in some individuals. Assessment of memory (CD45RA+) vs. naive (CD45RO+) CD4+ T cell subsets was more ambiguous, with subjects tending to group more as a flight crew. All subjects from one mission demonstrated an increased CD45RA:CD45RO ratio, while all subjects from another Mission demonstrated a decreased ratio. While no significant trend was seen in the monocyte population as defined by scatter, a decreased percentage of the CD14+ CD16+ monocyte subset was seen following spaceflight in all subjects tested. In general, most of the cellular changes described above which were assessed at R+O and compared to L-10 trended to pre-flight levels by R+3. Although no significant differences were seen in the expression of the cellular activation markers CD69 and CD25 following exposure to microgravity, significant alterations were seen in cytokine production in response to mitogenic activation for specific subsets. T cell (CD3+) production of IL-2 was significantly decreased

  15. Altered Ca2+-Homeostasis of Cisplatin-Treated and Low Level Resistant Non-Small-Cell and Small-Cell Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kathrin Schrödl

    2009-01-01

    Full Text Available Background: Chemotherapy often leads to encouraging responses in lung cancer. But, in the course of the treatment, resistance to chemotherapy ultimately limits the life expectancy of the patient. We aimed at investigating if treatment with cisplatin alters the intracellular Ca2+-homeostasis of lung cancer cells and how this may be related to cisplatin resistance.

  16. The Immunosuppressant Mycophenolic Acid Alters Nucleotide and Lipid Metabolism in an Intestinal Cell Model

    Science.gov (United States)

    Heischmann, Svenja; Dzieciatkowska, Monika; Hansen, Kirk; Leibfritz, Dieter; Christians, Uwe

    2017-01-01

    The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using 1H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 μM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 μM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 μM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity. PMID:28327659

  17. Short-term cigarette smoke exposure leads to metabolic alterations in lung alveolar cells.

    Science.gov (United States)

    Agarwal, Amit R; Yin, Fei; Cadenas, Enrique

    2014-08-01

    Cigarette smoke (CS)-induced alveolar destruction and energy metabolism changes are known contributors to the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examines the effect of CS exposure on metabolism in alveolar type II cells. Male A/J mice (8 wk old) were exposed to CS generated from a smoking machine for 4 or 8 weeks, and a recovery group was exposed to CS for 8 weeks and allowed to recover for 2 weeks. Alveolar type II cells were isolated from air- or CS- exposed mice. Acute CS exposure led to a reversible airspace enlargement in A/J mice as measured by the increase in mean linear intercept, indicative of alveolar destruction. The effect of CS exposure on cellular respiration was studied using the XF Extracellular Flux Analyzer. A decrease in respiration while metabolizing glucose was observed in the CS-exposed group, indicating altered glycolysis that was compensated by an increase in palmitate utilization; palmitate utilization was accompanied by an increase in the expression of CD36 and carnitine-palmitoyl transferase 1 in type II alveolar cells for the transport of palmitate into the cells and into mitochondria, respectively. The increase in palmitate use for energy production likely affects the surfactant biosynthesis pathway, as evidenced by the decrease in phosphatidylcholine levels and the increase in phospholipase A2 activity after CS exposure. These findings help our understanding of the mechanism underlying the surfactant deficiency observed in smokers and provide a target to delay the onset of COPD.

  18. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  19. Phenethylisothiocyanate alters site- and promoter-specific histone tail modifications in cancer cells.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Site-specific histone modifications are important epigenetic regulators of gene expression. As deregulation of genes often results in complex disorders, corrective modulation of site-specific histone marks could be a powerful therapeutic or disease-preventive strategy. However, such modulation by dietary compounds and the resulting impact on disease risk remain relatively unexplored. Here we examined phenethylisothiocyanate (PEITC, a common dietary compound derived from cruciferous vegetables with known chemopreventive properties under experimental conditions, as a possible modulator of histone modifications in human colon cancer cells. The present study reports novel, dynamic, site-specific chemical changes to histone H3 in a gene-promoter-specific manner, associated with PEITC exposure in human colon tumor-derived SW480 epithelial cells. In addition, PEITC attenuated cell proliferation in a concentration- and time-dependent manner, likely mediated by caspase-dependent apoptotic signalling. The effects of PEITC on histone modifications and gene expression changes were achieved at low, non-cytotoxic concentrations, in contrast to the higher concentrations necessary to halt cancer cell proliferation. Increased understanding of specific epigenetic alterations by dietary compounds may provide improved chemopreventive strategies for reducing the healthcare burden of cancer and other human diseases.

  20. PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression.

    Science.gov (United States)

    Vincent-Fabert, Christelle; Platet, Nadine; Vandevelde, Amelle; Poplineau, Mathilde; Koubi, Myriam; Finetti, Pascal; Tiberi, Guillaume; Imbert, Anne-Marie; Bertucci, François; Duprez, Estelle

    2016-04-14

    Hematopoietic stem cells (HSCs) give rise to all blood populations due to their long-term self-renewal and multipotent differentiation capacities. Because they have to persist throughout an organism's life span, HSCs tightly regulate the balance between proliferation and quiescence. Here, we investigated the role of the transcription factor promyelocytic leukemia zinc finger (plzf) in HSC fate using the Zbtb16(lu/lu)mouse model, which harbors a natural spontaneous mutation that inactivates plzf. Regenerative stress revealed that Zbtb16(lu/lu)HSCs had a lineage-skewing potential from lymphopoiesis toward myelopoiesis, an increase in the long-term-HSC pool, and a decreased repopulation potential. Furthermore, oldplzf-mutant HSCs present an amplified aging phenotype, suggesting that plzf controls age-related pathway. We found that Zbtb16(lu/lu)HSCs harbor a transcriptional signature associated with a loss of stemness and cell cycle deregulation. Lastly, cell cycle analyses revealed an important role for plzf in the regulation of the G1-S transition of HSCs. Our study reveals a new role for plzf in regulating HSC function that is linked to cell cycle regulation, and positions plzf as a key player in controlling HSC homeostasis.

  1. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial Epithelial Cells In Vitro

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examin...

  2. Diets high in fermentable protein and fibre alter tight junction protein composition with minor effects on barrier function in piglet colon.

    Science.gov (United States)

    Richter, Jan F; Pieper, Robert; Zakrzewski, Silke S; Günzel, Dorothee; Schulzke, Joerg D; Van Kessel, Andrew G

    2014-03-28

    Protein fermentation end products may damage the colonic mucosa, which could be counteracted by dietary inclusion of fermentable carbohydrates (fCHO). Although fermentable crude protein (fCP) and fCHO are known to affect microbial ecology, their interactive effects on epithelial barrier function are unknown. In the present study, in a 2 × 2 factorial experiment, thirty-two weaned piglets were fed low-fCP/low-fCHO (14·5 % crude protein (CP)/14·5 % total dietary fibre (TDF)), low-fCP/high-fCHO (14·8 % CP/16·6 % TDF), high-fCP/low-fCHO (19·8 % CP/14·5 % TDF) and high-fCP/high-fCHO (20·1 % CP/18·0 % TDF) diets. After 21-23 d, samples of proximal and distal colonic mucosae were investigated in Ussing chambers with respect to the paracellular and transcytotic passages of macromolecules and epithelial ion transport. The high-fCHO diets were found to reduce the permeability of the distal colon to the transcytotic marker horseradish peroxidase (HRP, 44 kDa; P ion transport), transepithelial resistance (barrier function) and charge selectivity were largely unaffected in both the segments. However, the high-fCP diets were found to suppress the aldosterone-induced epithelial Na channel activity (P composition in a compensatory way, so that colonic transport and barrier properties were only marginally affected.

  3. The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins.

    Science.gov (United States)

    van Herwaarden, Antonius E; Schinkel, Alfred H

    2006-01-01

    The breast cancer resistance protein [BCRP (also known as ABCG2)] belongs to the ATP binding cassette (ABC) family of transmembrane drug transporters. BCRP has a broad substrate specificity and actively extrudes a wide variety of drugs, carcinogens and dietary toxins from cells. Situated in the apical plasma membrane of epithelial cells of the small and large intestine and renal proximal tubules and in the bile canalicular membrane of hepatocytes, BCRP decreases the oral availability and systemic exposure of its substrates. In several blood-tissue barriers BCRP reduces tissue penetration of its substrates and it protects haematopoietic stem cells from cytotoxic substrates. Moreover, BCRP is expressed in mammary gland alveolar epithelial cells during pregnancy and lactation, where it actively secretes a variety of drugs, toxins and carcinogens into milk. In apparent contradiction with the detoxifying role of BCRP in mothers, this contamination of milk exposes suckling infants and dairy consumers to xenotoxins. BCRP thus affects many important aspects of pharmacology and toxicology.

  4. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    Energy Technology Data Exchange (ETDEWEB)

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  5. Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice

    Directory of Open Access Journals (Sweden)

    Bao Tim

    2009-09-01

    Full Text Available Abstract Background Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. Methods An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL-4 and IL-13 as markers of inflammation. Results Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13. At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics. Conclusion Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.

  6. Oxygen- and water-induced degradation of an inverted polymer solar cell: the barrier effect

    DEFF Research Database (Denmark)

    Vesterager Madsen, Morten; Norrman, Kion; Krebs, Frederik C

    2011-01-01

    , the barrier effect is reported in both a dry oxygen atmosphere and an oxygen-free humid atmosphere. The devices under study are comprised of a bulk heterojunction formed by poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester sandwiched between a layer of zinc oxide (electron transporting...

  7. Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle.

    Science.gov (United States)

    Woo, H-H; Hirsch, A M; Hawes, M C

    2004-07-01

    Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.

  8. HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus

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    Klein George

    2007-04-01

    Full Text Available Abstract The latency-associated nuclear antigen (LANA-1 of Human Herpes Virus 8 (HHV-8, alternatively called Kaposi Sarcoma Herpes Virus (KSHV is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin 1. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells 2. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3. The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2. The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.

  9. Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

    Science.gov (United States)

    Brummelman, Jolanda; Veerman, Rosanne E; Hamstra, Hendrik Jan; Deuss, Anna J M; Schuijt, Tim J; Sloots, Arjen; Kuipers, Betsy; van Els, Cécile A C M; van der Ley, Peter; Mooi, Frits R; Han, Wanda G H; Pinelli, Elena

    2015-01-01

    Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

  10. Tumor promoters alter the temporal program of adenovirus replication in human cells.

    Science.gov (United States)

    Fisher, P B; Young, C S; Weinstein, I B; Carter, T H

    1981-04-01

    In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

  11. Dielectric and diffusion barrier multilayer for Cu(In,Ga)Se{sub 2} solar cells integration on stainless steel sheet

    Energy Technology Data Exchange (ETDEWEB)

    Amouzou, Dodji, E-mail: dodji.amouzou@fundp.ac.be [Research Centre in Physics of Matter and Radiation (PMR), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur (Belgium); Guaino, Philippe; Fourdrinier, Lionel; Richir, Jean-Baptiste; Maseri, Fabrizio [CRM-Group, Boulevard de Colonster, B 57, 4000 Liège (Belgium); Sporken, Robert [Research Centre in Physics of Matter and Radiation (PMR), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur (Belgium)

    2013-09-02

    For the fabrication of monolithically integrated flexible Cu(In, Ga)Se{sub 2}, CIGS modules on stainless steel, individual photovoltaic cells must be insulated from metal substrates by a barrier layer that can sustain high thermal treatments. In this work, a combination of sol–gel (organosilane-sol) and sputtered SiAlxOy forming thin diffusion barrier layers (TDBL) was prepared on stainless steel substrates. The deposition of organosilane-sol dielectric layers on the commercial stainless steel (maximal roughness, Rz = 500 nm and Root Mean Square roughness, RMS = 56 nm) induces a planarization of the surface (RMS = 16.4 nm, Rz = 176 nm). The DC leakage current through the dielectric layers was measured for the metal-insulator-metal (MIM) junctions that act as capacitors. This method allowed us to assess the quality of our TDBL insulating layer and its lateral uniformity. Indeed, evaluating a ratio of the number of valid MIM capacitors to the number of tested MIM capacitors, a yield of ∼ 95% and 50% has been reached respectively with non-annealed and annealed samples based on sol–gel double layers. A yield of 100% was achieved for sol–gel double layers reinforced with a sputtered SiAlxOy coating and a third sol–gel monolayer. Since this yield is obtained on several samples, it can be extrapolated to any substrate size. Furthermore, according to Glow Discharge Optical Emission Spectroscopy and Time of Flight Secondary Ion Mass Spectroscopy measurements, these barrier layers exhibit excellent barrier properties against the diffusion of undesired atoms which could otherwise spoil the electronic and optical properties of CIGS photovoltaic cells. - Highlights: • We functionalize steel for monolithically integrated Cu(In,Ga)Se{sub 2} solar cells • Thin dielectric and diffusion barrier layers (TDDBL) prepared on steel • Reliability and breakdown voltage of dielectric layers have been studied. • Investigation of thermal treatment effect on dielectric

  12. PROGNOSTIC VALUE OF VHL GENE ALTERATION IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

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    D. A. Nosov

    2011-01-01

    Full Text Available Objective: to estimate the rate, predictive and prognostic value of VHL gene alterations in the population of patients with sporadic metastatic renal cell carcinoma (mRCC.Subjects and methods. Paraffin embedded tumor tissue blocks were available from 88 patients with mRCC who had undergone antitumor therapy in 1994- 2010. Of them, 53 patients received only immunotherapy regimens with interferon (IFN-α and 35 patients had targeted therapy with VEGFR inhibitors. VHL mutations were detected by polymerase chain reaction (PCR for exons of 1-3, single-strand conformation polymorphism analysis of PCR products, and further sequencing. VHL gene methylation was determined by methyl-sensitive PCR.Results. Somatic mutations and/or promoter hypermethylation of the VHL gene were found in 23 (26% patients; Of them, VHL gene mutations and promoter hypermethylation were found in 15 patients and 7 patients respectively. Mutation and promoter methylation VHL were simultaneously observed in one case. VHL gene mutations were detected only in patients with clear cell RCC while aberrant promoter methylation was seen in both clear cell and papillary RCC. With a median follow-up of 34 months (range, 2-127 months, the median time to progression (TTP and median overall survival (OS for the entire group of patients were 5.8 and 26.7 months, respectively. In patients with and without VHL gene alterations, the median TTP was 5.5 and 6.9 months, respectively (p = 0.15 and the median overall survival time was 22.0 and 34.5 months, respectively (p = 0.98. Moreover, the subgroup analysis revealed that VHL gene inactivation events had no impact on the objective response rate (ORR, TTP and OS in the subgroup of patients who received immunotherapy (n = 53 or antiangiogenic targeted therapy (n = 35 (p > 0.05.Conclusion. VHL gene mutations and/or promotor hypermethylation observed in 26% of patients with mRCC. These VHL gene alterations were neither prognostic nor predictive factors

  13. PROGNOSTIC VALUE OF VHL GENE ALTERATION IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

    Directory of Open Access Journals (Sweden)

    D. A. Nosov

    2014-08-01

    Full Text Available Objective: to estimate the rate, predictive and prognostic value of VHL gene alterations in the population of patients with sporadic metastatic renal cell carcinoma (mRCC.Subjects and methods. Paraffin embedded tumor tissue blocks were available from 88 patients with mRCC who had undergone antitumor therapy in 1994- 2010. Of them, 53 patients received only immunotherapy regimens with interferon (IFN-α and 35 patients had targeted therapy with VEGFR inhibitors. VHL mutations were detected by polymerase chain reaction (PCR for exons of 1-3, single-strand conformation polymorphism analysis of PCR products, and further sequencing. VHL gene methylation was determined by methyl-sensitive PCR.Results. Somatic mutations and/or promoter hypermethylation of the VHL gene were found in 23 (26% patients; Of them, VHL gene mutations and promoter hypermethylation were found in 15 patients and 7 patients respectively. Mutation and promoter methylation VHL were simultaneously observed in one case. VHL gene mutations were detected only in patients with clear cell RCC while aberrant promoter methylation was seen in both clear cell and papillary RCC. With a median follow-up of 34 months (range, 2-127 months, the median time to progression (TTP and median overall survival (OS for the entire group of patients were 5.8 and 26.7 months, respectively. In patients with and without VHL gene alterations, the median TTP was 5.5 and 6.9 months, respectively (p = 0.15 and the median overall survival time was 22.0 and 34.5 months, respectively (p = 0.98. Moreover, the subgroup analysis revealed that VHL gene inactivation events had no impact on the objective response rate (ORR, TTP and OS in the subgroup of patients who received immunotherapy (n = 53 or antiangiogenic targeted therapy (n = 35 (p > 0.05.Conclusion. VHL gene mutations and/or promotor hypermethylation observed in 26% of patients with mRCC. These VHL gene alterations were neither prognostic nor predictive factors

  14. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

    Science.gov (United States)

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

    2014-01-01

    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  15. Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

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    Ravi Shankar Akundi

    Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

  16. Growth of human bronchial epithelial cells at an air-liquid interface alters the response to particle exposure

    Science.gov (United States)

    Abstract: We tested the hypothesis that relative to submerged cells, airway epithelial cells grown at an air-liquid interface would have an altered response to particle exposure. RNA for IL-8, IL-6, heme oxygenase 1 and cyclooxygenase 2 increased following exposure of submer...

  17. The calpain, caspase 12, caspase 3 cascade leading to apoptosis is altered in F508del-CFTR expressing cells.

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    Mathieu Kerbiriou

    Full Text Available In cystic fibrosis (CF, the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER. We previously showed that the unfolded protein response (UPR may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.

  18. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  19. Red blood cells in Rett syndrome: oxidative stress, morphological changes and altered membrane organization.

    Science.gov (United States)

    Ciccoli, Lucia; De Felice, Claudio; Leoncini, Silvia; Signorini, Cinzia; Cortelazzo, Alessio; Zollo, Gloria; Pecorelli, Alessandra; Rossi, Marcello; Hayek, Joussef

    2015-11-01

    In this review, we summarize the current evidence on the erythrocyte as a previously unrecognized target cell in Rett syndrome, a rare (1:10 000 females) and devastating neurodevelopmental disorder caused by loss-of-function mutations in a single gene (i.e. MeCP2, CDKL5, or rarely FOXG1). In particular, we focus on morphological changes, membrane oxidative damage, altered membrane fatty acid profile, and aberrant skeletal organization in erythrocytes from patients with typical Rett syndrome and MeCP2 gene mutations. The beneficial effects of ω-3 polyunsaturated fatty acids (PUFAs) are also summarized for this condition to be considered as a 'model' condition for autism spectrum disorders.

  20. Arsenic alters ATP-dependent Ca²+ signaling in human airway epithelial cell wound response.

    Science.gov (United States)

    Sherwood, Cara L; Lantz, R Clark; Burgess, Jefferey L; Boitano, Scott

    2011-05-01

    Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca²+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease.

  1. HPV detection and p53 alteration in squamous cell verrucous malignancies of the lower genital tract.

    Science.gov (United States)

    Pilotti, S; Donghi, R; D'Amato, L; Giarola, M; Longoni, A; Della Torre, G; De Palo, G; Pierotti, M A; Rilke, F

    1993-12-01

    We examined five cases of verrucous carcinoma (VC) and two cases of giant condyloma of Buschke-Löwenstein (GCBL) associated with invasive squamous cell carcinoma (ISCC), by immunocytochemistry and molecular techniques. Neither human papillomavirus (HPV) footprints nor p53-altered expression and/or mutation were observed among the cases of VC. By contrast, both cases of GCBL with ISCC turned out to be HPV 6 or 11 positive, showed overexpression of p53 and, one of the two, a mutation in the nucleotide sequence of this tumor suppressor gene. The results point out that VC and GCBL with ISCC, in spite of some morphologic similarities, are two distinct entities, the former being unrelated to both HPV and p53 inactivation and the latter related to both. Regarding p53, immunocytochemical and molecular data on GCBL with ISCC suggest a role of mutant p53 in the progression of malignancy into invasion.

  2. Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate

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    Priya Srikanth

    2015-09-01

    Full Text Available Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1 as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11 translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

  3. Olfactory aversive conditioning alters olfactory bulb mitral/tufted cell glomerular odor responses

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    Max L Fletcher

    2012-03-01

    Full Text Available The anatomical organization of receptor neuron input into the olfactory bulb (OB allows odor information to be transformed into an odorant-specific spatial map of mitral/tufted cell glomerular activity at the upper level of the olfactory bulb. In other sensory systems, neuronal representations of stimuli can be reorganized or enhanced following learning. While the mammalian OB has been shown to undergo experience-dependent plasticity at the glomerular level, it is still unclear if similar representational change occurs within mitral/tufted cell glomerular odor representations following learning. To address this, odorant-evoked glomerular activity patterns were imaged in mice expressing a GFP-based calcium indicator (GCaMP2 in OB mitral/tufted cells. Glomerular odor responses were imaged before and after olfactory associative conditioning to aversive foot shock. Following conditioning, we found no overall reorganization of the glomerular representation. Training, however, did significantly alter the amplitudes of individual glomeruli within the representation in mice in which the odor was presented together with foot shock. Further, the specific pairing of foot shock with odor presentations lead to increased responses primarily in initially weakly activated glomeruli. Overall, these results suggest that associative conditioning can enhance the initial representation of odors within the olfactory bulb by enhancing responses to the learned odor in some glomeruli.

  4. Alterations of hippocampal place cells in foraging rats facing a "predatory" threat.

    Science.gov (United States)

    Kim, Eun Joo; Park, Mijeong; Kong, Mi-Seon; Park, Sang Geon; Cho, Jeiwon; Kim, Jeansok J

    2015-05-18

    Fear is an adaptive mechanism evolved to influence the primal decisions of foragers in "approach resource-avoid predator" conflicts. To survive and reproduce, animals must attain the basic needs (food, water, shelter, and mate) while avoiding the ultimate cost of predation. Consistent with this view, ecological studies have found that predatory threats cause animals to limit foraging to fewer places in their habitat and/or to restricted times. However, the neurophysiological basis through which animals alter their foraging boundaries when confronted with danger remains largely unknown. Here, we investigated place cells in the hippocampus, implicated in processing spatial information and memory, in male Long-Evans rats foraging for food under risky situations that would be common in nature. Specifically, place cells from dorsal cornu ammonis field 1 (CA1) were recorded while rats searched for food in a semi-naturalistic apparatus (consisting of a nest and a relatively large open area) before, during, and after encountering a "predatory" robot situated remotely from the nest. The looming robot induced remapping of place fields and increased the theta rhythm as the animals advanced toward the vicinity of threat, but not when they were around the safety of the nest. These neurophysiological effects on the hippocampus were prevented by lesioning of the amygdala. Based on these findings, we suggest that the amygdalar signaling of fear influences the stability of hippocampal place cells as a function of threat distance in rats foraging for food.

  5. Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

    Science.gov (United States)

    Lee, W-J; Lee, J-H; Jeon, R-H; Jang, S-J; Lee, S-C; Park, J-S; Lee, S-L; King, W-A; Rho, G-J

    2017-02-13

    Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

  6. The pluralization of the international: Resistance and alter-standardization in regenerative stem cell medicine.

    Science.gov (United States)

    Rosemann, Achim; Chaisinthop, Nattaka

    2016-02-01

    The article explores the formation of an international politics of resistance and 'alterstandardization' in regenerative stem cell medicine. The absence of internationally harmonized regulatory frameworks in the clinical stem cell field and the presence of lucrative business opportunities have resulted in the formation of transnational networks adopting alternative research standards and practices. These oppose, as a universal global standard, strict evidence-based medicine clinical research protocols as defined by scientists and regulatory agencies in highly developed countries. The emergence of transnational spaces of alter-standardization is closely linked to scientific advances in rapidly developing countries such as China and India, but calls for more flexible regulatory frameworks, and the legitimization of experimental for-profit applications outside of evidence-based medical care, are emerging increasingly also within more stringently regulated countries, such as the United States and countries in the European Union. We can observe, then, a trend toward the pluralization of the standards, practices, and concepts in the stem cell field.

  7. Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction.

    Science.gov (United States)

    Coelho, N Miranda; González-García, C; Planell, J A; Salmerón-Sánchez, M; Altankov, G

    2010-06-09

    Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl)silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50microg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine - nearly single molecular size - network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both alpha1 and alpha2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  8. Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction

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    NM Coelho

    2010-06-01

    Full Text Available Considering the structural role of type IV collagen (Col IV in the assembly of the basement membrane (BM and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass and hydrophobic trichloro(octadecylsilane (ODS surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50μg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine – nearly single molecular size – network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC attach less efficiently to the aggregated Col IV (on ODS, as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both α1 and α2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  9. Altered FGF Signaling Pathways Impair Cell Proliferation and Elevation of Palate Shelves.

    Directory of Open Access Journals (Sweden)

    Weijie Wu

    Full Text Available In palatogenesis, palatal shelves are patterned along the mediolateral axis as well as the anteroposterior axis before the onset of palatal fusion. Fgf10 specifically expressed in lateral mesenchyme of palate maintains Shh transcription in lateral epithelium, while Fgf7 activated in medial mesenchyme by Dlx5, suppressed the expansion of Shh expression to medial epithelium. How FGF signaling pathways regulate the cell behaviors of developing palate remains elusive. In our study, we found that when Fgf8 is ectopically expressed in the embryonic palatal mesenchyme, the elevation of palatal shelves is impaired and the posterior palatal shelves are enlarged, especially in the medial side. The palatal deformity results from the drastic increase of cell proliferation in posterior mesenchyme and decrease of cell proliferation in epithelium. The expression of mesenchymal Fgf10 and epithelial Shh in the lateral palate, as well as the Dlx5 and Fgf7 transcription in the medial mesenchyme are all interrupted, indicating that the epithelial-mesenchymal interactions during palatogenesis are disrupted by the ectopic activation of mesenchymal Fgf8. Besides the altered Fgf7, Fgf10, Dlx5 and Shh expression pattern, the reduced Osr2 expression domain in the lateral mesenchyme also suggests an impaired mediolateral patterning of posterior palate. Moreover, the ectopic Fgf8 expression up-regulates pJak1 throughout the palatal mesenchyme and pErk in the medial mesenchyme, but down-regulates pJak2 in the epithelium, suggesting that during normal palatogenesis, the medial mesenchymal cell proliferation is stimulated by FGF/Erk pathway, while the epithelial cell proliferation is maintained through FGF/Jak2 pathway.

  10. Transcorneal electrical stimulation alters morphology and survival of retinal ganglion cells after optic nerve damage.

    Science.gov (United States)

    Henrich-Noack, Petra; Voigt, Nadine; Prilloff, Sylvia; Fedorov, Anton; Sabel, Bernhard A

    2013-05-24

    Traumatic optic nerve injury leads to retrograde death of retinal ganglion cells (RGCs), but transcorneal electrical stimulation (TES) can increase the cell survival rate. To understand the mechanisms and to further define the TES-induced effects we monitored in living animals RGC morphology and survival after optic nerve crush (ONC) in real time by using in vivo confocal neuroimaging (ICON) of the retina. ONC was performed in rats and ICON was performed before crush and on post-lesion days 3, 7 and 15 which allowed us to repeatedly record RGC number and size. TES or sham-stimulation were performed immediately after the crush and on post-injury day 11. Three days after ONC we detected a higher percentage of surviving RGCs in the TES group as compared to sham-treated controls. However, the difference was below significance level on day 7 and disappeared completely by day 15. The death rate was more variable amongst the TES-treated rats than in the control group. Morphological analysis revealed that average cell size changed significantly in the control group but not in stimulated animals and the morphological alterations of surviving neurons were smaller in TES-treated compared to control cells. In conclusion, TES delays post-traumatic cell death significantly. Moreover, we found "responder animals" which also benefited in the long-term from the treatment. Our in vivo cellular imaging results provide evidence that TES reduces ONC-associated neuronal swelling and shrinkage especially in RGCs which survived long-term. Further studies are now needed to determine the differences of responders vs. non-responders.

  11. Smooth muscle cell phenotype alters cocultured endothelial cell response to biomaterial-pretreated leukocytes.

    Science.gov (United States)

    Rose, Stacey L; Babensee, Julia E

    2008-03-01

    Model in vitro culturing systems were developed to analyze roles of biomaterial-induced leukocyte activation on endothelial cell (EC) and smooth muscle cell (SMC) phenotype, and their crosstalk. Isolated monocytes or neutrophils were pretreated with model biomaterial beads and applied directly to "more secretory" (cultured in media containing 5% fetal bovine serum) or forced contractile (serum and growth factor starved) human aortic SMCs (HASMCs), or to the human aortic EC (HAEC) surface of HAEC/HASMC cocultures (HASMC phenotype varied to be "more or less secretory") for 5 or 24 h of static culture. Surface expression of proinflammatory [ICAM-1, VCAM-1, E-selectin], procoagulant (tissue factor), and anticoagulant (thrombomodulin) markers, as well as HAEC proliferation, were assessed by flow cytometry. Incubation of HAEC with biomaterial-pretreated monocytes (and neutrophils to lesser degree) suppressed HAEC proliferation and induced a proinflammatory/procoagulant HAEC phenotype. This HAEC phenotype was amplified in coculture with "more secretory" HASMCs and subdued in coculture with "less secretory" HASMCs. Direct incubation of biomaterial-pretreated monocytes or neutrophils with "more secretory" HASMCs further increased HASMC ICAM-1 and tissue factor expression. Direct incubation of biomaterial-pretreated monocytes or neutrophils with forced contractile HASMCs upregulated ICAM-1, VCAM-1, and tissue factor expression above the presence of serum-containing media alone.

  12. DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4(+) T-Cell Population Structure

    OpenAIRE

    Nestor, Colm E; Fredrik Barrenäs; Hui Wang; Antonio Lentini; Huan Zhang; Sören Bruhn; Rebecka Jörnsten; Langston, Michael A.; Gary Rogers; Mika Gustafsson; Mikael Benson

    2014-01-01

    Altered DNA methylation patterns in CD4(+) T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N(patients) = 8, N(controls) = 8) and gene expression (N(patients) ...

  13. Perioperative dynamic alterations in peripheral regulatory T and B cells in patients with hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Chen Tianxiang

    2012-01-01

    Full Text Available Abstract Background Intratumoral and circulating regulatory T cells (Tregs have been shown to be critical in the pathogenesis of hepatocellular carcinoma (HCC. However there is limited knowledge on the alterations of regulatory B cells (Bregs. We here investigated perioperative dynamic alterations of peripheral circulating Tregs and Bregs in HCC patients to reveal the relationship between regulatory lymphocytes and its clinical implications. Methods 36 patients with HCC, 6 with chronic hepatitis B infection and 10 healthy donors were enrolled for this study. Frequencies of peripheral Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD19 and IL-10 before, and after radical surgery. Then, clinical informatics of HCC patients was achieved through Digital Evaluation Score System (DESS for the assessment of disease severity. Finally, we analysed correlations between digitalized clinical features and kinetics of circulating regulatory lymphocytes. Results Level of circulating CD4+CD25+CD127- Tregs in HCC patients was significantly lower than that in healthy donors and patients with chronic hepatitis B infection before surgery, but was increased after surgery. Preoperative level of CD19+ IL-10+ Bregs in HCC patients was also significantly lower than the other groups. However it dramatically was elevated right after surgery and remained elevated compared to controls (about 7 days after surgery, P = 0.04. Frequency of circulating Tregs was correlated with circulating leukocytes, ferritin, and clinical features suggesting tumor aggressiveness including portal vein thrombosis, hepatic vein involvement and advanced clinical stages. Frequency of circulating Bregs was associated with Hepatitis B e Antigen (HBeAg and Hepatitis B virus (HBV DNA copy number. In addition, DESS was significantly and positively correlated with other staging systems. Conclusion Frequencies of peripheral Tregs and Bregs in HCC patients

  14. Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.

    Science.gov (United States)

    Burga, Laura N; Tung, Nadine M; Troyan, Susan L; Bostina, Mihnea; Konstantinopoulos, Panagiotis A; Fountzilas, Helena; Spentzos, Dimitrios; Miron, Alexander; Yassin, Yosuf A; Lee, Bernard T; Wulf, Gerburg M

    2009-02-15

    Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

  15. Prenatal stress is a vulnerability factor for altered morphology and biological activity of microglia cells.

    Directory of Open Access Journals (Sweden)

    Joanna eŚlusarczyk

    2015-03-01

    Full Text Available Several lines of evidence suggest that the dysregulation of the immune system is an important factor in the development of depression. Microglia are the resident macrophages of the central nervous system and a key player in innate immunity of the brain. We hypothesized that prenatal stress (an animal model of depression as a priming factor could affect microglial cells and might lead to depressive-like disturbances in adult male rat offspring. We investigated the behavioral changes (sucrose preference test, Porsolt test, the expression of C1q and CD40 mRNA and the level of microglia (Iba1 positive in 3 month old control and prenatally stressed male offspring rats. In addition, we characterized the morphological and biochemical parameters of potentially harmful (NO, iNOS, IL-1β, IL-18, IL-6, TNF-α, CCL2, CXCL12, CCR2, CXCR4 and beneficial (IGF-1, BDNF phenotypes in cultures of microglia obtained from the cortices of 1-2 days old control and prenatally stressed pups. The adult prenatally stressed rats showed behavioral (anhedonic- and depression-like disturbances, enhanced expression of microglial activation markers and an increased number of Iba1-immunopositive cells in the hippocampus and frontal cortex. The morphology of glia was altered in cultures from prenatally stressed rats, as demonstrated by immunofluorescence microscopy. Moreover, in these cultures, we observed enhanced expression of CD40 and MHC II and release of pro-inflammatory cytokines, including IL-1β, IL-18, TNF-α and IL-6. Prenatal stress significantly up-regulated levels of the chemokines CCL2, CXCL12 and altered expression of their receptors, CCR2 and CXCR4 while IGF-1 production was suppressed in cultures of microglia from prenatally stressed rats.Our results suggest that prenatal stress may lead to excessive microglia activation and contribute to the behavioral changes observed in depression in adulthood.

  16. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    Science.gov (United States)

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis.

  17. Inhibition of Adhesion, Proliferation, and Invasion of Primary Endometriosis and Endometrial Stromal and Ovarian Carcinoma Cells by a Nonhyaluronan Adhesion Barrier Gel

    Directory of Open Access Journals (Sweden)

    Stefan P. Renner

    2015-01-01

    Full Text Available Endometriosis is a chronic disease of women in the reproductive age, defined as endometrial cells growing outside of the uterine cavity and associated with relapses. Relapses are hypothesized to correlate with incomplete surgical excision or result from nonrandom implantation of new endometrial implants in adjacent peritoneum. Thus, surgical excision could lead to free endometriotic cells or tissue residues, which readhere, grow, and invade into recurrent lesions. Barrier agents are frequently used to prevent postoperative adhesions. We tested if the absorbable cell adhesion barrier gel Intercoat consisting of polyethylene oxide and sodium carboxymethyl cellulose could inhibit cellular adhesion, proliferation, and invasion of primary endometriosis and endometrial cells. Due to an association of endometriosis with ovarian carcinoma, we tested two ovarian carcinoma cell lines. Prior to cell seeding, a drop of the barrier gel was placed in cell culture wells in order to test inhibition of adherence and proliferation or coated over a polymerized collagen gel to assay for prevention of invasion. Results showed that the barrier gel significantly inhibited cell adherence, proliferation, and invasion of endometriosis and endometrial stromal cells as well as ovarian carcinoma cells in culture. Our findings could help to prevent local cell growth/invasion and possible consequent recurrences.

  18. Amine-containing film deposited in pulsed dielectric barrier discharge at a high pressure and its cell adsorption behaviours

    Institute of Scientific and Technical Information of China (English)

    Hu Wen-Juan; Xie Fen-Yan; Chen Qiang; Weng Jing

    2009-01-01

    With monomer allylamine, amine-containing functional films were prepared in alternative current pulsed dielectric barrier discharge (DBD) at a high pressure. This paper analyses in detail the film properties and structures, such as hydrophilicity, compounds and microstructures as well as amine density by the water contact angle, Fourier transform infrared spectroscopy, atomic force microscopy, and ultraviolet-visible measurement. The influence of discharge param-eters, in particular applied power, on amine density was investigated. As an application the cell adsorption behaviours on plasma polymerization films was performed in-vitro. The results show that at a high pressure pulsed DBD plasma can polymerize films with sufficient amine group on surface, through which the very efficient cell adsorption behaviours was demonstrated, and the high rate of cell proliferation was visualized.

  19. Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging

    Science.gov (United States)

    Sahay, Peeyush; Shukla, Pradeep K.; Ghimire, Hemendra M.; Almabadi, Huda M.; Tripathi, Vibha; Mohanty, Samarendra K.; Rao, Radhakrishna; Pradhan, Prabhakar

    2017-04-01

    Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10–12 week-old mice fed a Lieber–DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.

  20. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  1. Specific N-glycan alterations are coupled in epithelial-mesenchymal transition induced by EGF in GE11 epithelial cells.

    Science.gov (United States)

    Xu, Qingsong; Qu, Chen; Wang, Wenjing; Gu, Jianguo; Du, Yuguang; Song, Linsheng

    2017-02-01

    Epithelial-mesenchymal transition (EMT) is a phenomenon in cancer progression during which cancer cells undergo remarkable alteration acquiring highly invasive property. The aim of this study was to evaluate specific N-glycan alterations during EMT induced by epidermal growth factor (EGF) in GE11 epithelial cells. Herein, we demonstrated that EGF activated epidermal growth factor receptor (EGFR)/Akt/extracellular signal-regulated kinase (ERK) phosphorylation and promoted GE11 cell proliferation. Meanwhile, EGF stimulated the epithelial cells to undergo morphological alteration, destroying cell-cell inter-contact and exhibiting mesenchymal cells higher metastatic potential. A wound-healing assay showed the migratory ability increased 1.5-fold after EGF treatment. Moreover, the relative intensity of N-cadherin versus E-cadherin increased 2.6-fold, and the E-cadherin distribution in cell-cell junctions became jagged and faint after EGF incubation for 72 h. Interestingly, the amounts of bisecting GlcNAc structure were dramatically declined, by contrast, the formation of β1,6 GlcNAc branches on cell surface was upregulated during EMT induced by EGF. To understand the roles of N-glycans in EGF-induced EMT, the cells were stably transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the bisecting GlcNAc structure formation. As the markers for EMT, EGF-induced E-cadherin decrease and fibronectin increase were delayed in GnT-III-overexpressing cells. Taken together, these results demonstrated that specific N-glycan alterations were coupled in EMT induced by EGF, which might be contributed to diagnosis and therapy of tumor metastasis.

  2. Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

    Science.gov (United States)

    Mankame, T; Hokanson, R; Fudge, R; Chowdhary, R; Busbee, D

    2006-05-01

    Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

  3. Alterations of T-cell surface markers in older women with persistent human papillomavirus infection

    Science.gov (United States)

    Rodríguez, Ana Cecilia; García-Piñeres, Alfonso J; Hildesheim, Allan; Herrero, Rolando; Trivett, Matthew; Williams, Marcus; Atmella, Ivannia; Ramírez, Margarita; Villegas, Maricela; Schiffman, Mark; Burk, Robert; Freer, Enrique; Bonilla, José; Bratti, Concepción; Pinto, Ligia A

    2012-01-01

    We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case-control study within a 10,049-woman population-based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46 to 74 and with HPV results at their 5th year anniversary visit were considered, and all women (n=87) with persistent HPV infections, all women (n=196) with transient HPV infections and a random sample of HPV DNA-negative women (n=261) frequency-matched to cases on age were selected for this study. A median of 3 years after the case-control matching visit, cervical cells were collected for liquid-based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for three marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95%CI=2.2–13.3) for CD69+CD4+, 2.6 (95%CI=1.2–5.9) for HLADR+CD3+CD4+ and 2.3 (95%CI=1.1–4.7) for CD45RO+CD27−CD8+. A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO+CD27+CD4+ (OR=0.36; 95%CI = 0.17–0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies. PMID:20473864

  4. Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons

    Science.gov (United States)

    Merianda, Tanuja T.; Jin, Ying

    2017-01-01

    Abstract The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons. PMID:28197547

  5. Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

    Science.gov (United States)

    Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID

  6. Repeated stress-induced stimulation of catecholamine response is not followed by altered immune cell redistribution.

    Science.gov (United States)

    Imrich, Richard; Tibenska, Elena; Koska, Juraj; Ksinantova, Lucia; Kvetnansky, Richard; Bergendiova-Sedlackova, Katarina; Blazicek, Pavol; Vigas, Milan

    2004-06-01

    Stress response is considered an important factor in the modulation of immune function. Neuroendocrine hormones, including catecholamines, affect the process of immune cell redistribution, important for cell-mediated immunity. This longitudinal investigation was aimed at evaluating the effect of repeated stress-induced elevation of catecholamines on immune cell redistribution and expression of adhesive molecules. We assessed the responses of epinephrine (EPI), norepinephrine (NE), cortisol, changes in lymphocytes subpopulations, and percentages of CD11a+, CD11b+, and CD62L+ lymphocytes to a 20-min treadmill exercise of an intensity equal to 80% of the individual's Vo(2)max. The exercise was performed before and after 6 weeks of endurance training consisting of a 1-h run 4 times a week (ET) and after 5 days of bed rest (HDBR) in 10 healthy males. We did not observe any significant changes in the basal levels of EPI, NE, and cortisol in the plasma, nor in the immune parameters after ET and HDBR. The exercise test led to a significant (P <.001) elevation of EPI and NE levels after both ET and HDBR, a significant elevation (P <.01) of cortisol after HDBR, an increase in the absolute numbers of leukocytes, granulocytes, monocytes, CD3+, CD4+, CD8+, CD16+, CD19+ lymphocytes, percentage of CD11a+ and CD11b+ lymphocytes, and to a decrease of CD62L1 before, after ET, and after HDBR. We found comparable changes in all measured immune parameters after ET and HDBR. In conclusion, repeated stress-induced elevation of EPI and NE was not associated with an alteration in immune cell redistribution found in response to the single bout of exercise.

  7. Dietary Phenethyl Isothiocyanate Alters Gene Expression in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Young Jin Moon

    2011-01-01

    Full Text Available Phenethyl isothiocyanate (PEITC, a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3 μM or the vehicle. Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc. that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH, were utilized. Arrays from treated and control cells (n=4 per group were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53, cyclin-dependent kinase inhibitor 1C (p57 Kip2, breast cancer Type 2 early onset (BRCA2, cAMP responsive element binding protein 2 (ATF-2, interleukin 2 (IL-2, heat shock 27 KD protein (hsp27, and CYP19 (aromatase. Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.

  8. The role of the spray pyrolysed Al2O3 barrier layer in achieving high efficiency solar cells on flexible steel substrates

    Science.gov (United States)

    Gledhill, Sophie E.; Zykov, Anton; Rissom, Thorsten; Caballero, Raquel; Kaufmann, Christian A.; Fischer, Christian-Herbert; Lux-Steiner, Martha; Efimova, Varvara; Hoffmann, Volker; Oswald, Steffen

    2011-07-01

    Thin film chalcopyrite solar cells grown on light-weight, flexible steel substrates are poised to enter the photovoltaic market. To guarantee good solar cell performance, the diffusion of iron from the steel into the CIGSe absorber material must be hindered during layer deposition. A barrier layer is thus required to isolate the solar module from the metal substrate, both electronically and chemically. Ideally the barrier layer would be deposited by a cheap roll-to-roll process suitable to coat flexible steel substrates. Aluminium oxide deposited by spray pyrolysis matches the criteria. The coating is homogeneous over rough substrates allowing comparatively thin barrier layers to be utilized. In this article, solar cell results are presented contrasting the device performance made with a barrier layer to that without a barrier layer. Secondary Ion Mass spectrometry (SIMS) measurements show that the spray pyrolysed barrier layer diminishes iron diffusion to the chalcopyrite absorber layer. The role of sodium, imperative for the growth of high efficiency chalcopyrite solar cells, and how it interacts with Al2O3 is discussed.

  9. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α

    Directory of Open Access Journals (Sweden)

    Elizabeth K Lucas

    2015-01-01

    Full Text Available Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α -/- mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV, a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2, structural (neurofilament heavy chain, Nefh, and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1 functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α -/- mice. We observed a significant loss of Purkinje cells by six weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α’s actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency.

  10. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α

    Science.gov (United States)

    Lucas, Elizabeth K.; Reid, Courtney S.; McMeekin, Laura J.; Dougherty, Sarah E.; Floyd, Candace L.; Cowell, Rita M.

    2014-01-01

    Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α) have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α−/− mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV), a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2), structural (neurofilament heavy chain, Nefh), and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1) functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α−/− mice. We observed a significant loss of Purkinje cells by 6 weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α's actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency. PMID

  11. Treatment of poly(ethylene terephthalate) foils by atmospheric pressure air dielectric barrier discharge and its influence on cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Kuzminova, Anna [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Vandrovcová, Marta [Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4 (Czech Republic); Shelemin, Artem [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Kylián, Ondřej, E-mail: ondrej.kylian@gmail.com [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Choukourov, Andrei; Hanuš, Jan [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic); Bačáková, Lucie [Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4 (Czech Republic); Slavínská, Danka; Biederman, Hynek [Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University, V Holešovickách 2, 180 00 Praha 8 (Czech Republic)

    2015-12-01

    Highlights: • Effect of atmospheric pressure DBD plasma on PET foils was investigated. • DBD treatment causes increase in surface density of O-containing functional groups. • DBD plasma causes increase of wettability, roughness and complex modulus of PET. • DBD treatment positively influences cells growth on PET. • Enhancement of cell growth on treated PET depends on the cell type. - Abstract: In this contribution an effect of dielectric barrier discharge (DBD) sustained in air at atmospheric pressure on surface properties of poly(ethylene terephthalate) (PET) foils is studied. It is found that exposure of PET to DBD plasma leads to rapid changes of surface chemical composition, wettability, surface morphology as well as mechanical properties of PET surface. In addition, based on biological tests that were performed using two cell types (Saos-2 human osteoblast-like cells and HUVEC human umbilical vein endothelial cells), it may be concluded that DBD plasma treatment positively influences cell growth on PET. This effect was found to be connected predominantly with increased surface energy and oxygen content of the surface of treated PET foils.

  12. Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Alterations of membrane lipid biophysical properties of sensitiveA549 and resistant A549/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A549 cell and 0.194 for the resistant A549/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A549/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that altera-tions of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A549 cell membranes was looser than that of the A549/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A549/DDP cells was also lower than that of A549 cells. Taken together, it was proposed that the al-teration of membrane lipid biophysical state may be involved in the resistance of A549/DDP cells to cisplatin.

  13. Effect of uranium on proliferation and mortality of the major constitutive cell types of the skin: influence on skin barrier integrity

    Energy Technology Data Exchange (ETDEWEB)

    Petitot, F.; Spessotto, S.; Paquet, F. [Inst. de Radioprotection et de Surete Nucleaire, Lab. de Radiotoxicologie Experimentale, IRSN/DRPH/SRBE/LRTOX, Site du Tricastin - Pierrelatte (France)

    2005-07-01

    The skin is the initial barrier against mechanical, chemical or biological external stresses. It is a complex, multilayered organ. The upper layer is the epidermis that is mainly constituted by keratinocytes. The fibroblast is the major cell type of the dermis which is underlying the epidermis. In the case of an external contamination, uranium is able to diffuse through the skin [1-3] and can affect skin barrier integrity after chronic topical exposure[1, 3]. Our study tried to elucidate the cellular mechanisms leading to this skin alteration after uranyl nitrate contamination. Proliferation rate and mortality of primary cultures of rat skin fibroblasts and keratinocytes contaminated in vitro with different concentration of depleted-uranyl nitrate or 233-uranyl nitrate were measured. The huge difference between {sup 233}U and depleted-U specific activities, respectively 3.57 x 10{sup 8}Bq.g{sup -1} and 1.45 x 10{sup 4}Bq.g{sup -1}', allowed to distinguish cellular radiotoxicity and chemotoxicity of uranium. Concerning fibroblasts, a significant radiotoxicity of the emitted alpha particles of {sup 233}U was observed with no chemotoxicity for the lowest concentrations, i.e. 2{mu}M and 4{mu}M, of uranyl nitrate. Keratinocytes were more sensitive to both uranium radiotoxicity and chemotoxicity than fibroblasts. This can be explained by the about three times higher ability of keratinocytes to incorporate uranium compared to fibroblasts. This greater capacity of epidermal cells than dermal cells to incorporate uranium was confirmed in vivo for the hairless rat following a uranyl nitrate topical contamination. As a conclusion, the important toxic effect of uranium on keratinocyte demonstrated in our study can explain the previous observations [1, 3] that epidermis was atrophied and so skin permeability increased after an in vivo chronic topical exposure of rat skin to uranyl nitrate. These results are of great importance concerning radiation protection of exposed

  14. NF-Y activates genes of metabolic pathways altered in cancer cells

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A.; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-01

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells. PMID:26646448

  15. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus

    Directory of Open Access Journals (Sweden)

    Jennifer eKim

    2012-07-01

    Full Text Available Temperature has multiple effects on neurons, yet little is known about the effects of high temperature on the physiology of mammalian central neurons. Hyperthermia can influence behavior and cause febrile seizures. We studied the effects of acute hyperthermia on the immature hippocampus in vitro by recording from pyramidal neurons and inhibitory oriens-lacunosum moleculare (O-LM interneurons (identified by green fluorescent protein expression in the GIN mouse line. Warming to 41°C caused depolarization, spontaneous action potentials, reduced input resistance and membrane time constant, and increased spontaneous synaptic activity of most pyramidal cells and O-LM interneurons. Pyramidal neurons of area CA3 were more strongly excited by hyperthermia than those of area CA1. About 90% of O-LM interneurons in both CA1 and CA3 increased their firing rates at hyperthermic temperatures; interneurons in CA3 fired faster than those in CA1 on average. Blockade of fast synaptic transmission did not abolish the effect of hyperthermia on neuronal excitability. Our results suggest that hyperthermia increases hippocampal excitability, particularly in seizure-prone area CA3, by altering the intrinsic membrane properties of pyramidal cells and interneurons.

  16. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus.

    Science.gov (United States)

    Kim, Jennifer A; Connors, Barry W

    2012-01-01

    Temperature has multiple effects on neurons, yet little is known about the effects of high temperature on the physiology of mammalian central neurons. Hyperthermia can influence behavior and cause febrile seizures. We studied the effects of acute hyperthermia on the immature hippocampus in vitro by recording from pyramidal neurons and inhibitory oriens-lacunosum moleculare (O-LM) interneurons (identified by green fluorescent protein (GFP) expression in the GIN mouse line). Warming to 41°C caused depolarization, spontaneous action potentials, reduced input resistance and membrane time constant, and increased spontaneous synaptic activity of most pyramidal cells and O-LM interneurons. Pyramidal neurons of area CA3 were more strongly excited by hyperthermia than those of area CA1. About 90% of O-LM interneurons in both CA1 and CA3 increased their firing rates at hyperthermic temperatures; interneurons in CA3 fired faster than those in CA1 on average. Blockade of fast synaptic transmission did not abolish the effect of hyperthermia on neuronal excitability. Our results suggest that hyperthermia increases hippocampal excitability, particularly in seizure-prone area CA3, by altering the intrinsic membrane properties of pyramidal cells and interneurons.

  17. Ablation of p120-Catenin Altering the Activity of Small GTPase in Human Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nan LIU

    2009-05-01

    Full Text Available Background and objective p120-catenin (p120ctn, a member of the Armadillo gene family, has emerged as an important modulator of small GTPase activities. Therefore, it plays novel roles in tumor malignant phenotype, such as invasion and metastasis, whose mechanism are not well clarified yet. The aim of this study is to explore the roles of p120ctn on the regulation of small GTP family members in lung cancer and the effects to lung cancer invasions andmetastasis. Methods After p120ctn was knocked down by siRNA, in vivo and in vitro analysis was applied to investigate the role and possible mechanism of p120ctn in lung cancer, such as Western Blot, pull-down analysis, and nude mice models. Results p120ctn depletion inactivated RhoA, with the the activity of Cdc42 and Rac1 increased, the invasiveness of lung cancer cells was promoted both in vitro and in vivo . Conclusion p120ctn gene knockdown enhances the metastasis of lung cancer cells, probably by altering expression of small GTPase, such as inactivation of RhoA and activation of Cdc42/Rac1.

  18. Eugenol alters the integrity of cell membrane and acts against the nosocomial pathogen Proteus mirabilis.

    Science.gov (United States)

    Devi, K Pandima; Sakthivel, R; Nisha, S Arif; Suganthy, N; Pandian, S Karutha

    2013-03-01

    Eugenol, a member of the phenylpropanoids class of chemical compounds, is a clear to pale yellow oily liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, and bay leaf. The antibacterial activity of eugenol and its mechanism of bactericidal action against Proteus mirabilis were evaluated. Treatment with eugenol at their minimum inhibitory concentration [0.125 % (v/v)] and minimum bactericidal concentration [0.25 % (v/v)] reduced the viability and resulted in complete inhibition of P. mirabilis. A strong bactericidal effect on P. mirabilis was also evident, as eugenol inactivated the bacterial population within 30 min exposure. Chemo-attractant property and the observance of highest antibacterial activity at alkaline pH suggest that eugenol can work more effectively when given in vivo. Eugenol inhibits the virulence factors produced by P. mirabilis as observed by swimming motility, swarming behavior and urease activity. It interacts with cellular membrane of P. mirabilis and makes it highly permeable, forming nonspecific pores on plasma membrane, which in turn directs the release of 260 nm absorbing materials and uptake of more crystal violet from the medium into the cells. SDS-polyacrylamide gel, scanning electron microscopy and Fourier transform infrared analysis further proves the disruptive action of eugenol on the plasma membrane of P. mirabilis. The findings reveal that eugenol shows an excellent bactericidal activity against P. mirabilis by altering the integrity of cell membrane.

  19. Alterations of Thymic Epithelial Cells in Lipopolysaccharide-induced Neonatal Thymus Involution

    Directory of Open Access Journals (Sweden)

    Yong-Jie Zhou

    2016-01-01

    Full Text Available Background: Vascular endothelial growth factor (VEGF in the thymus was mainly produced by the thymic epithelial cells (TECs, the predominant component of the thymic microenvironment. The progression of TECs and the roles of VEGF in the neonatal thymus during sepsis have not been reported. This study aimed to explore the alterations of TECs and VEGF level in the neonatal thymus involution and to explore the possible mechanisms at the cellular level. Methods: By establishing a model of clinical sepsis, the changes of TECs were measured by hematoxylin-eosin staining, confocal microscopy, and flow cytometry. Moreover, the levels of VEGF in serum and thymus were assessed based on enzyme-linked immunosorbent assay and Western blotting. Results: The number of thymocytes and TECs was significantly decreased 24 h after lipopolysaccharide (LPS challenge, (2.40 ± 0.46×10 7 vs. (3.93 ± 0.66×10 7 and (1.16 ± 0.14×10 5 vs. (2.20 ± 0.19×10 5 , P < 0.05, respectively. Cortical TECs and medullary TECs in the LPS-treated mice were decreased 1.5-fold and 3.9-fold, P < 0.05, respectively, lower than those in the controls. The number of thymic epithelial progenitors was also decreased. VEGF expression in TECs was down-regulated in a time-dependent manner. Conclusion: VEGF in thymic cells subsets might contribute to the development of TECs in neonatal sepsis.

  20. Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

    Science.gov (United States)

    Bhave, Madhura; Papanikou, Effrosyni; Iyer, Prasanna; Pandya, Koushal; Jain, Bhawik Kumar; Ganguly, Abira; Sharma, Chandrakala; Pawar, Ketakee; Austin, Jotham; Day, Kasey J.; Rossanese, Olivia W.; Glick, Benjamin S.; Bhattacharyya, Dibyendu

    2014-01-01

    ABSTRACT Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number. PMID:24190882

  1. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jose F Abisambra

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

  2. Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

    Science.gov (United States)

    Surmacz, L; Wiejak, J; Wyroba, E

    2001-01-01

    SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

  3. 闭孔泡沫铝声屏障设计%Design of Closed-Cell Aluminum Foam Sound Barriers

    Institute of Scientific and Technical Information of China (English)

    梁李斯; 姚广春; 穆永亮; 华中胜

    2011-01-01

    为改变高速路两侧噪声污染严重,为设计性能更加优越的公路声屏障,结合沈阳市东西快速干道高架桥声屏障建设的工程要求,设计闭孔泡沫铝材的吸声共振型声屏障.使用噪声频谱分析仪实地测量噪声污染情况,利用驻波管法测试打孔闭孔泡沫铝板的吸声系数值.设计的声屏障主要吸声结构为密度0.3 g/cm3,厚度10 mm,打孔率3%的闭孔泡沫铝吸声板,背后设置30 mm厚空腔,其吸声特性为:吸声主频率为300~400 Hz,对500 Hz以下的低频噪声吸声率可达到60 %~90 %.%To reduce noise pollution of expressway and design better-performance sound barriers, an acoustic resonance type sound barrier is designed using closed-cell aluminum foam.The noise spectrum analyzer is used to measure the noise pollution.The sound absorption coefficient is measured using stationary-wave method.The closed-cell aluminum foam panel with 0.3g/cm3 mass density, 10mm thickness and 3% perforation rate are used for the noise barrier.A 30mm thick cavity is designed on its back.The results of measurement show that the main sound absorption frequency range is from 300 to 400Hz, the sound absorption coefficient can reach 60%-90% for the noise frequencies lower than 500Hz.

  4. Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

    Science.gov (United States)

    Vidau, Cyril; Brunet, Jean-Luc; Badiou, Alexandra; Belzunces, Luc P

    2009-06-01

    Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

  5. Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate.

    Science.gov (United States)

    Panzarini, Elisa; Tenuzzo, Bernadette; Palazzo, Fabio; Chionna, Alfonsina; Dini, Luciana

    2006-04-01

    The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.

  6. Silica-sol-based spin-coating barrier layer against phosphorous diffusion for crystalline silicon solar cells.

    Science.gov (United States)

    Uzum, Abdullah; Fukatsu, Ken; Kanda, Hiroyuki; Kimura, Yutaka; Tanimoto, Kenji; Yoshinaga, Seiya; Jiang, Yunjian; Ishikawa, Yasuaki; Uraoka, Yukiharu; Ito, Seigo

    2014-01-01

    The phosphorus barrier layers at the doping procedure of silicon wafers were fabricated using a spin-coating method with a mixture of silica-sol and tetramethylammonium hydroxide, which can be formed at the rear surface prior to the front phosphorus spin-on-demand (SOD) diffusion and directly annealed simultaneously with the front phosphorus layer. The optimization of coating thickness was obtained by changing the applied spin-coating speed; from 2,000 to 8,000 rpm. The CZ-Si p-type silicon solar cells were fabricated with/without using the rear silica-sol layer after taking the sheet resistance measurements, SIMS analysis, and SEM measurements of the silica-sol material evaluations into consideration. For the fabrication of solar cells, a spin-coating phosphorus source was used to form the n(+) emitter and was then diffused at 930°C for 35 min. The out-gas diffusion of phosphorus could be completely prevented by spin-coated silica-sol film placed on the rear side of the wafers coated prior to the diffusion process. A roughly 2% improvement in the conversion efficiency was observed when silica-sol was utilized during the phosphorus diffusion step. These results can suggest that the silica-sol material can be an attractive candidate for low-cost and easily applicable spin-coating barrier for any masking purpose involving phosphorus diffusion.

  7. Target or barrier? The cell wall of early- and later- diverging plants vs cadmium toxicity: differences in the response mechanisms

    Directory of Open Access Journals (Sweden)

    Luigi eParrotta

    2015-03-01

    Full Text Available Increasing industrialization and urbanization result in emission of pollutants in the environment including toxic heavy metals, as cadmium and lead. Among the different heavy metals contaminating the environment, cadmium raises great concern, as it is ecotoxic and as such can heavily impact ecosystems. The cell wall is the first structure of plant cells to come in contact with heavy metals. Its composition, characterized by proteins, polysaccharides and in some instances lignin and other phenolic compounds, confers the ability to bind non-covalently and/or covalently heavy metals via functional groups. A strong body of evidence in the literature has shown the role of the cell wall in heavy metal response: it sequesters heavy metals, but at the same time its synthesis and composition can be severely affected. The present review analyzes the dual property of plant cell walls, i.e. barrier and target of heavy metals, by taking Cd toxicity as example. Following a summary of the known physiological and biochemical responses of plants to Cd, the review compares the wall-related mechanisms in early- and later-diverging land plants, by considering the diversity in cell wall composition. By doing so, common as well as unique response mechanisms to metal/cadmium toxicity are identified among plant phyla and discussed. After discussing the role of hyperaccumulators’ cell walls as a particular case, the review concludes by considering important aspects for plant engineering.

  8. Immunogenicity of in vitro maintained and matured populations: potential barriers to engraftment of human pluripotent stem cell derivatives.

    Science.gov (United States)

    Tang, Chad; Weissman, Irving L; Drukker, Micha

    2013-01-01

    The potential to develop into any cell type makes human pluripotent stem cells (hPSCs) one of the most promising sources for regenerative treatments. Hurdles to their clinical applications include (1) formation of heterogeneously differentiated cultures, (2) the risk of teratoma formation from residual undifferentiated cells, and (3) immune rejection of engrafted cells. The recent production of human isogenic (genetically identical) induced PSCs (hiPSCs) has been proposed as a "solution" to the histocompatibility barrier. In theory, differentiated cells derived from patient-specific hiPSC lines should be histocompatible to their donor/recipient. However, propagation, maintenance, and non-physiologic differentiation of hPSCs in vitro may produce other, likely less powerful, immune responses. In light of recent progress towards the clinical application of hPSCs, this review focuses on two antigen presentation phenomena that may lead to rejection of isogenic hPSC derivates: namely, the expression of aberrant antigens as a result of long-term in vitro maintenance conditions or incomplete somatic cell reprogramming, and the unbalanced presentation of receptors and ligands involved in immune recognition due to accelerated differentiation. Finally, we discuss immunosuppressive approaches that could potentially address these immunological concerns.

  9. Methylglyoxal alters glucose metabolism and increases AGEs content in C6 glioma cells.

    Science.gov (United States)

    Hansen, Fernanda; de Souza, Daniela Fraga; Silveira, Simone da Luz; Hoefel, Ana Lúcia; Fontoura, Júlia Bijoldo; Tramontina, Ana Carolina; Bobermin, Larissa Daniele; Leite, Marina Concli; Perry, Marcos Luiz Santos; Gonçalves, Carlos Alberto

    2012-12-01

    Methylglyoxal is a dicarbonyl compound that is physiologically produced by enzymatic and non-enzymatic reactions. It can lead to cytotoxicity, which is mainly related to Advanced Glycation End Products (AGEs) formation. Methylglyoxal and AGEs are involved in the pathogenesis of Neurodegenerative Diseases (ND) and, in these situations, can cause the impairment of energetic metabolism. Astroglial cells play critical roles in brain metabolism and the appropriate functioning of astrocytes is essential for the survival and function of neurons. However, there are only a few studies evaluating the effect of methylglyoxal on astroglial cells. The aim of this study was to evaluate the effect of methylglyoxal exposure, over short (1 and 3 h) and long term (24 h) periods, on glucose, glycine and lactate metabolism in C6 glioma cells, as well as investigate the glyoxalase system and AGEs formation. Glucose uptake and glucose oxidation to CO(2) increased in 1 h and the conversion of glucose to lipids increased at 3 h. In addition, glycine oxidation to CO(2) and conversion of glycine to lipids increased at 1 h, whereas the incorporation of glycine in proteins decreased at 1 and 3 h. Methylglyoxal decreased glyoxalase I and II activities and increased AGEs content within 24 h. Lactate oxidation and lactate levels were not modified by methylglyoxal exposure. These data provide evidence that methylglyoxal may impair glucose metabolism and can affect glyoxalase activity. In periods of increased methylglyoxal exposure, such alterations could be exacerbated, leading to further increases in intracellular methylglyoxal and AGEs, and therefore triggering and/or worsening ND.

  10. PLAG1 alterations in lipoblastoma: involvement in varied mesenchymal cell types and evidence for alternative oncogenic mechanisms.

    Science.gov (United States)

    Gisselsson, D; Hibbard, M K; Dal Cin, P; Sciot, R; Hsi, B L; Kozakewich, H P; Fletcher, J A

    2001-09-01

    Lipoblastomas are rare soft tissue tumors that occur primarily in young children. They typically contain variably differentiated adipocytes, primitive mesenchymal cells, myxoid matrix, and fibrous trabeculae. Abnormalities in chromosome 8, leading to rearrangements of the PLAG1 gene, were demonstrated recently in four lipoblastomas. In the present report, we determine the frequency of PLAG1 alterations in 16 lipoblastomas from children aged 13 years or younger, and we also evaluate the stages of lipoblastoma differentiation at which PLAG1 genomic alterations are found. Eleven lipoblastomas (69%), including those with either classic or lipoma-like histology, had rearrangements of the 8q12 PLAG1 region. Another three lipoblastomas had polysomy for chromosome 8 in the absence of PLAG1 rearrangement. Only two cases (13%) lacked a chromosome 8 abnormality. Notably, the lipoblastomas with chromosome 8 polysomy had up to five copies of chromosome 8 as an isolated cytogenetic finding in an otherwise diploid cell. We also demonstrate that PLAG1 alterations are found in a spectrum of mesenchymal cell types in lipoblastomas, including lipoblasts, mature adipocytes, primitive mesenchymal cells, and fibroblast-like cells. This finding is consistent with neoplastic origin in a primitive mesenchymal precursor and with variable differentiation to a mature adipocyte end-point. Hence, our studies provide biological validation for the clinical observation that lipoblastomas can evolve into mature, lipoma-like, lesions. They also suggest that PLAG1 dosage alterations caused by polysomy 8 might represent an alternative oncogenic mechanism in lipoblastoma.

  11. Study of Nickel Silicide as a Copper Diffusion Barrier in Monocrystalline Silicon Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Kale, Abhijit; Beese, Emily; Saenz, Theresa; Warren, Emily; Nemeth, William; Young, David; Marshall, Alexander; Florent, Karine; Kurinec, Santosh K.; Agarwal, Sumit; Stradins, Pauls

    2016-11-21

    NiSi as a conductive diffusion barrier to silicon has been studied. We demonstrate that the NiSi films formed using the single step annealing process are as good as the two step process using XRD and Raman. Quality of NiSi films formed using e-beam Ni and electroless Ni process has been compared. Incomplete surface coverage and presence of constituents other than Ni are the main challenges with electroless Ni. We also demonstrate that Cu reduces the thermal stability of NiSi films. The detection of Cu has proven to be difficult due to temperature limitations.

  12. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

    Directory of Open Access Journals (Sweden)

    Valentina Cigliola

    Full Text Available Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36, which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D. GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123 associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.

  13. Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa

    2013-06-01

    Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

  14. Contaminant resistant molten carbonate fuel cell: Annual report, June 1986--June 1987. [Ni hydrogen-permeablel barriers

    Energy Technology Data Exchange (ETDEWEB)

    Remick, R.J.; Jewulski, J.R.; Lu, S.H.

    1987-06-01

    This report summarizes the results of a year-long program evaluating the application of solid nickel foils as hydrogen-permeable barriers to contaminants (H/sub 2/S, HCl, NH/sub 3/) in molten carbonate fuel cells. A parametric study was conducted using 2.5 to 7.5 ..mu..m thick nickel foils in both laboratory-scale and bench-scale fuel cell tests. Two design configurations were evaluated, one in which the foil was placed adjacent to the electrolyte matrix and one in which the foil was placed between two porous metal plaques. In both cases the foil served as a barrier for contaminants. Post-test analysis of electrolyte matrices indicated that both configurations retarded or prevented contaminants from reaching the electrolyte. However, problems were encountered with the first configuration in that gaseous products built up on the electrolyte side of the anode, substantially increasing cell polarization. The second configuration performed significantly better than the first, delivering a performance nearly equal to that of a standard porous metal anode structure. However, the flux of hydrogen crossing the foil in this configuration proved to be sensitive to sulfur contaminants in the fuel. As a consequence, a reduction in current density at constant cell voltage was observed when H/sub 2/S was present in the fuel, despite the fact that no H/sub 2/S reached the three-phase region where electrode, fuel, and electrolyte meet. This behavior, however, may be overcome by using a foil other than pure nickel. 36 refs., 30 figs., 22 tabs.

  15. The effect of altered gravity on immune cells (Ground studies: TRIPLE LUX-A BIOLAB experiment)

    Science.gov (United States)

    Horn, Astrid; Huber, Kathrin; Kuebler, Ulrich; Briganti, Luca; Baerwalde, Sven; Zander, Vanja; Ullrich, Oliver; Hemmersbach, Ruth

    The experiment TRIPLE LUX A, whose performance on Biolab is foreseen for 2010, aims to increase the information about the functioning of immune cells during space flight. Thus, we investigate the impact of altered gravity -microgravity and hypergravity conditions -on the immune response of mammalian macrophages. Previous studies had already demonstrated that phagocytosis in macrophages, an essential step in the innate immune response, is decreased on a fast rotating clinostat. Now, the production of ROS (reactive oxygen species) within the oxidative burst reaction, was measured by means of a luminol assay (luminescence + photo-multiplier technique) comparable to the set up which will be used in the TRIPLE LUX flight hardware. The kinetics of the ROS production was investigated a) under 1 g conditions, b) on a clinostat (with one rotation axis) under varied rotational speed c) in short-term real micro-gravity on a parabolic flight and d) in hypergravity (1.8 g) on the Short Arm Human Centrifuge (SAHC) at DLR Cologne. By means of a photomultiplier clinostat online kinetic luminescent measurements during clinorotation were possible. Permanent fast clinorotation (60 rpm) leads to a dramatic reduction of the oxidative burst signal by up to 60% compared to the signal at 1 g. Slower rotation (30 rpm to 2 rpm) reduces the signal strength even more by up to 90% of the original strength. 60 rpm clinorotation as well as short-term real microgravity (22 s) during parabolic flight likewise decreases the signal of the oxidative burst to a comparable amount, thus the term "simulated weightlessness" is valid for the chosen experimental condi-tion. In contrast, hypergravity leads to a significant signal increase. The results demonstrate a clear effect of altered gravity on the immune response of the macrophages. In the upcoming ISS experiment the established test system (oxidative burst of macrophages) will be tested in continues microgravity within the Biolab hardware, designed by

  16. Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

    NARCIS (Netherlands)

    Rychter, J.; van Nassauw, L.; Brown, J.K.; van Marck, E.; Knight, P.A.; Miller, H.R.P.; Kroese, A.; Timmermans, J.P.

    2010-01-01

    Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMC

  17. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These